In this issue, the JOURNAL initiates a newfeature, “Images from the History of Lep-rosy.” Of all the major maladies of man-kind, few have a history as extraordinary andas well-documented as leprosy. We believethat this JOURNAL provides an appropriateplace in which to collect a series of imagesportraying the depth and richness of this his-tory, and issue an open invitation to mem-bers of the International Leprosy Associa-tion, as well as to other physicians, friends,and institutions who may have in their pos-session valuable images from this multi-faceted history.

Of the greatest interest for this feature arephotographs, sketches, or other images thatillustrate events, discoveries, institutions,and ideas that have played a significant rolein the history of leprosy. In order to avoidhaving this feature become only a gallery ofphysicians and scientists who have workedon leprosy, we do not wish to encourage sub-mission of photographs of these individuals,believing that their contributions are moreappropriately recognized in other ways.

Two major criteria will be applied for theselection of images for this feature: first,

that the subject is important in the history ofleprosy, and second, that the image itself isof high quality. Those who submit imagesfor consideration are asked to provide asmuch documentation as possible concern-ing the subject of the image, as well as doc-umentation about the image itself (includ-ing the source or artist, medium, and datesof creation or publication if known). If youwould like to contribute an image for con-sideration, please communicate first withthe JOURNAL office (ijl@lsu.edu) to describethe nature of the image. Please do not sendoriginals; we will provide contributors withinformation about the preferred methods ofelectronic (or other) reproduction for publi-cation.

This series begins with the haunting por-trait of a 14-year-old girl published in thelandmark atlas of Daniellsen and Boeck in1847. The artist has carefully recorded theclinical details of the macular lesions on hercheeks, but has also captured in her eyes thebewildered look of sadness and apprehen-sion that is familiar to generations of physi-cians who have had the task of advisingtheir patients of this diagnosis.

INTERNATIONAL JOURNAL OF LEPROSY Volume 72, Number 2Printed in the U.S.A.

(ISSN 0148-916X)

INTERNATIONAL JOURNAL OF LEPROSY

and Other Mycobacterial Diseases

VOLUME 72, NUMBER 2 JUNE 2004

Images from the History of Leprosy

119

72, 2 Images from the History of Leprosy 123

IMAGES FROM THE HISTORYOF LEPROSY

Previous page: A young girl with macular lesions of leprosy, 1847.

Reproduced here is Planche IX from the original Atlas Colorié de Spedalskhed [Atlas ofLeprosy] by D. C. Daniellsen and C. W. Boeck. This Atlas is a landmark in the medical his-tory of leprosy, as it represents the beginning of the modern understanding and classifica-tion of this disease.

The Atlas was printed by Trykt i Prahls Lithographs in Bergen, Norway, in 1847. Theimage here is reproduced electronically from an original chromolithograph made from apainting by J. L. Losting. The lithograph measures 49.5 cm × 33.0 cm.

This image and documentation were contributed by the Section of Rare Books—LibraryLuiza Keffer—Instituto Lauro de Souza Lima, Bauru, Brazil.

This image may be viewed in color in the electronic edition of the Journal. Please visitour web-site at leprosy-ila.org, and click on the Journal icon.

1 Received for publication on 24 September 2002. Accepted for publication on 11 March 2004.2 B. Kumar, M.D., MNAMS; S. Dogra, M.D., DNB, MNAMS; I. Kaur, M.D., MNAMS, Department of Der-

matology, Venereology and Leprology, Postgraduate Institute of Medical Education and Research, Chandigarh,India.

Reprint requests to: Prof. Bhushan Kumar, Dept. of Dermatology, Venereology and Leprology, PostgraduateInstitute of Medical Education and Research, Chandigarh-160 012, India. E-mail: kumarbhushan@hotmail.com

INTERNATIONAL JOURNAL OF LEPROSY Volume 72, Number 2Printed in the U.S.A.

(ISSN 0148-916X)

INTERNATIONAL JOURNAL OF LEPROSY

and Other Mycobacterial Diseases

VOLUME 72, NUMBER 2 JUNE 2004

Epidemiological Characteristics of Leprosy Reactions:

15 Years Experience from North India1

Bhushan Kumar, Sunil Dogra, and Inderjeet Kaur2

ABSTRACTA retrospective analysis of patient’s leprosy clinic records at PGIMER, Chandigarh, In-

dia for the period 1983 to 1998 was undertaken to study the frequency, time of onset, andrisk factors for leprosy reactions. Of the 2600 cases analyzed, 1494 were multibacillary and1106 had paucibacillary disease. Presentation with reaction was common with 30.9% of ourpatients having reactions at the time of first visit. The incidence of reversal reaction (RR)was highest during 6 to 12 months after starting multi-drug therapy (MDT), thereafter de-clining gradually. Late RR occurred in 9.5% of all cases and was noted up to 7 years aftertreatment. Female gender, widespread disease, and multibacillary disease were identified asrisk factors for RR. Erythema nodosum leprosum (ENL) reactions were noted to occurmostly during second or third year after starting MDT. Of the total number of patients whoexperienced ENL, 64.3% had recurrent episodes which continued for up to 8 years after thestart of treatment. Lepromatous leprosy, female gender, and high Bacterial Index (≥3) wererecognized as risk factors for developing ENL. Occurrence of recurrent and late reactions,even though of mild severity, highlights the importance of recognizing and treating thempromptly to prevent or reduce morbidity, complications, and further deterioration in the dis-ability status. Although it is hoped that leprosy will have been eliminated at all levels by2005, the recognition and management of these reactions will continue to be the most es-sential/significant task in the post elimination era.

RÉSUMÉUne analyse rétrospective des dossiers cliniques de patients traités à la clinique contre la

lèpre du PGIMER de Chandigarh aux Indes fut conduite de 1983 à 1998, afin d’étudier lafréquence, la date d’apparition et les facteurs de risque des réactions lépreuses. Parmi les2600 cas analysés, 1494 souffraient de maladie multibacillaires et 1106 de maladie pau-cibacillaire. Les réactions étaient fréquentes au moment de la première visite, étant de30,9%. L’incidence de réactions reverses (RR) fut la plus élevée pendant les 6 à 12 moissuivant le début de la polychimiothérapie (PCT), puis déclinèrent graduellement. Des RRtardives apparurent dans 9,5% du nombre total de cas et furent détectées jusqu’à 7 annéesaprès traitement. Le sexe féminin, maladie disséminée et maladie multibacillaires furent

125

126 International Journal of Leprosy 2004

With the success of multi-drug therapy(MDT) in the treatment of leprosy, attentionhas focused on the problem of leprosy reac-tions, which are now the most significant is-sue in the management of individual pa-tients. Despite a large burden of leprosycases in India, very limited data has beenpublished on the epidemiology of reactionsfrom this part of the world. The availableinformation about the epidemiology of lep-rosy reactions is incomplete and scanty, de-spite a growing amount of literature on itstreatment.

Postgraduate Institute of Medical Educa-tion and Research (PGIMER), Chandigarh,is a tertiary care institute in Northern India,which is a low endemic area for leprosy.Many patients with leprosy are self-referredand some are referred by a doctor or clinic,citing the better quality of care available atour center. In addition to the population ofthis region, the institute also caters to a

large migrant population from variousstates of the country where leprosy is en-demic. In the leprosy clinic at the institute,a good record keeping system combinedwith regular evaluation of patients has gen-erated a very large and useful database forretrospective analysis of reactions in lep-rosy. Systematic analysis of these recordswas carried out to determine the incidenceof leprosy reactions in our patients, and toidentify risk factors if any. In this paper, wediscuss the incidence and risk factors forleprosy reactions over the last 15 years.

MATERIALS AND METHODSA total of 2867 new, previously untreated

patients enrolled in the leprosy clinic atPGIMER during the period from 1983 to1998 were included in the study. Patientswith pure neuritic leprosy were excludedfrom this analysis. Until 1987 all patientswith Bacterial Index (BI) ≥2 were classified

identifiés comme des facteurs de risques de la RR. L’érythème noueux lépreux (ENL) futprincipalement détecté durant les deuxième et troisième années après le début de la PCT.Parmi les patients ayant déclarés un ENL, 64,3% ont souffert d’épisodes cliniques récurrentset ce, pendant parfois plus de 8 années après le début du traitement. Une lèpre lépromateuse,un sexe féminin et un index bactérioscopique élevé (≥3) furent identifiés comme des facteursde risque pour le développement d’un ENL. La survenue de réactions récurrentes ou tar-dives, même si elles sont de faible sévérité, souligne l’importance de les reconnaître et de lestraiter rapidement afin de prévenir ou réduire la morbidité, les complications et une plusgrande détérioration de l’état de handicap. Cependant, l’espoir de voir la lèpre éliminée àtous les niveaux vers 2005 est terni par l’enjeu essentiel, qui se devra d’être significatif, dereconnaître et de traiter ces réactions dans l’ère de l’après éradication.

RESUMENSe realizó un estudio retrospectivo de los expedientes clínicos de los pacientes con lepra

en el PGIMER de Chandigarh, India, del periodo 1983 a 1998, para analizar la frecuencia,el tiempo de aparición, y los factores de riesgo de las reacciones leprosas (RL). De los 2600casos analizados, 1494 fueron multibacilares y 1106 paucibacilares. La presencia de reac-ción leprosa fue común; 30.9% de los pacientes tuvieron RL en el tiempo de su primeravisita. La incidencia de reacción reversa (RR) fue más alta entre los 6 y 12 meses despuésde haber iniciado el tratamiento con poliquimioterapia (PQT) y después declinó gradual-mente. La RR tardía ocurrió en el 9.5% de todos los casos y se observó hasta 7 años despuésdel tratamiento. El género femenino, la enfermedad diseminada y la enfermedad bacilar, seidentificaron como factores de riesgo para la RR. Las reacciones tipo eritema nudoso leproso(ENL) se observaron más frecuentemente durante el segundo y tercer año del inicio de laPQT. Del total de los pacientes que desarrollaron ENL, el 64.3% tuvieron episodios recur-rentes que continuaron hasta 8 años después de haber iniciado el tratamiento. La enfermedadlepromatosa, el género femenino y los IB altos (≥3) se reconocieron como factores de riesgopara el ENL. La ocurrencia de las reacciones recurrentes y tardías, aunque sean de severidadmoderada, subraya la importancia de reconocerlas y tratarlas prontamente para prevenir opara reducir la morbilidad, las complicaciones y el progreso de las deformidades incapaci-tantes. Aunque se espera que la lepra se haya podido eliminar en todos sus niveles hacia elaño 2005, el reconocimiento y manejo de las reacciones leprosas continuará siendo el retomás importante y esencial en la era de la post-eliminación de la enfermedad.

72, 2 Kumar, et al.: Epidemiology of Leprosy Reactions 127

as multibacillary (MB), and <2 as pau-cibacillary (PB). From 1988 to 1998, all pa-tients having positive slit skin smear wereassigned to the MB group. Until 1994, allMB cases were treated with WHO MDTMB regimen for a minimum period of 2 yrsor until skin smear negativity, whicheveroccurred last. From 1994 to 1998, theywere treated with fixed duration (24months) MDT MB regimen.

After release from treatment (RFT), pa-tients were seen at intervals of 3 to 6months for the initial 2 years and later onceor twice a year. Apart from this, patientswere instructed on features of reactions/re-lapse, and told to attend the clinic immedi-ately if they ever experience such symp-toms or any other problems. A detailed clin-ical examination was done on each visit andskin smears are taken at least once a year.Details about time of onset and clinical fea-tures of reactions are recorded in the pa-tient’s record file.

Reversal reaction (RR) involving skinwas diagnosed if the patient had rednessand swelling of (already existing) lesions,or the appearance of a few new lesionsclose to the existing lesions or at distantsites, with or without tenderness of lesions.Neuritis as a part of reaction was defined asspontaneous pain (shooting, tingling, orburning), or tenderness of the nerves withnerve function impairment (NFI). In mostpatients with reactions, features of con-comitant skin and nerve involvement werepresent. Erythema nodosum leprosum(ENL) was diagnosed if a patient developedmultiple, usually small, tender, evanescentnodules, with or without ulceration, whichwere usually associated with constitutionalsymptoms. Wherever indicated, reactionalepisodes were treated with oral pred-nisolone (40–60 mg/day) tapered to a stopover 12 weeks. Occurrence of a RR aftersix months of stopping MDT was labeled aslate RR. Recurrent episodes of RR or ENLwere defined as having a recurrence ofsymptoms of either type of reaction morethan six weeks after the completion of treat-ment for reaction. Recurrence of a reac-tional episode earlier than this was consid-ered to be possibly due to inadequate orabrupt stoppage of treatment. The exactdefinitions of the spectrum of the diseaseand duration of treatment with MDT were

not uniform in all the patients due to chang-ing definitions of PB/MB cases by theWHO; however, from a purely academic/research point of view we have also tried toanalyze trends of reactional events atpresentation, during MDT, and after releasefrom treatment, according to Ridley Joplingclassification (5) among subgroups of PB/MBcases. For validation of our supposition andbetter understanding, we have used certainterminologies like “disseminated disease”and “late ENL” because of the absence (ornot very satisfactory) definitions about re-actions. Patients were labelled as having“disseminated disease,” if there were ≥3body areas involved, 6 or more skin lesions,or involvement of at least 2 peripheralnerve trunks. We have used the terminology“late ENL” for occurrence of ENL reaction2 years after MDT completion.

The chi square test was used to analyzethe difference between proportions and toidentify trends. The difference between twounpaired sample means was tested using thestudent’s t-test. The significance of variousrisk factors for developing reactions wasanalyzed with Cox’s proportional hazardsregression, and the results were expressedas rate ratios. Of the ratio, the 95% confi-dence interval is given.

RESULTSPatients. Out of these 2867 patients, 62

patients were excluded, either because thediagnosis was changed or records were notcomplete. Another 205 patients were lost tofollow-up or left the area after diagnosis. Atotal of 2600 patients with follow-up avail-able up to 13 yrs (at least 3 yrs) were eligi-ble for analysis. Average period of follow-up was 74 months (range 36 to 156months). There were 1634 males (mean age36 ± 3.2 yrs) and 966 females (mean age 39± 2.3 yrs). Out of them, 1494 (57.5%) hadMB and 1106 (42.5%) had PB disease.

Incidence. The incidence of reversal re-actions (RR) and erythema nodosum lepro-sum (ENL), and their distribution inPB/MB cases at the time of first presenta-tion to the clinic is given in Table 1. The in-cidence of RR was 24.1% (627/2600 pa-tients) and the figure for ENL in MB caseswas 11.8% (177/1494 patients). Of the 627patients with RR, 169 (27%) had evidenceof reaction in skin lesions only and the re-

128 International Journal of Leprosy 2004

maining 458 (73%) had involvement ofboth skin and nerves, whereas in patientshaving ENL, 86 (48.6%) had only cuta-neous involvement and 91 (51.4%) had in-volvement of both skin and nerves.

In the total period of observation, 858 pa-tients experienced RRs and 337/1494 MBcases had ENL either at the time of registra-tion, during, or after release from treatmentwith cumulative incidence of 33% and22.5%, respectively. Altogether, there were1356 episodes of RR in 858 patients (1.6 re-action episodes/patient) and 885 episodesof ENL in 337 patients (2.6 reactionepisodes/patient). Of these 337 MB cases,203 were treated before 1994 and the restreceived fixed duration (24 months) MDT(p <0.05).

Time of onset. Figures about the reac-tional episodes according to the period oftime they occurred are given in Table 2. Agreat majority of RRs occurred during thefirst 6 months after starting MDT whereas,

the episodes of ENL occurred at a higherfrequency in the second or third year afterstarting MDT.

Late reactions. Late reversal reactionswere seen in 4.2% (47/1106) of PB patientsand 5.3% (79/1494 patients) with MB dis-ease. The majority of late reactions in themultibacillary group were observed in pa-tients with borderline lepromatous (BL)disease. The incidence of late RR was high-est during the first 2 yrs after being releasedfrom treatment (RFT). Even though the de-cline in the incidence of RR began early af-ter the initiation of therapy, and most reac-tions occurred during the first 2 yrs, a fewcontinued to occur until 7 yrs after RFT.

Late ENL reactions were seen in only 3%(45/1494) of MB [BL and polar leproma-tous (LL)] patients. Late ENL, though mildand usually not associated with significantconstitutional symptoms, continued to oc-cur for up to 8 yrs after completion ofMDT. Of these patients, 15 were treated be-

TABLE 2. Time of onset of reactional episodes.

Pauibacillary Multibacillary (N = 1494)

Period BT (N = 1106) BB (N = 82) BL (N = 902) LL (N = 510)

No. % No. % No. % No. %

Reversal reactions

At registration 228 44.5 18 27.3 379 49.0 2 400–6 months 144 28.1 20 30.3 191 24.7 3 607–12 months 65 12.6 15 22.7 114 14.7 — —2nd year 46 9.0 13 19.7 46 5.9 — —≥3 years 29 5.7 0 0 43 5.5 — —

Total 512 10000 66 10000 773 10000 5 100

ENL Reactions

At registration — — — — 35 19.4 142 00 20.10–6 months — — — — 19 10.5 91 00 12.97–12 months — — — 31 00 17.2 12300 00 17.42nd year — — — — 59 32.8 192 00 27.2≥3 years — — — — 36 20.0 157 00 21.8

Total 180 100% 705 100%

TABLE 1. Prevalence of reactions at the time of first presentation.

Reversal reaction ENLClassification No. of

Skin Skin + Total Skin Skin + Totalcasesonly nerves (%) only nerves (%)

MB 1494 108 291 399 (26.7) 86 91 177 (11.8)PB 1106 61 167 228 (20.6) 0 0 170 (11.8)

Total 2600 169 458 627 (24.1) 86 91 177 (11.8)

72, 2 Kumar, et al.: Epidemiology of Leprosy Reactions 129

fore 1994 and the remaining 30 with fixedduration regimen (24 months) (p <0.01).However, there was no statistically signifi-cant difference among the number of MBpatients manifesting late RRs treated before1994 (43/79) and those treated with fixedduration regimen (36/79) (p >0.1).

Recurrent reactions. Of the 858 patientswho manifested reversal reactions, 252(29.4%) had ≥2 episodes and the remaining606 (70.6%) had only a single episode ofRR. Cumulative incidence of recurrent re-versal reaction was 9.7% (252/2600)among all of the patients. The presence of areversal reaction at the time of initial pre-sentation or during the first year of treat-ment was more frequently associated withan increased risk of having another episodeoccurring later on.

Out of the total patients who experiencedENL, 217/337 (64.4%) experienced morethan one episode. The median number ofepisodes was 4 and the time between thefirst and last episode averaged 34 months(range 5 to 96 months). A considerablenumber of patients (51/217, 23.5%) hadmore than ≥4 episodes of ENL over a pe-riod of observation varying from 3 years to8 years, with no other identified risk factorexcept a higher BI (BI ≥3).

Risk factors. Female gender, multibacil-lary disease, and widespread disease (≥3body areas involved, ≥6 skin lesions, or ≥2peripheral nerve trunks involvement) werestatistically significant risk factors for de-

veloping reversal reactions. The strongestassociation was observed between the ex-tent of clinical disease and the risk of devel-oping RR. Of the 858 patients manifestingRRs, 544 (63.4%) had ≥3 body areas in-volved and/or ≥6 skin lesions, and 523(61%) had ≥2 nerve trunk involvement.Age was not found to be a significant riskfactor for reversal reactions (Table 3).

Risk factors for developing ENL reac-tions are given in Table 4. Lepromatous lep-rosy, female gender and higher BI (≥3)were significant risk factors, whereas agewas not.

DISCUSSIONIncidence. Much is known about the epi-

demiology of reactions, but their incidencein the period after MDT is less well docu-mented because of lack of long term follow-up (6). Various estimates of the frequency ofreversal reactions have been given by sev-eral authors (1, 6, 17). Published reports indi-cate that the frequency of RR at the time ofdiagnosis varies between 2.6% and 6.4% (6)though a much higher figure of 28% was re-ported in a hospital-based study from Nepal(17). In our study, this figure was 24.1%.Other studies have reported relatively lowertotal figures of 9.1% from Hyderabad, India(9) and 16.5% from Ethiopia (14), probablyreflecting a variable proportion of PB/MBcases and the use of mixed case definitionsused. Figures for the percentage of patientsmanifesting RRs at any time vary from

TABLE 3. Risk factors for developing reversal reactions (RR).

Patient groupRisk factors PB/MB/Both No. Rate ratio* (95%CI) p value

Spectrum PB 314/11060.69 (0.58–0.81) p <0.01

MB 544/1494

Age (years)≤20 Both 120/357

1.0 (0.81–1.3) p >0.1>20 Both 738/2243

SexMale Both 510/1634

0.80 (0.68–0.95) p <0.05Female Both 348/966

Extent of clinical disease≥6 skin lesions Both 544/858 2.5 (2.1–2.9) p <0.01≥2 nerves involved Both 523/858 2.4 (2.0–2.8) p <0.01≥3 Body areas involved Both 544/858 2.8 (2.2–3.1) p <0.01

*Rate ratio adjusted for the influence of age and sex

130 International Journal of Leprosy 2004

3.5% among PB cases in Malawi (1) to47.5% among MB cases in Zaire (6). Due tothe use of widely different case definitions,it is difficult to compare the frequencies ofRR in PB and MB patient groups in studiespublished from different centers. Other fac-tors contributing to such variation could bethe time between diagnosis and beginningMDT, duration of MDT, duration of steroidregimen, and quality of the local leprosycontrol program. Overall 33% of all pa-tients in our study developed RR at sometime during treatment and follow-up, in-cluding those with a reaction at presenta-tion.

ENL reactions were reported to occur inmore than 50% of lepromatous leprosy(LL) cases and in about 25% of borderlinelepromatous (BL) cases in the pre-MDT era(7). The incidence of ENL reactions appearsto have fallen with the introduction ofMDT, possibly due to the combined bacte-ricidal effect of rifampicin and the anti-inflammatory effect of clofazimine in sup-pressing ENL (8). A hospital-based studyfrom Nepal reported a high frequency ofENL reactions (28.6%) in LL, but only7.5% in BL cases (5). In the present study,47.4% of LL cases and 10.5% of BL casesmanifested ENL reactions. The lower inci-dence of ENL among patients treated be-fore 1994 could be because of the persistinganti-inflammatory effect of clofazimine tillbacterial clearance was achieved. Fieldstudies have reported a rather lower inci-dence of ENL such as 12% in LL and 3.6%in BL from Ethiopia (9), and 2.1% of allMB patients from Bangladesh (10). The var-ied frequency of ENL in reported studiescould be due to patients in the fieldscreened for reactions vs. patients reporting

to the hospitals for reactions. Most of ourpatients were self-reporting having reac-tions severe enough to force them to seektreatment from a hospital rather than field-based clinics. This could be one reason forrelatively higher figures for ENL in ourcenter.

In reactions (RR or ENL) involvement ofthe skin and nerves occurred either singlyor together. Of the total number of reactionsat the time of presentation, 31.7% had onlycutaneous involvement, whereas 68.3% hadinvolvement of both skin and nerves. In aretrospective analysis of reversal reactionsin a study from Hyderabad, India, 43.1%had only skin lesions, 31.8% had only neu-ritis, and 22.7% had both skin lesions andneuritis (6). Such observations emphasizethat neuritis can occur along with inflam-matory skin lesions or independently.Therefore, even very mild symptoms sug-gestive of neuritis should be taken seriouslyand nerves should be palpated on each visitto detect early signs of nerve inflammation,regardless of presence or absence of reac-tion involving skin.

Time of onset of reactions. Signifi-cantly, 30.9% of our patients presented tous because of reactions, in spite of havingsymptoms suggestive of leprosy for monthsor years. In a hospital-based study from Hy-derabad, India, Lockwood, et al. (6) notedreactions in a strikingly high percentage oftheir patients (41.3%) at the time of presen-tation. It is obvious that many patients seektreatment only when they get frightened bythe sudden development of such lesionsparticularly over face, or painful symptomsof neuritis due to reaction.

Although it is known that the reversal re-actions occur most frequently within 6 to 12

TABLE 4. Risk factors for development of ENL reactions.

Factors Variables No. Rate ratio* p value

Disease group BL 95/902 .12 (0.09–0.17) p <0.01LL 242/510

Age <20 34/238 1.1 (0.75–1.6) p >0.1>20 303/2322

Sex Male 192/1634 .75 (0.59–0.95) p <0.05Female 145/966

BI <3 120/2109 07 (0.05–0.09) p <0.01≥3 217/491

*Rate ratio adjusted for the influence of age, sex and bacteriological index

72, 2 Kumar, et al.: Epidemiology of Leprosy Reactions 131

months after starting treatment (4, 11), mostprevious studies did not report long termfollow-up. In the AMFES data (Ethiopia),RR were reported to occur as late as 5 yrsafter the start of treatment in both PB andMB patients (12). Our study also supportsthe fact that though the incidence of RRwas found to decline gradually, reactionscontinued to occur for 7 years, though invery few patients. However, in India thelongest interval reported between treatmentand reaction is 6.5 years (6).

The majority of ENL reactions after start-ing MDT occurred in the second or thirdyear and the recurrence of attacks wasmostly noted in the same period, though thereactions continued to occur up to 8 yrs af-ter RFT.

Late reactions. The percentage of pa-tients developing late RR in both PB andMB groups was 4.2% and 5.3%, respec-tively. In a study from Thailand, 2.7% ofPB patients and 9% of MB patients devel-oped a late reversal reaction (15). In Malawi,3.5% of PB patients developed late reac-tions during the first 4 yrs of follow-up (2).In a cohort of MB patients (treated withMDT for 2 yrs or longer until split-skinsmear tested negative) from Karigiri, India,only 1.1% experienced reversal reactionsduring almost 10 yrs of surveillance (18).Late RR is known to occur mostly withinthe first 3 to 4 yrs after RFT (1, 15) as wasalso observed by us. In the absence of aclear definition for late RR and differentMDT regimens used in various studies, anexact comparison of the frequency of lateRRs is not possible.

Late ENL reactions were recorded in 3%of our MB cases. The majority of theseepisodes consisted of a few ENL lesions,which were mostly associated with onlymild cutaneous and constitutional symp-toms.

Recurrent reactions. Recurrent episodesof reversal reactions are an important clini-cal phenomenon, which may result in con-tinuing nerve damage and add on to the de-gree and number of impairments. In ourstudy, 9.7% patients developed recurrentepisodes of reversal reaction. Almost halfof these episodes occurred within 3 monthsof stopping the course of prednisolone thathad been administered for the previous re-action. Strikingly, in a hospital-based study

from Hyderabad, India, 33% of patientswith RRs had recurrent episodes (9). Theimmune suppression in some of these pa-tients may have been for of too short a du-ration, or hospitals may be more likely toget the severely affected problem patients,which require still longer treatment withsteroids or some other adjuvant therapy.Possibly, Naafs, et al. may be right whenthey suggest that immunosuppressive treat-ment should continue throughout the periodwhen the antigen load is sufficient to triggerthe cell mediated immune response (10, 11).

Of all patients who manifested ENL,64.3% had recurrent episodes. In theAMFES cohort (Ethopia), 63% of all caseswith ENL had more than one episode, and31% of all ENL cases manifested 5 or moreepisodes over a period of more than 2 yrs.In general, recurrence in episodes of reac-tions is more common in ENL than in RR,and approximately one-third of patientswith ENL reactions go on to have recurrentepisodes (14, 16). Patients must be warned ofthis possibility and be educated to return forfollow-up, and clinicians should also beaware to diagnose even very late reactionsin post-elimination era. Though it could bein variance to other proposed definitions(Transactions of 16th International LeprosyCongress), we suggest that a patient couldbe labeled as having “chronic ENL” if heneeds continued antireaction treatment for aperiod of 6 months or more.

Risk factors. Risk factors for RR identi-fied to be significant in this study werefemale gender, and disseminated disease(extent of clinical disease measured by in-volvement of a number of body areas,nerves, and skin lesions) at the time of di-agnosis. PB patients had less risk of devel-oping reversal reactions than MB patients.Patients with three or more body areas in-volved or having ≥6 skin lesions had abouttwice the risk of developing RR than thosewith limited disease (63.4% vs. 36.6%).Similar observations (>10 skin lesions)have been made by Van Brakel, et al. (17),which suggested that this can be of consid-erable importance for control programs, toidentify patients at risk of developing reac-tions. They argued that “body area” mayjust be a proxy indicator for the bacterio-logical index or multibacillary end of theleprosy spectrum. Like our observation,

132 International Journal of Leprosy 2004

they also noted similar association withinthe borderline tuberculoid (BT) patient sub-group, indicating that body area count isuseful indicator of the risk of developingRR. Pregnancy and lactation are reported tobe risk factors for RR and ENL (6, 8), but theassociation has not been quantified and re-mains unclear. Leprosy lesions over theface have also been observed to developRR more frequently (3). However, the rela-tion between pregnancy or lactation andleprosy reactions, the significance of apatch over the face as a risk factor for de-veloping reversal reactions, could not bestatistically analyzed in the present studydue to lack of complete information in therecords.

For ENL, the risk factors identified in ourstudy were lepromatous leprosy, female gen-der, and higher bacteriological index (≥3).For recurrent ENL, a number of risk factorslike age, sex, spectrum of disease were ana-lyzed, but only high BI (≥3) was found to bestatistically significant. In AMFES cohort(Ethopia), no specific risk factor for recur-rent ENL could be identified except age, be-tween 20 and 45 yrs (14). In our study,though the majority of cases were in thisage range, correlation with any age groupcould not be confirmed. ENL reactions arereported to occur throughout pregnancy andlactation, and may be severe and recurrent;however, because of incomplete informa-tion as stated above, this relationship couldnot be anlyzed in our cohort.

In conclusion, this is the largest series ofpatients with long term follow-up, delineat-ing the epidemiology of reactions in leprosyfrom India. RR and ENL are common com-plications in leprosy patients in India. Fe-male gender, multibacillary leprosy, and ex-tensive disease were found to be major riskfactors for occurrence of RRs. The majorityof RRs occurred within 12 months of start-ing MDT, and then the incidence declinedgradually but the reactions continued to oc-cur until 7 yrs after RFT. For ENL, lepro-matous leprosy, female gender and higherbacteriological index (≥3). Significant riskfactors were approximately one-third of pa-tients with ENL reactions go on to manifestrecurrent episodes spread out over a periodof more than 2 yrs requiring specialized ex-pertise to manage them. Though leprosy isexpected to be eliminated from all nations

by 2005, even those patients who have suc-cessfully completed their treatment willcontinue to manifest with late or recurrentreactions in settings of poorly available ex-pertise or services to manage theseepisodes.

REFERENCES1. BECX-BLEUMINK, M., and BERHE, D. Occurrence

of reactions, their diagnosis and management inleprosy patients treated with multidrug therapy:experience in the Leprosy Control Program of theAll Africa leprosy and Rehabilitation TrainingCenter (ALERT) in Ethiopia. Int. J. Lepr. OtherMycobact. 60 (1992) 173–184.

2. BOERRIGTER, G., PONNIGHAUS, J. M., FINE, P. E.M., and WILSON, R. J. Four years follow-up re-sults of a WHO-recommended multidrug regimenin paucibacillary patients in Malawi. Int. J. Lepr.Other Mycobact. Dis. 59 (1991) 255–261.

3. BRITON, W. J. The management of leprosy rever-sal reactions (Editorial). Lepr. Rev. 69 (1998)225–234.

4. DE RIJK, A. J., GEBRE, S., BYASS, P., and BERHANU, T.Field evaluation of WHO-MDT of fixed duration atALERT, Ethiopia: the AMFES project, Part 2. Reac-tions and neuritis during and after MDT in PB andMB leprosy patients. Lepr. Rev. 65 (1994) 320–332.

5. JOPLING, W. H., and MCDOUGALL, A. C. In:Handbook of Leprosy, 5th edn. New Delhi: CBSPublishers. 1996, pp. 10–47.

6. LIENHARDT, C., and FINE, P. E. M. Type 1 reac-tion, neuritis and disability in leprosy. What is thecurrent epidemiological situation? Lepr. Rev. 65(1994) 9–33.

7. LOCKWOOD, D. N. J. The management of ery-thema nodosum leprosum: current and future op-tions (Editorial). Lepr. Rev. 67 (1996) 253–259.

8. LOCKWOOD, D. N. J., and SINHA, H. H. Pregnancyand leprosy: a comprehensive literature review.Int. J. Lepr. Other Mycobact. Dis. 67 (1999) 6–12.

9. LOCKWOOD, D. N. J., VINAYAKUMAR, S., STANLEY,J. N. A., MCADAM, P. W. J., and COLSTON, M. J.Clinical features and outcome of reversal (type 1)reactions in Hyderabad, India. Int. J. Lepr. OtherMycobact. Dis. 61 (1993) 8–15.

10. NAAFS, B. Treatment of reactions and nerve dam-age. Int. J. Lepr Other Mycobact. Dis. 64 (1996)S21–S28.

11. NAAFS, B., PEARSON, J., and WHEATE, H. Reversalreaction: The prevention of permanent nerve dam-age. Comparison of short and long term steroidtreatment. Int. J. Lepr. Other Mycobact. Dis. 47(1979) 7–12.

12. RICHARDUS, P., FINLAY, K. M., CROFT, R. P., andSMITH, W. C. Nerve Function impairment in lep-rosy at diagnosis and at completion of MDT: a ret-rospective cohort study of 786 patients inBangladesh. Lepr. Rev. 67 (1996) 297–305.

13. ROSE, P., and WALTERS, M. F. Reversal reactions

in leprosy and their management (Editorial) Lepr.Rev. 62 (1991) 131–121.

14. SAUNDERSON, P., GEBRE, S., and BYASS, P. Rever-sal reactions in the skin lesions of AMFES pa-tients: incidence and risk factors. Lepr. Rev. 71(2000) 309–317.

15. SCHREUDER, P. A. M. The occurrence of reactionsand impairments in leprosy: experience in the lep-rosy control program of three provinces in North-eastern Thailand, 1978–1995. II. Reactions. Int. J.Lepr. Other Mycobact. Dis. 66 (1998) 159–169.

16. SCOLLARD, D. M., SMITH, T., BHOOPAT, L., THEETRA-NONT, C., RANGDAENG, S., and MORENS, D. M. Epi-demiologic characteristics of leprosy reactions. Int.J. Lepr. Other Mycobact. Dis. 62 (1994) 559–567.

17. VAN BRAKEL, W. H., KHAWAS, I. B., and LUCAS, S.Reactions in leprosy: an epidemiological study of386 patients in West Nepal. Lepr. Rev. 65 (1994)190–203.

18. VIJAYKUMARAN, V., MANIMOZHI, N., and JESU-DASAN, K. Incidence of late lepra reaction amongmultibacillary leprosy after MDT. Int. J. Lepr.Other Mycobact. Dis. 63 (1995) 18–22.

19. WEMAMBU, S. N., TURK, J. L., WATERS, M. F., andREES, R. J. Erythema nodosum leprosum. A clini-cal manifestation of the Arthus phenomenon.Lancet 2 (1969) 933–935.

20. WHO EXPERT COMMITTEE ON LEPROSY. Seventhreport. Geneva: World Health Organization, 1998.Tech. Rep. Ser. 874.

72, 2 Kumar, et al.: Epidemiology of Leprosy Reactions 133

1 Received for publication on 15 October 2003. Accepted for publication on 24 February 2004.2 P. R. N. A. G. Stump, M.D.; R. Baccarelli, F. T., Ph.D.; Lúcia H. S. C. Marciano, O.T., M.S.; S. Ura, M.D.;

M. C. L. Virmond, M.D., Ph.D., Dept. of Rehabilitation, Instituto Lauro de Souza Lima, Bauru, Brazil; J. R. P.Lauris, M.D., Ph.D., Dentistry School, U.S.P., Bauru, Brazil; M. J. Teixeira, M.D., Ph.D., School of Medicine,U.S.P., São Paulo, Brazil.

Reprint requests to: Marcos Virmond, CP 3021 Bauru–SP, Brazil. 17034-971. E-mail: mvirmond@ilsl.br

Neuropathic Pain in Leprosy Patients1

Patrick R. N. A. G. Stump, Rosemari Baccarelli, Lúcia H. S. C. Marciano, José R. P. Lauris, Manoel Jacobsen Teixeira, Somei Ura, and Marcos C. L. Virmond2

ABSTRACTThe introduction of multidrug therapy by the World Health Organization has dramatically

reduced the world prevalence of leprosy but the disease is still a public health problem inmany countries, with a world prevalence of almost 600,000 cases in 2001. Damage to pe-ripheral nerves is a key component of leprosy and the sensory and motor loss that follows isthe basis for many of the classical features of this disease, such as skin wounds, cracks, plan-tar ulcers, clawed hands, drop foot, and incomplete closure of the eyelids. One of the mostremarkable aspects of leprosy to lay persons and health care workers alike is that patients arereputed to feel no pain. However, neuropathic pain is arising as a major problem among lep-rosy patients. It can be nociceptive due to tissue inflammation, which mostly occurs duringepisodes of immune activation or neuropathic due to damage or dysfunction of the nervoussystem. This study, conducted among 358 leprosy patients, reveals a considerable preva-lence of neuropathic pain and presents evidence that this common problem should be a highpriority of those in charge of leprosy control programs.

RÉSUMÉL’introduction de la poly-chimiothérapie par l’Organisation Mondiale de la Santé a

diminué de façon drastique la prévalence mondiale de la lèpre mais la maladie est encore unproblème de santé publique dans plusieurs pays, avec une prévalence mondiale de presque600 000 cas en 2001. L’atteinte des nerfs est une composante clef de la lèpre et les pertessensorielles et motrices qui s’ensuivent forment la base des caractères classiques de cettemaladie, comme les blessures cutanées, les fissures, les ulcères de la plante des pieds, lesmains en crochets, les pieds tombants et la fermeture incomplète des paupières. Un des as-pects les plus remarquables de la lèpre pour le grand public comme pour le prestataire desoins de santé est que les patients ont la réputation de ne pas ressentir de douleur. Cependant,les douleurs neurogènes sont en train d’émerger comme un problème majeur parmi les pa-tients hanséniens. Elle peut être nociceptive, due à l’inflammation qui apparaît principale-ment durant les épisodes d’activation immunologique, ou bien neurogène, causée par une at-teinte ou une dysfonction du système nerveux. Cette étude, menée chez 358 patients lépreux,révèle une prévalence considérable de douleurs neurogènes et présente des arguments afinque les responsables de santé publique et de contrôle de la lèpre considèrent ce problèmecomme une priorité.

RESUMENNo obstante que la introducción de la poliquimioterapia por la Organización Mundial de

la Salud ha reducido dramáticamente la prevalencia de la lepra a nivel mundial, la enfer-medad es todavía un problema de salud pública en muchos países, con una prevalencia decasi 600,000 casos en 2001. El daño a los nervios periféricos es un componente crítico de lalepra y la pérdida sensorial y motora que le siguen es la base de muchas de las característi-cas clásicas de la enfermedad que incluyen heridas en la piel, cuarteaduras, úlcerasplantares, manos en garra, pie caído y cierre incompleto de los párpados. Uno de los aspec-tos más remarcables de la lepra es la creencia general de que los pacientes no sienten dolor.Sin embargo, el dolor neuropático se está manifestando como un problema cada vez mayorentre los pacientes con lepra. El dolor puede ser enmascarado por la inflamación del tejidoque ocurre principalmente durante los episodios de activación inmune o neuropática asoci-

134

INTERNATIONAL JOURNAL OF LEPROSY Volume 72, Number 2Printed in the U.S.A.

(ISSN 0148-916X)

Since the introduction of an effectivetreatment based upon a multi-drug therapy(MDT) of dapsone, clofazimine, and ri-fampicin as recommended by the WorldHealth Organization (WHO), the preva-lence of leprosy has dramatically decreased(13). However, the disease is still a publichealth problem in many countries with anestimated global prevalence of near600,000 cases as for the year 2003 (14).

Damage to peripheral nerves is a keycomponent of leprosy and, together withtypical skin lesions, accounts for the majortraditional clinical features of the disease.Little is known about the mechanism bywhich the mycobacteria infect Schwanncells, but recently some evidence hasemerged. A glycoprotein (α-dystroglican)that binds to the surface of Mycobacteriumleprae also binds to a molecule on the sur-face of the Schwann cell surface and pro-vides a potential mechanism for internaliza-tion of the bacilli by Schwann cells (1, 9).

The sensory and motor loss that followsnerve damage in leprosy is the basis formany of the classical features of the diseasesuch as skin wounds, cracks, plantar ulcers,clawed hands, drop foot, and lagophtalmos.Sensory damage includes an early loss ofpain and temperature perception followedby compromise of tactile and pressuresenses. The distribution and onset of nervedamage can vary according to the type ofleprosy, being more disseminated and grad-ual in the lepromatous cases, or localizedand acute in tuberculoid and borderlinecases. The indeterminate type is an initialpresentation of the disease in which majornerve damage has not yet developed.

One of the most remarkable aspects ofleprosy to lay persons and health care work-ers alike is that patients are reputed to feelno pain. This wide-spread impression israpidly changing as many patients whohave completed their WHO MDT are nowreporting complaints of stimulus-independentongoing pain, and seeking relief. Indeed,pain in leprosy can be nociceptive due totissue inflammation, which mostly occurs

during episodes of immune activation [“re-versal reaction” (RR) and “erythema no-dosum leprosum” (ENL)] or neuropathicdue to damage or dysfunction of the ner-vous system. Nociceptive pain is due toactivation of peripheral nociceptors on A-delta and C-fibers, secondary to nervetissue injury during ENL or RR. There is arelease of bradykinin, serotonin, substanceP, histamine and prostaglandin, which facil-itate the transmission of pain impulses fromthe periphery to the spinal cord.

The complex and stigmatizing burden ofbeing diagnosed with leprosy may compelpatients to focus solely upon curing the dis-ease, which is actually accomplished withthe WHO drug regimen, and to accept theirsymptoms as an inevitable concomitant orresidual of the disease. However, the num-ber of cases with pain problems seemed tous to be substantial (4). The aims of thisstudy, conducted in a country where leprosyis endemic, were to estimate the prevalenceof pain in patients with leprosy and to de-termine the main characteristics of theirpain.

MATERIALS AND METHODSThe study was conducted at the Instituto

Lauro de Souza Lima, Bauru, Brazil, a na-tional referral center for leprosy patients.Brazil has a prevalence of 77.676 cases per100,000, and an important detection rate of24.1/100,000 (41.070 new cases in 2000),which makes it the second largest endemiccountry for leprosy in the world after India(14).

The study included 358 patients with lep-rosy who presented to the Dermatologicalclinic of Instituto Lauro de Souza Limafrom October 1, 2001 to March 31, 2002.Among them, 215 were male (60.1%) and143 were female (39.9%). The mean agewas 54.8 yrs (S.D. = 16 yrs), with a rangeof 11 to 87 yrs.

The mean time from initial diagnosis was18.3 yrs (range, 10 months to 68 yrs, S.D. =18.5 yrs), and 178 (49.7%) patients were di-agnosed over 10 years earlier. According to

ada al daño o disfunción del sistema nervioso. El presente estudio, realizado en 358 pa-cientes, revela una considerable prevalencia de dolor neuropático, y presenta evidencias deque este es un fenómeno común que debe ser de alta prioridad para aquellos encargados delos programas de control de la lepra.

72, 2 Stump, et al.: Neuropathic Pain 135

136 International Journal of Leprosy 2004

the Madrid Classification (8), 207 patients(57.8%) were lepromatous, 92 (25.7%) bor-derline, 54 (15.1%) tuberculoid, and only 5(1.4%) were indeterminate. All cases werereceiving treatment except for 283 (79.1%)patients who had concluded their standardcourse by the time of the study. Treatmentregimens included the WHO MDT, dapsoneplus rifampicin, or dapsone as monotherapyin some previously treated cases.

Two hundred and one (56.1%) of the pa-tients reported past or current moderate tosevere chronic neuropathic pain that in-terfered with activities of daily living ordisturbed sleep. None of these cases re-vealed signs or symptoms of RR or ENL asassessed by an experienced clinician. Thesecases underwent clinical neurological exam-ination by trained health workers, includingdetailed anamnesis assessment focusing onthe occurrence of pain, its localization, dura-tion, pattern of symptoms onset, quality, andquantity. Localization of pain refers to theanatomical distribution and trunk of themost relevant affected peripheral nerve.Duration of pain was classified as less orgreater than 6 months. The pattern of symp-tom onset was categorized as abrupt, insid-ious, or in repetitive bursts. Assessment ofquality of pain was based upon the Brazil-ian Portuguese version of the McGill PainQuestionnaire (12). In addition, we inquiredwhether present pain was experienced assuperficial, deep, or mixed. Pain intensitywas verbally rated by patients as mild,moderate, or severe and was also rated on agraphic scale (empty to full water glass).

RESULTSUsing the day of interview and examina-

tion as the reference point, 148 (73.6%) pa-tients reported episodes of pain only in thepast and 53 (26.4%) had complaints at pres-ent. In the 148 patients with past pain only,leprosy had been diagnosed less than 10years earlier in 59 cases (39.8%) and morethan 10 years earlier in 89 cases (60.2%). Inthose with present pain, only 14 (26.4%)cases had been diagnosed over 10 years ear-lier. Pain had been present for 6 months orless in 55 cases (27.4%), whereas 141(70.1%) reported pain for longer than 6months. Only 5 patients (2.5%) could notestimate the duration of their pain. Thenerve most often affected by pain was the

ulnar (59.2%), followed by the tibial(30.3%), fibular (18.9%), median (4.5%),radial (2.0%), and trigeminal (1.5%). Thesepercentages sum to greater than 100 be-cause patients were free to indicate pain inthe distribution of more than one nerve.Glove (22.4%) and stocking (24.9%) distri-bution of pain were also quite common(Fig. 1). The onset of episodes was reportedas abrupt by 39 patients (19.4%), as insidi-ous by 73 patients (36.3%), and as recurrentbursts by 89 (44.3%) patients. The assess-ment of quality of pain can be seen in Fig. 2.

In the 53 patients with pain present at thetime of interview, the most affectedanatomical layer was deep in 30 (56.6%)patients, superficial in 8 (15.1%) and mixedin 15 (28.3%). In these patients, pain wasconstant in 34 (64.2%) and episodic in 19(35.8%). Verbal ratings of present painwere severe in 29 (54.7%), moderate in 17(32.1%), and mild in only 7 patients(13.2%). On the graphic scale, 22 (41.5%)patients rated their pain as severe, 21(39.6%) as moderate, and 10 (18.9%) asmild. These pain characteristics are summa-rized in The Table.

DISCUSSIONLack of sensation is a paradigm of

leprosy, and the diagnosis of this chronicinfectious disease is assured by the pres-ence of skin lesions (usually patchy) withmarked sensory loss as assessed bySemmes-Weinstein monofilaments or aballpoint pen tip. Abnormalities includeloss of touch, temperature, and pressuresensation. Although clinical consensus re-gards leprosy as painless, in reality nervepain in leprosy is often present during neu-ritis, a feature that accompanies acute lep-rosy reactions. These reactive episodes in-clude entrapment of the nerve in selectedsites (most often the ulnar canal at the el-bow) due to edema from acute and severeinflammation of the nerve. In such a situa-tion, activation of the nervi nervorum maybe the main contributor to pain. Anotherpossibility is that the acute neural inflam-mation can excite and sensitize nociceptors.In some cases, there is severe destruction ofnerve fibers (2) and the partial regenerationthat follows may produce discharges,diminution of stimulus thresholds, and ex-aggerated responses of nociceptors.

72, 2 Stump, et al.: Neuropathic Pain 137

This study reveals that neuropathic painnot directly associated with an acute reactiveepisode may be present in a considerableproportion of patients with leprosy. In fact,out of 358 patients presenting to the outpa-tient dermatological clinic for other reasons,56.1% reported prior or current episodes ofneuropathic pain. Most of these patients re-ported that the intensity was severe and suffi-cient to interfere with activities of daily life orsleep.

According to the literature (3, 5), the mostcommon nerve affected in leprosy is the ul-nar nerve, and we confirmed the frequencyof this as a painful site. However, a gloveand stocking distribution of pain was alsofrequently reported by our patients. Al-though involvement of nerve trunks in lep-rosy is common, the superficial branches

and their rami may be also compromised,particularly in lepromatous and borderlinecases in which dissemination of the diseaseis a characteristic feature.

It is important to note that, among pa-tients with present pain, 40 patients (75.4%)had completed antimicrobial treatment and,according to the present policy of leprosycontrol, are discharged from further follow-up. Such patients receive little further care.Therefore, a significant number of patientsdid not have ongoing access to care andcould not seek assistance for relief of neu-ropathic pain and improvement of qualityof life. In addition, 130 patients (87.8%)with past pain had already completed treat-ment by the time their pain occurred. Thus,successful completion of antimicrobialtreatment does not appear to prevent occur-

THE TABLE. Some characteristics of neuropathic pain among 201 cases of leprosy withpast (148) or present (53) pain.

Groups Past pain* % Past pain* %

Clinical form Lepromatous 94 63 26 49Borderline 29 20 20 38Tuberculoid 24 16 7 13Indeterminate 1 0.6 0 0

Onset Abrupt 30 20 9 17Insidious 47 32 26 49Bursts 71 48 18 34

Duration in months <6 months 49 33 6 11>6 months 94 63 47 89Not known 5 3 0 0

Time of leprosy diagnosis <10 years 59 40 39 74>10 years 89 60 14 26

Treatment completion Yes 130 88 40 75No 18 12 13 25

Pain intensity Mild — 10 19Moderate — 21 40Severe — 22 41

Verbal rating of pain intensity Mild — 7 13Moderate — 17 32Severe — 29 55

Time of worst pain Morning — 7 13Afternoon — 9 17Evening — 15 28Not specific — 22 41

Anatomical layer of involvement Superficial — 8 15Deep — 30 57Mixed — 15 28

Evolution Worsening — 20 43Stable — 24 51Remitting — 9 19

Character Episodic — 19 36Constant — 34 64

*Number of patients

138 International Journal of Leprosy 2004

rence of neuropathic pain. As a matter offact, the teams caring for patients with lep-rosy are not generally aware of the problemof neuropathic pain. Furthermore, whenconsidering the indication for classicaldrugs for both RR and ENL, there appearsto be some misunderstanding as to what isresponsible for the pain component. Some-times, it appears that health personnel be-lieve that thalidomide or steroids can act asanalgesics, which is not true. Of course,their action on reducing edema and attenu-ating immunological aspects of reaction canlead to a reduction in pain. Conversely, inpure neurophatic pain there is no room forsuch drugs. Therefore, it is of utmost im-portance that control of neuropathic pain inleprosy be included as an issue to be dealtwith by leprosy control program managers.Control and treatment of neuropathic painin leprosy has not yet been well studied.However, the experience with the treatmentof such pain in other conditions has stimu-lated the use of some standard drugs incases of leprosy (6, 7, 10, 11). In this regard,some good results have been reported whileusing tryciclic antidepressant drugs such asaminotriptyline and imipramine. Anticon-vulsant agents can also be used, such as car-bamazepine and gabapentin. It is importantto note that these drugs are analgesic with acentral action and, therefore, do not interferein the nerve damage process, either in its re-covery or worsening.

CONCLUSIONThis study reveals a considerable preva-

lence of neuropathic pain among patientswith leprosy and presents evidence that thiscommon problem should be a high priorityamong those in charge of leprosy controlprograms. In fact, given the present publichealth policy of shorter regimens and imme-diate discharge of patients after completionof treatment, this problem could worsen fur-ther. Thus, at present there is a strong needto review the concept of leprosy care to pro-vide adequate attention to this disablingcomplication, and plenty of room for stud-

ies on new therapies to cope with this previ-ously ignored clinical problem.

REFERENCES1. FREEDMAN, V. H., WEINSTEIN, D. E., and KAPLAN,

G. How Mycobacterium leprae infects peripheralnerves. Lepr. Rev. 70 (1999) 136–139.

2. GARBINO, J. A. Manejo clínico das diferentes for-mas de comprometimento da neuropatia han-sênica. Hansen Int. special (1998) 93–99.

3. HASTINGS, R. C. Leprosy, 2nd ed. New York:Churchill Livingstone, 1993.

4. HIETAHARJU, A., CROFT, R., ALAM, R., BIRCH, P.,MONG, A., and HAANPAA, M. Chronic neuropathicpain in treated leprosy. Lancet 356 (2000)1080–1081.

5. JOPLING, W. H., and MCDOUGALL, A. C. Hand-book of Leprosy, 5th ed. New Delhi: CBS Publish-ers and Distributors, 1996.

6. KLOKE, M., HOFFKEN, K., OLBRICH, H., andSCHMIDT, C. G. Antidepressants and anticonvulsantsfor the treatment of neuropathic pain syndromes incancer patients. Onkologie 14 (1991) 40–43.

7. MAX, M. B., CULNANE, M., SCHAFER, S. C.,GRACELY, R. H., WALTHER, D. J., SMOLLER, B., andDUBNER, R. Amitriptyline relieves diabetic neu-ropathy pain with normal or depressed mood.Neurology 37 (1987) 589–596.

8. OPROMOLLA, D. V. A. Noções de Hansenologia.Bauru: Centro de Estudos Reynaldo Quagliatto,2000.

9. RAMBUKKANA, A., SALZER, J. L., YURCHENCO, P.D., and TUOMANEN, E. I. Neural targeting of My-cobacterium leprae mediated by the G domain ofthe laminin-alpha 2 chain. Cell 88 (1997) 881–821.

10. ROCKLIFF, B. W., and DAVIS, E. H. Controlled se-quential trials of carbamazepine in trigeminal neu-ralgia. Arch. Neurol. 15 (1966) 129–136.

11. ROWBOTHAM, M., HARDEN, N., STACY, B., BERN-STEIN, P., and MAGNUS-MILLER, L. Gabapentinfor the treatment of postherpetic neuralgia: a mul-ticenter double-blind, placebo-controlled study.JAMA 280 (1998) 1837–1842.

12. TEIXIRA, M. J., and PIMENTA, C. A. M. Avaliaçãodo doente com dor. In: Dor: epidemiologia, fi-siopatologia, avaliação, síndromes dolorosas etratamento. São Paulo: Moreira Jr., 2001. pp.56–68.

13. WORLD HEALTH ORGANIZATION. Guide to Elimi-nate Leprosy as a Public Health Problem.Geneva: World Health Organization, 2000.

14. WORLD HEALTH ORGANIZATION. Weekly Epi-demiol. Rec. 77 (2002).

1 Received for publication on 19 September 2003. Accepted for publication on 14 January 2004.2 E. Jayaseelan, DNB; and V. V. Aithal, DVD, DNB, Department of Dermatology, St. John’s Medical College

and Hospital, Bangalore, India.Reprint requests to: Dr. Vijay V. Aithal, Lecturer, Department of Dermatology, St. John’s Medical College and

Hospital, Sarjapur Road, Bangalore-560 034, India. E-mail : vijay_aithal@hotmail.com, or vijay_aithal@rediffmail.com

Pinch Skin Grafting in Non-Healing Leprous Ulcers1

Elizabeth Jayaseelan and Vijay V. Aithal2

ABSTRACTTreatment of leprous ulcers has remained inadequate, owing to the fact that most of these

ulcers are still being managed conservatively especially in developing nations, probably dueto financial constraints. Pinch skin grafting, though obsolete now (2), tries to bridge this gapbetween cost and effectiveness. It is a simple office-based technique, not requiring much ex-pertise or investment, and can be done in a simple set-up such as a side room (3). Also, pinchskin grafting has an added advantage over single grafts, in that even if one graft is rejected,there are other grafts, which successfully heal, and epidermize to the surrounding. Moreover,if the ulcer is draining, the discharge flows out in between the grafts, thus preventing thewhole graft from being rejected. The only disadvantage to pinch skin grafting is the finalcosmetic appearance, which might not be most pleasing.

We had very good results with all four patients who underwent this procedure in our in-stitution. The procedure and the final result are described in detail in this report.

RÉSUMÉLe traitement des ulcères lèpreux est resté insuffisant, parce que la majorité de ces ulcères

est encore appréhendée de manière conservatoire, en particulier dans les pays en voie dedéveloppement, qui souffrent de contraintes financières encore importantes. La greffe depeau par pincement, quoique obsolète de nos jours (2), essaye de relier l’incompatibilité en-tre faible coût et bonne efficacité. C’est une technique simple de pratique courante, ne né-cessitant que peu d’expertise et d’investissement, qui peut être réalisée dans une salle desoin (3). De plus, la greffe de peau par pincement présente l’avantage par rapport à la greffesimple d’être formée de plusieurs greffes, si bien que si une d’entre elles est rejetée, la cica-trisation et l’épidermisation peut être assurée par les autres si elles réussissent. Enfin, si l’ul-cère est fistulisé dû à un drainage, celui-ci peut s’effectuer entre les greffes, évitant ainsi àtoute l’aire greffée d’être rejetée. Le seul désavantage à la greffe par pincement est l’aspectcosmétique final, qui n’est pas toujours très plaisant.

Nous avons eu de très bons résultats chez 4/4 patients qui ont subi cette intervention ànotre institution. La procédure et le résultat final sont présentés en détail dans cet article.

RESUMENEl tratamiento de las úlceras leprosas se ha mantenido inadecuado debido a que la may-

oría de éstas todavía se manejan de manera conservadora especialmente en los países en víasde desarrollo, probablemente por dificultades económicas. El procedimiento de injerto devarios fragmentos de piel aunque obsoleto ahora (2), trata de cubrir este hueco entre costo yefectividad. Se trata de una técnica simple que no requiere mucha experiencia o inversión, yque puede hacerse casi en cualquier área adaptada del consultorio (3). El procedimiento deinjertos múltiples también tiene la ventaja sobre la aplicación de injertos únicos, de que auncuando uno de estos injertos se rechace, todavía quedan otros injertos que generalmentesanan exitosamente y epidermizan la zona a su alrededor. Además, si la úlcera estádrenando, la descarga fluye entre los injertos, evitando así que estos sean rechazados. Laúnica desventaja de la técnica de injertos múltiples de piel es la apariencia cosmética final,la cual puede ser no muy agradable.

Nosotros hemos tenido muy buenos resultados en 4 pacientes sometidos a este proced-imiento en nuestra institución. El procedimiento y el resultado final se describen en esteartículo.

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140 International Journal of Leprosy 2004

Non-healing ulcers resulting from lep-rosy pose a great threat not only due to theirdisability, but also their morbidity. A simpletechnique like pinch grafting would reducethis morbidity. As compared to single sheetfull thickness skin grafts, pinch grafts areeasy, inexpensive, and can be done in a mi-nor operating theater, or as an office-basedprocedure.

Most pinch grafts have been described assplit-thickness grafts (2), but they are full-thickness centrally and thin split-thicknessat the periphery (3). The proceedure in-volves placing small slices of split skin overthe dermabraded ulcer site, and allowing itto heal by epithelialization.

MATERIALS AND METHODSThis technique was done on 4 leprosy pa-

tients with non-healing ulcers, located atvarious sites and of differing duration (TheTable). All 4 were selected from the leprosyrehabilitation unit of the Dermatology de-partment at St. John’s Medical CollegeHospital, Bangalore, India.

Inclusion criteria: (i) Chronic non-healing ulcers due to leprosy (of more than2 months duration); (ii) A clean ulcer bedwith healthy granulation tissue and goodvascularity.

Exclusion criteria: (i) Systemic diseasesnot under control, such as uncontrolled ane-mia and diabetes mellitus; (ii) Ulcers notprimarily due to leprosy.

Serous discharge, though copious, wasnot a contraindication. Also, those with ahyperkeratotic edge were taken up, aftersaucerizing the edge, to increase vascularity(Fig. 1)

The donor area was prepared by cleaningthe skin with Povidone-Iodine solution(Betadine®) and surgical spirit. The lateralaspect of the mid-thigh was selected as thedonor area, although abdomen and buttockshave also been used as donor sites (5). Afield block was given by injecting Ligno-caine 2%, so as to cover the entire donorarea, an area approximately 5 cm × 5 cm forall 10 grafts.

Although various techniques have beendescribed for taking pinch grafts (1, 5),werecommend the shave technique using a hy-podermic needle (Fig. 2), which has beendescribed earlier, though some authors con-sider it obsolete (4). A 23 or 24 gauge needlewas inserted into the skin at the donor area,and brought out 1 cm away, so that the skintaken would involve an area of 1cm2. Theneedle was pushed at a plane, as superfi-cially as possible. The skin was then slicedinto a thin layer using a scalpel, just belowthe needle, at the approximate level of thepapillary dermis. This not only ensuresminimal blood loss, but also enhances thechance of healing by way of epithelializa-tion. The grafts were then placed in a sterilecontainer containing normal saline and aguaze pad. Adequate numbers of graftswere taken so as to cover the entire ulcer.The donor area was then closed with an an-tibiotic-impregnated pre-sterilized gauzetulle (Sofratulle®), gauze pads, and an adhe-sive elastic plaster (Dynaplast®).

The recipient area, usually anesthetic,did not require local anesthetic infiltration.The ulcer bed was dermabraded, using amanual dermabrader. Once the area startedbleeding, the grafts were placed over the re-

FIG. 1. Clean ulcer without secondary infection,ready to be grafted.

FIG. 2. Shave technique of harvesting the pinchgraft donor site.

72, 2 Jayaseelan and Aithal: Pinch Skin Grafting for Ulcers 141

cipient site, making sure the dermal sidefaced downwards. The grafts were placedas close to one another as possible (Fig. 3).After the whole area was covered, it wasclosed with an antibiotic-impregnated pre-sterilized gauze tulle (Sofratulle®), gauzepads, and an adhesive elastic plaster (Dy-naplast®). Joint areas and areas with a highdegree of mobility were immobilized in asplint. The dressing was kept for a week,with a change in dressing once every 3 to 4days. Postoperative care included immobi-lization, avoidance of wetting the grafted

site, a course of antibiotics, analgesics, sup-plementary zinc, and vitamins.

After a week the dressing was removed,and the grafts looked pinkish-yellow and sod-den due to the collection of serous dischargeunderneath the dressing. It took another weekor two for inter-space epithelialization to becompleted (Fig. 4). After 4 to 6 weeks, thewhole ulcer area was covered by skin.

RESULTSIn all 4 patients, the ulcers healed com-

pletely (The Table and Fig. 4). Two of the

FIG. 3. Grafts placed in the recipient ulcer site. FIG. 4. Two weeks post-surgery, inter-graft epithe-lialization was completed.

THE TABLE. Details of patients who underwent Pinch skin grafting.

Site & DurationPatient Age/ Duration area of of Treatment Time for

No. Sex of ulcer the ulcer leprosy received healingRemarks

1 42 year 2 months Lt. lat. 18 years Monotherapy- 1 month Immediate responseF maleolus, Dapsone for 2 years not very good.

3 cm × 3 cm Antibiotics, Gradual healing in analgesic for 1 month.the ulcer.

2 49 year 6 months Lt. shin. 10 cm 20 years No treatment for 11⁄2 OsteomyelitisM × 5 cm Leprosy. months (clinically,

Antibiotics, radiologically, andanalgesics for culture-proved), the ulcer. treated adequately.

Anemia corrected. Gradual healing in 11⁄2 months.

3 38 year 1 year Rt. ankle antr. 6 years MDT(?)-2 years. No 15 days Gradual healingM 3 cm × 5 cm treatment for ulcer in 15 days

4 40 year 1 year Rt. knee antr. 10 years MDT-for 2 years. 15 days Immobilized theM 4 cm × 4 cm Antibiotics, recipient area in a

analgesics for Bohler’s iron the ulcer. splint. Gradual

healing in 15 days.

142 International Journal of Leprosy 2004

patients had ulcers over difficult sites, viz.prepatellar and anterior to the ankle. One ofthe patients who had underlying osteo-myelitis proven clinically and radiologi-cally, and culture-positive for aerobic andanaerobic micro-organisms, also did wellunder cover of appropriate antibiotics forthe osteomyelitis.

DISCUSSIONPinch skin grafting for non-healing ulcers

is a simple procedure, which can be doneby any doctor with minimal training (1). Tothe best of our knowledge, this techniquehas not been previously reported for leprousulcers. Single sheet split-thickness skingrafts have been described for the treatmentof non-healing ulcers; however, pinch graft-ing seems to have certain advantages oversingle sheet grafting.

Advantages of pinch skin graft oversingle sheet skin graft (split or full thick-ness) (1). (i) Pinch grafting can be done inulcers having unfavorable local factors,such as those with copious serous dis-charge. If a single sheet of skin is placed insuch a situation, the whole graft tends to getthrown off, due to collection of the dis-charge underneath. In contrast, in pinchgrafts the discharge is allowed to drain fromthe inter-space. (ii) Even if one graft getsrejected, the other grafts would still take upand the rejected area would epithelializefrom the adjacent grafts. (iii) This simple

technique can be done by anyone, with littletraining and experience. (iv) The set-up re-quired is very simple and inexpensive.

Disadvantages (5). The only disadvan-tage is that it results in cobbling, scarring,and depigmentation (1).

CONCLUSIONPinch skin grafting is a safe, simple, and

inexpensive technique, which can be easilymastered, with minimal training and experi-ence. We recommend it to be used in all un-complicated leprous ulcers, which cannotbe brought under control with drugs anddressing alone. In the future, we plan to ex-tend this study to include pinch grafting onplantar ulcers.

REFERENCES1. HILL, T. G. Skin grafts. In: Cutaneous Surgery, 1st

edn. Philadelphia: W. B. Saunders Company, 1987.pp. 587–589.

2. KRIZEK, T. J. Grafts and flaps. In: Plastic Surgery:A core curriculum. Missouri: Mosby Year Book,Inc., 1994. pp. 36–64.

3. SAVANT, S. S. Pinch skin grafting for non healingulcers. In: Textbook and Atlas of Dermatosurgeryand Cosmetology, 1st edn. Mumbai: A.S.C.A.D.,1998. pp. 252–254.

4. STEGMAN, S. J., TROMOCITCH, T. A., and GLOGAN,R. G. Basics of Dermatologic Surgery. Chicago:Year book Medical, 1982. pp. 105–106.

5. WHEELAND, R. G. The technique and current statusof pinch grafting. J. Dermat. Surg. Oncol. 13(1987) 873–880.

1 Received for publication on 19 November 2002. Accepted for publication on 13 January 2004.2 P. R. Vanderborght, M.Sci., Leprosy Laboratory, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro, Brazil; H.

J. Matos, Ph.D., Informatica Medica, Faculty of Medical Science, State University of Rio de Janeiro, Brazil; A.M. Salles, M. D.; S. E. Vasconcellos, B.Sci; and Valcemir F. Silva-Filho, B.Sci., Leprosy Laboratory, OswaldoCruz Institute, Fiocruz, Rio de Janeiro, Brazil; T. W. J. Huizinga, Ph.D.; and Tom H. M. Ottenhoff, Ph.D., De-partment of Rheumatology and Department of Immunohematology and Blood Transfusion, Leiden UniversityMedical Center, Netherlands; E. P. Sampaio, M.D.; E. N. Sarno, M.D.; A. R. Santos, Ph.D.; and Milton O.Moraes, Ph.D., Leprosy Laboratory, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro, Brazil.

Reprint request to: Dr. Adalberto Rezende Santos, Leprosy Laboratory, Oswaldo Cruz Institute, FIOCRUZ,Av. Brasil 4365 Manguinhos, 21045-900 Rio de Janeiro, RJ, Brazil. E-mail: adalbertorezende@hotmail.com

Single Nucleotide Polymorphisms (SNPs) at

-238 and -308 Positions in the TNFα Promoter:

Clinical and Bacteriological Evaluation in Leprosy1

Patrícia R. Vanderborght, Haroldo J. Matos, Ana M. Salles, Sidra E. Vasconcellos,Valcemir F. Silva-Filho, Tom W. J. Huizinga, Tom H. M. Ottenhoff, Elisabeth P.

Sampaio, Euzenir N. Sarno, Adalberto R. Santos, and Milton O. Moraes2

ABSTRACTTumor necrosis factor alpha (TNFα) plays a key role in orchestrating the complex events

involved in inflammation and immune response. The presence of single nucleotide poly-morphisms (SNPs) within the promoter region of the TNFa gene has been associated with anumber of diseases. The aim of this study was to investigate the distribution of polymorph-isms at positions -238 (G/A) and -308 (G/A) at the TNFα promoter, and its association to theoutcome of different clinical forms of leprosy. Furthermore, the bacteriological index (BI)was evaluated among genotyped multibacillary (MB) patients in order to investigate the pos-sible influence of each polymorphism on the bacterial load. This study included a total of631 leprosy patients being 401 MB and 230 paucibacillary (PB), that was further separatedaccording to its ethnicity (Afro- and Euro-Brazilians). The combination of SNPs in haplo-types generated three different arrangements: TNFG-G, TNFG-A and TNFA-G. In spite ofthe marked differences observed in the frequency of the haplotypes along the ethnic groups,no statistical differences were observed in haplotype frequencies between MB and PB pa-tients. The BI analyses showed a lower bacteriological index among the -308 carriers, whilethe BI of the -238 carriers was higher. Although no significance has been achieved in thisanalysis regarding the influence of the polymorphisms to the development of the clinicaloutcome, it seems that in a different stage (among the MB patients) the polymorphismscould contribute to the degree of severity observed.

RÉSUMÉLe facteur alpha de nécrose tumorale (TNFα) joue un rôle important dans l’ajustement

des évènements complexes qui régulent l’inflammation et la réponse immunitaire. Laprésence de polymorphismes mono-nucléotidiques (SNPs) au sein de la région promotricedu gène codant pour TNFα a été associée à un certain nombre de maladies. Le but de cet ar-ticle était d’explorer la distribution de polymorphismes aux positions -238 (G/A) et –308(G/A) du promoteur de TNFα et son association aux résultats phénotypiques des différentesformes de lèpre. De plus, l’index bactérioscopique (IB) a été évalué parmi les patients multi-bacillaires (MB) génotypés dans le but d’évaluer la possible influence de chaque polymor-phisme sur la charge bactérienne. Cette étude a porté sur 631 lépreux comportant 401 MB et230 PB, qui furent encore séparés par ethnie (Afro et Euro-brésiliens). La combinaison desSNPs en haplotypes a généré 3 arrangements différents : TNF-G, TNF-A et TNFA-G. Endépit de différences marquées observées dans les fréquences haplotypiques entre les groupesethniques, aucune différence statistiquement significative ne fut observée entre les patientsMB et PB. Les analyses de IB ont montré un index bactérioscopique plus faible parmi lesporteurs –308, tandis que le IB des porteurs -238 était plus élevé. Bien que cette analyse depolymorphismes n’ait pas démontré de différence significative sur l’issue clinique de la

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Leprosy, a chronic human disease, is theresult of infection by Mycobacterium lep-rae. The clinical spectrum of the disease in-cludes two polar (lepromatous-LL andtuberculoid-TT) and three borderline forms(tuberculoid-BT, borderline-BB, and lepro-matous-BL) (19). The multibacillary (MB)form, including LL, BB, and BL, is charac-terized by low immune responsiveness andhigh bacterial load. The paucibacillary (PB)form, including TT and BT, is marked bystrong cell-mediated immunity against thebacillus (22). Much evidence has implicatedcytokines in the immune response of lep-rosy (16), mainly the tumor necrosis factor-alpha ( TNFα), which has a beneficial func-tion in host defense but, if produced in highlevels, contributes to tissue damage (25).

The presence of single nucleotide poly-morphisms (SNPs) in the TNFα promoterregion and their association with autoim-mune and infectious diseases has been ex-tensively studied (30). The polymorphismdetected at position -308 (G/A) within thepromoter region of the TNFα gene (29) wasthe first one found to be associated with dis-ease (18). This polymorphism has been ob-served in association with several other in-fectious diseases where excessive TNFαproduction seems to play a role, such as incerebral malaria (12) and mucocutaneousleishmaniasis (2). An association of the -308polymorphism with the development of lep-romatous leprosy was previously reported

in an Indian population (21). However, inBrazilian leprosy patients the association ofthis polymorphism with protection has beendescribed (23, 24, 27). A plausible explanationfor these divergent findings may involve theethnicity, where the allelic frequency ofTNFα -308A in leprosy patients range from7.0 to 10.8 in Indian and Brazilian, respec-tively (21, 24). This allelic frequency varia-tion is wide among populations such asCaucasian Irish (23.0), African Zulu (22.1),Arabian Omani (8.1), Singapore Chinese(12.0) and Mexican Mestizos (2.5) (13).Studies at another G/A transition polymor-phism (-238) in the TNTα promoter region(4) has shown that the A allele was in-creased among patients with chronic hepati-tis B and C, suggesting association to dis-ease susceptibility (7, 8) while in cancer aprotective effect was observed (9). In fact,the complex relationship between SNPs inthe human genome and disease associationindicates the need for the construction ofhaplotypes (specific combination of SNPson the same chromosome) on the locusstudied because they are more informativethan any single SNP (1, 26).

In the recent analyses from Brazilian lep-rosy patients we have shown in paucibacil-lary forms an increased allelic frequency ofthe TNF-308A in comparison to the multi-bacillary (0.14 and 0.09, respectively) witha borderline significance (χ2 = 3.47; p =0.06) (24). This data did not define if TNF

lèpre, il semble qu’à un certain stade de la maladie des patients multibacillaires, ces poly-morphismes pourraient contribuer au degré de sévérité observé.

RESUMENEl factor de necrosis tumoral alfa (TNFα) juega un papel importante en la orquestación

de los complejos eventos que ocurren en la inflamación y la respuesta inmunitaria. La pres-encia de polimorfismos simples en polinucleótidos (SNPs) dentro de la región promotora delgene TNFα ha sido asociada a numerosas enfermedades. El objetivo del estudio fue el in-vestigar la distribución de los polimorfismos en las posiciones -238 (G/A) y -308 (G/A) delpromotor del TNFα, y su asociación con las diferentes formas clínicas de la lepra. Se evaluó,además, el índice bacteriológico entre los pacientes MB estudiados para poder investigar laposible influencia de cada polimorfismo sobre la carga bacilar. En el estudio se incluyeron631 pacientes con lepra (401 MB y 230 PB) los cuales fueron adicionalmente separados deacuerdo a su etnicidad como Afro- y Euro-brasileños. La combinación de SNPs en los hap-lotipos generó 3 diferentes arreglos: TNFG-G, TNFG-A y TNFA-G. No obstante las mar-cadas diferencias en la frecuencia de los haplotipos entre los grupos étnicos, no se obser-varon diferencias estadísticas en las frecuencias de los haplotipos entre los pacientes MB yPB. Los análisis mostraron un menor índice bacteriológico entre los portadores-308 que en-tre los portadores-238 donde el IB fue mayor. Aunque en este análisis sobre la relación en-tre los polimorfismos y el tipo clínico de la enfermedad no se llegó a observar significanciaestadística, parecería ser que al menos entre los pacientes MB los polimorfismos puedencontribuir al grado de severidad observado.

72, 2 Vanderborght, et al.: SNP’s in the TNFα Promoter 145

-308A was a trend marker for protection,i.e., allele frequency in control > PB > MBor whether TNF-308A discriminates be-tween cases and controls only, being a re-sistance locus of leprosy per se. Thus, theaim of this study was to describe whetherthere was a difference between PB and MBusing an increased number of patients. Toovercome a possible cryptic stratificationthat would impact the SNP frequency andmask an association effect, patients wereseparated according to ethnicity. Besides,the -308 and -238 SNPs were combined inhaplotypes that better analyze the TNF pro-moter region. In addition, to understand theimpact of polymorphisms of TNFα pro-moter in relation to progression of the dis-ease, the comparison between SNPs in -308and -238 position and the bacteriologicalindex (BI) was set out in MB patients.

MATERIALS AND METHODSPatients. Six hundred and thirty one lep-

rosy patients from the Leprosy Out-PatientUnit, Oswaldo Cruz Foundation (Rio deJaneiro, Brazil) were included in this study.They were diagnosed on the basis of clini-cal and bacteriological criteria and classi-fied according to the Ridley and JoplingScale (20). Four hundred one MB and 230PB patients were studied, including 405males and 226 females. Brazilians wereclassified according to their ethnic originafter careful inspection of facial morpholog-ical features, hair type and skin color. Twogroups were ascertained: Afro-Braziliansand Euro-Brazilians with N = 251 and N =235, respectively. Asians and Amerindiansare not commonly represented in the popu-lation of Rio de Janeiro and were not ob-served among the individuals inspected.

Bacteriological index determination.Bacteriological index (BI) was determinedaccording to Ridley, 1964 (19) among themultibacillary patients in slit skin smearsfrom six different anatomic sites and rangedfrom 0.16 to 5.33 (mean = 2.50 ± 1.46).

DNA extraction and SNPs genotyping.Genomic DNA was prepared from frozenwhole blood collected with sodium citratebuffer by a commercially available DNAzolextraction kit (Invitrogen Life Technolo-gies, Gaithersburg, MD, USA). Genotypingof the TNFα promoter region for analysisof polymorphisms at the -238 and -308 po-sitions was performed by polymerase chain

reaction (PCR)-restriction fragment lengthpolymorphism (RFLP), a single PCR stepand further restriction analysis (6, 29). Re-stricted amplified products were visualizedby electrophoresis in 3% agarose gel andethidium bromide staining.

Statistical analysis. The statistical sig-nificance of TNFα promoter polymorphismdistributions was analyzed by way of the χ2

test Odds-ratio (OR) and 95% confidenceintervals (CI). Yates’ correction or Fisher’sexact test was used when appropriate (EpiInfo 6: CDC, Atlanta, GA). The signifi-cance level adopted was p <0.05. The hap-lotype frequencies were estimated using theEH program (28).

Generalized additive model. the vari-ability of the bacteriological index (BI) inrelation of the presence of the mutation inthe TNFα promoter was studied through ageneralized additive model (GAM). TheGAM is a special regression model, inwhich some of regression assumptions arerelaxed. So, GAM models may have advan-tages in many biological phenomena, andmay replace traditional linear or logistic re-gression models. In mathematical terms,GAM models allow predictors (or covari-ates) to be replaced by arbitrary smoothfunctions. For instance, B-splines (poly-nomials that can be adjusted to a set ofpoints) could be used as a smoothing func-tion. In this context, a GAM model wasused to study the association between theoccurrence of the G/A substitution in thepositions -308 and -238 of the promotergene of TNFα, and the baciloscopic index,calculated as previously described. For theadjustment of the model, one should indi-cate the number of nodes necessary. A nodeis the number of points used for the curve tobe adjusted. The SPLUS 2000 program,Professional Release 3, MathSoft, Inc. wasused to perform these analyses.

RESULTSAnalyses of TNFα haplotypes frequen-

cies in leprosy patients in differentBrazilian ethnic groups. Haplotypes ofTNFα were estimated using a maximum-likelihood probability test from -308 and -238 SNP genotypes of unrelated patientsstratified in Euro- and Afro-Brazilian (TheTable). Three differents haplotypes on theTNFα gene have been identified (TNFG-G,TNFA-G, TNFG-A). No statistical differ-

146 International Journal of Leprosy 2004

ences were observed in haplotypes fromTNFα promoter between MB and PB pa-tients irrespective to the ethnic group ana-lyzed. The TNFG-G haplotype in both PBand MB forms presented the highest fre-quency among patients in both ethnicgroups. Marked differences between Euro-and Afro-Brazilians were observed whenother haplotypes were analyzed. Among PBpatients, an increased frequency of theTNFA-G haplotype in Euro-Brazilian wasdetected in comparison to Afro-Brazilian(0.16 and 0.08, respectively) was detected.

Analyses of the bacteriological index(BI) from multibacillary patients geno-typed for SNPs at position -308 and -238.Bacteriological index (BI) variability in re-lation to the presence of the -238 (N = 343)and -308 (N = 341) polymorphisms was an-alyzed via the GAM (Fig. 1A, B). The GAMregression model was performed to study theassociation between the presence of the Aallele at the positions -238 and -308 of thepromoter gene of TNFα, as the dependentvariable, and the variability of the bacilo-scopic index, as a predictor variable. Forthe model using the substitution at position-238, a regression polynomial spline with1,5 nodes was used. It was observed thatthe probability for the occurrence of the Aallele at the -238 position increases withhigher bacteriological index (Fig. 1A).

For the model using the substitution at po-sition -308, a regression polynomial splinewith 1,1 nodes was used. In contrast to theresult obtained with -238, the probability forthe occurence of the A allele at the -308 po-sistion is greater with a low BI (Fig. 1B).

DISCUSSIONStudies using the frequency of single nu-

cleotide polymorphisms in candidate genesare interesting approaches to the investiga-tion of the susceptibility and severity of dis-eases. It is believed that SNPs are relevant

since they can be used as genotypic markersof specific disease phenotypes or can regu-late biological phenomena influencingmRNA expression, thereby altering mRNAisoforms (unravelling cryptic splicing sites)or modifying enzymatic activity of genes(11). The problem is that SNP frequenciesvary enormously among populations (13),especially Brazilians who originated from avariety of ethnicities, mainly Portugueseexplorers mixed with native Amerindiansand Africans (3). The outcome of this ad-mixtured colonization is a dense populationwithout a clear genetic/morphological eth-nic cut off (17). However, some cryptic strat-ification may still be functioning as con-founding factors in Brazilians in popula-tion-based studies. Indeed, the separationaccording to the morphological features ofthe patients better discriminate Afro- andEuro-Brazilians, where a difference in the -308A/-238G haplotype frequency fromAfro-Brazilian (9%) to Euro-Brazilian (13%)patients was observed. Still, no statisticaldifferences were observed when PB andMB were compared, demonstrating that ifthere is some cryptic stratification due toadmixture, it is not being detected by con-ventional morphological inspection in ourpatient population. Thus, to scrutinize thestratification in Brazilian population-basedstudies it would be necessary to use ge-nomic controls (5). Moreover, a recent studyperformed in Gambian and Malawianpopulations studying SNPs spanning 4.4kbof the TNFα /LTα locus demonstrated theneed to Type 8 in Gambians, and 7 out 12SNPs in Malawians, to detect the haplo-typic structure and informative SNPs in thisregion due to the high frequency of recom-bination (1). We do not have data for theBrazilians but it seems to be necessary toenlarge the focused region of the TNF locusto capture more information about severityin leprosy.

THE TABLE. Haplotypes frequencies of the TNFα promoter polymorphisms among MBand PB leprosy patients within distinct ethnic groups.

Haplotypes Brazilian Euro-Brazilian Afro-Brazilian

-308/-238 MB PB MB PB MB PB

G-G 0.83 0.81 0.82 0.78 0.84 0.86G-A 0.05 0.05 0.05 0.04 0.04 0.04A-G 0.10 0.12 0.12 0.16 0.10 0.08

72, 2 Vanderborght, et al.: SNP’s in the TNFα Promoter 147

On the other hand, the analysis of BI andits association with polymorphisms at po-sitions -308 and -238 in TNFα suggestedthese polymorphisms are functionally rele-vant. We previously demonstrated thatTNFα -308A was associated with astronger response in Mitsuda reaction (15).In this study, GAM analyses in -308A, re-vealed that such patients have lower BIs.The results of our previous findings (15)with this new data is that TNF -308A couldbe upregulating the secretion of TNFα that,in turn, induces a stronger DTH skin re-sponse in paucibacillary patients and restrictsM. leprae growth in multibacillary patients.

The opposite was verified concerning thestudy of -238A and BIs. In this case, the pres-ence of the A allele was more frequent inmultibacillary patients with higher BIs. This

data is in accordance with the literature,where it has been demonstrated that the -238A polymorphism is associated withlower levels of TNFα (10).

The possibility of using slit skin smearsis one of the few alternatives for in vivo de-termination of the bacterial load. The BI isone of the clinical parameters indicatingdisease progression and severity, represent-ing a clear risk factor for the developmentof the acute inflammatory episodes in lep-rosy (14). Thus, by way of the adjustedmodel (GAM), the existence of a clinicalsignificance for the variability of BI in rela-tion to the presence of polymorphisms inthe TNFα promoter suggests a functionaldichotomy between the -308 and -238SNPs in relation to TNF regulation and lep-rosy progression.

Acknowledgment. We are grateful to the attendingphysicians at the Leprosy Out-Patient Unit, FIOCRUZ,to Edson Albuquerque, for his technical assistance, andto Judy Grevan for the English revision. Patrícia Van-derborght is supported by a grant from CAPES (Coor-denação de Aperfeiçoamento de Pessoal de Nível Su-perior), Brazil. This study was financially supported bythe World Health Organization/World Bank/TDR, ID930063, by the EU (INCO-DC), grant number ERBIC18CT 980377, and by The Netherlands Leprosy Foun-dation (NLR).

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FIG. 1. Analyses of the bacteriological index (BI)variability of TNFα promoter polymorphism -238 (a)and -308 (b) by Generalized Additive Model (GAM)in 341 and 343 multibacillary genotyped patients, re-spectively. The “y” axis means the probability of “A”allele occurrence (homozygous or heterozygous) andBI at “x” axis, range from 0.16 to 5.33. Each indi-vidual BI is represented as a small bar.

148 International Journal of Leprosy 2004

7. HÖHLER, T., KRUGER, A., GERKEN, G., SCHNEIDER, P.M., BÜSCHENFELDE, K.-H., and RITTNER, C. A tumor necrosis factor-alpha (TNFα) promoterpolymorphism is associated with chronic hepatitis Binfection. Clin. Exp. Immunol. 111 (1998) 579–582.

8. HÖHLER, T., KRUGER, A., GERKEN, G., SCHNEIDER,P. M., BÜSCHENFELDE, K.-H., and RITTNER, C. Tu-mor necrosis factor alpha promoter polymorphismat position -238 is associated with chronic activehepatitis C infection. J. Med. Virol. 54 (1998)173–177.

9. JANG, W. H., YANG, Y. I., YEA, S. S., LEE, Y. J.,CHUN, J. H., KIM, H. I., KIM, M. S., and PAIK, K.H. The -238 tumor necrosis factor-alpha promoterpolymorphism is associated with decreased sus-ceptibility to cancers. Cancer Lett. 166 (2001)41–46.

10. KALUZA, W., REUSS, E., GROSSMANN, S., HUG, R.,SCHOPF, R. E., GALLE, P. R., MAERKER-HERMANN,E., and HOEHLER, T. Different transcriptional ac-tivity and in vitro TNF-alpha production in psori-asis patients carrying the TNF-alpha 238A pro-moter polymorphism. J. Invest. Dermatol. 114(2000) 1180–1183.

11. LAI, E. Application of SNP technologies in medi-cine: lessons learned and future challenges.Genome Res. 11 (2001) 927–929.

12. MCGUIRE, W., HILL, A. V. S., ALLSOPP, C. E. M.,GREENWOOD, B. M., and KWIATKOWSKI, K. Varia-tion in the TNFα promoter region associated withsusceptibility to cerebral malaria. Nature 371(1994) 508–511.

13. MEENAGH, A., WILLIAMS, F., ROSS, O. A., PATTER-SON, C., GORODEZKY, C., HAMMOND, M., LEHENY,W. A., and MIDDLETON, D. Frequency of cytokinepolymorphisms in populations from western Eu-rope, Africa, Asia, the Middle East, and SouthAmerica. Hum. Immunol. 11 (2002) 1055–1061.

14. MEYERSON, M. S. Erythema nodosum leprosum.Int. J. Dermatol. 35 (1996) 389–392.

15. MORAES, M. O., DUPPRE, N. C., SUFFYS, P. N.,SANTOS, A. R., ALMEIDA, A. S., NERY, J. A., SAM-PAIO, E. P., and SARNO, E. N. Tumor necrosis factor-alpha promoter polymorphism TNF2 is associatedwith a stronger delayed-type hypersensitivity reac-tion in the skin of borderline tuberculoid leprosypatients. Immunogenetics 53 (2001) 45–47.

16. MORAES, M. O., SARNO, E. N., ALMEIDA, A. S.,SARAIVA, B. C., NERY, J. A., MARTINS, R. C., andSAMPAIO, E. P. Cytokine mRNA expression inleprosy: a possible role for interferon-gamma andinterleukin-12 in reactions (RR and ENL). Scand.J. Immunol. 50 (1999) 541–549.

17. PARRA, F. C., AMADO, R. C., LAMBERTUCCI, J. R.,ROCHA, J., ANTUNES, C. M., and PENA, S. D.Color and genomic ancestry in Brazilians. Proc.Natl. Acad. Sci. U.S.A. 100 (2003) 177–182.

18. POCIOT, F., WILSON, A. G., NERUP, J., and DUFF, G.W. No independent association between a tumor

necrosis factor-alpha promoter region polymor-phism and insulin-dependent diabetes mellitus.Eur. J. Immunol. 23 (1993) 3050–3053.

19. RIDLEY, D. S. Bacterial indices. In: Leprosy inTheory and Practice. 2nd edn. Bristol: JohnWright, 1964, pp. 620–621.

20. RIDLEY, D. S., and JOPLING, W. H. Classificationof leprosy according to immunity: a five-groupsystem. Int. J. Lepr. 34 (1966) 255–273.

21. ROY, S., MCGUIRE, W., MASCIE TAYLOR, C. G.,SAHA, B., HAZRA, S. K., HILL, A. V., andKWIATKOWSKI, D. Tumor necrosis factor promoterpolymorphism and susceptibility to lepromatousleprosy. J. Infect. Dis. 176 (1997) 530–532.

22. SAMPAIO, E. P, MORAES, M. O., PESSOLANI, M. C. V.,and SARNO, E. N. Role of Th1 host defenses againstMycobacterium leprae. Cytokines and Chemokines.In: Infectious Diseases Handbook 1st edn. NewJersey: Humana Press, 2003, pp. 163–188.

23. SANTOS, A. R., ALMEIDA, A. S., SUFFYS, P. N.,MORAES, M. O., FILHO, V. F., MATTOS, H. J., NERY,J. A., CABELLO, P. H., SAMPAIO, E. P., and SARNO,E. N. Tumor necrosis factor promoter polymor-phism (TNF2) seems to protect against develop-ment of severe forms of leprosy in a pilot study inBrazilian patients. Int. J. Lepr. Other Mycobact.Dis. 68 (2000) 325–327.

24. SANTOS, A. R., SUFFYS, P. N., VANDERBORGHT, P. R.,MORAES, M. O., VIEIRA, L. M., CABELLO, P. H.,BAKKER, A. M., MATOS, H. J., HUIZINGA, T. W., OT-TENHOFF, T. H., SAMPAIO, E. P., and SARNO, E. N.Role of tumor necrosis factor-alpha and interleukin-10 promotor gene polymorphisms in leprosy. J. In-fect. Dis. 186(11) 1687–1691.

25. SARNO, E. N., GRAU, G. E., VIEIRA, L. M., and NERY,J. A. C. Serum levels of tumor necrosis factor-alphaand interleukin 1β during leprosy reactional states.Clin. Exp. Immunol. 84 (1991) 103–108.

26. SCHAID, D. J. Relative efficiency of ambiguousvs. directly measured haplotype frequencies.Genet. Epidemiol. 4 (2002) 426–443.

27. SHAW, M. A., DONALDSON, I. J., COLLINS, A., PEA-COCK, C. S., LINS-LAINSON, Z., SHAW, J. J., RAMOS,F., SILVEIRA, F., and BLACKWELL, J. M. Associa-tion and linkage of leprosy phenotypes with HLAclass II and tumour necrosis factor genes. GenesImmun. 2 (2001) 196–204.

28. TERWILLIGER, J. D., and OTT, J. Handbook of Hu-man Genetic Linkage. Baltimore and London: TheJohns Hopkins University Press, 1994, p. 307.

29. WILSON, A. G., DI GIOVINE, F. S, BLAKEMORE, A. I.F., and DUFF, G. W. Single base polymorphism inthe human tumor necrosis factor (TNF) alpha genedetectable by NcoI restriction of PCR product.Hum. Mol. Gen. 1 (1992) 353.

30. WILSON, A. G., DI GIOVINE, F. S., and DUFF, G. W.Genetics of tumor necrosis factor-α in autoim-mune, infectious, and neoplastic diseases. J. In-flam. 45 (1995) 1–12.

1 Received for publication on 17 October 2003. Accepted for publication on 24 February 2004.2 K. K. Mohanty, Ph.D.; Beenu Joshi, Ph.D.; Kiran Katoch, M.D., Clinical Divison; and Utpal Sengupta,

Ph.D., Department of Immunology, Central JALMA Institute for Leprosy, Tajganj, Agra 282 001 (India).Reprint requests to: Utpal Sengupta, Department of Immunology, Central JALMA Institute for Leprosy, Taj-

ganj, Agra 282 001 (India).

Leprosy Reactions: Humoral and Cellular Immune

Responses to M. leprae, 65kDa, 28kDa, and

18 kDa Antigens1

Keshar K. Mohanty, Beenu Joshi, Kiran Katoch, and Utpal Sengupta2

ABSTRACTThis study examines the immune responses against some stress proteins of Mycobacte-

rium leprae in leprosy patients with and without leprosy reactions. Leprosy patients showeda higher level of antibodies to all antigens compared to healthy controls. The antibody re-sponse to 18kDa antigen was significantly higher in patients with Type 1 reaction comparedto those of TT or borderline patients without Type 1 reaction, or those with Type 2 reaction.Borderline (BT/BL), lepromatous (LL) and patients with reactions (Type 1 and Type 2) hadhigher levels of antibodies to M. leprae soluble extract (MLSE) and 65kDa than those of thetuberculoid (TT) group. LL, borderline patients, and patients with Type 1 reaction had ahigher level of antibody to 28kDa than those of healthy controls. However, no significantdifferences could be observed in antibody response to these antigens (MLSE, 65kDa, and28kDa) between patients with reaction and without reaction. A significant proportion ofTT/BT patients showed positive lymphoproliferative response to MLSE compared to BL/LLpatients. In addition, the lymphoproliferative response to MLSE was significantly greater inpatients with Type 1 reaction compared to patients without reaction. No difference in prolif-erative response to 65kDa could be observed in any of these groups. The finding of high lev-els of antibodies against stress proteins in patients with Type 1 reactions, especially to 18kDa antigen, along with a heightened lymphoproliferative response to MLSE is suggestiveof a coexistence of cell mediated and humoral immunity in leprosy patients during Type 1reactions. On the other hand, in Type 2 reactions no significant role of stress proteins couldbe demonstrated except a heightened lymphoproliferative response to the 28 kDa antigen.

RÉSUMÉCette étude présente les réponses immunitaires chez les patients hanséniens et les patients

souffrant de réactions, contre les protéines de stress de Mycobacterium leprae. Les patientshanséniens ont montré de plus haut niveaux d’anticorps dirigés contre tous les antigènes queles personnes témoins en bonne santé. La réponse sérique dirigée contre l’antigène de 18kDaétait significativement plus élevée chez les patients souffrant de réaction de type 1 comparéeà celles des patients TT ou borderline, ou celle des patients avec réaction de type 2. De plus,un plus grand pourcentage de patients avec réaction reverse avaient une réponse détectablepour cet anticorps, comparé à celui des patients sans réaction. Les patients borderline(BT/BL), lépromateux (LL) et les patients avec réactions (type 1 et type 2) présentaient deplus hauts niveaux d’anticorps dirigés contre l’extrait soluble de M. leprae (MLSE) et laprotéine 65kDa que les patients tuberculoïdes (TT). Les patients LL, borderline et les pa-tients présentant une réaction de type 1 présentaient de plus haut niveaux d’anticorps contrela protéine 28kDa par rapport aux témoins en bonne santé. Cependant, aucune différencesignificative ne fut observée entre les patients avec et sans réaction dans la réponse sériquecontre les antigènes MLSE, 65kDa et 28kDa. Comparé aux patients BL/LL, une proportionsignificative de patients TT/BT montraient une réponse lymphoproliférative positive contreMLSE. De plus, comparé aux patients sans réaction en cours, les patients de type 1 mon-traient une réponse lymphoproliférative plus élevée contre MLSE. Aucune différence deréponse proliférative contre 65kDa ne fut observée entre les groupes. La mise en évidencede hauts niveaux d’anticorps dirigés contre les protéines de stress de M. leprae chez les pa-

149

INTERNATIONAL JOURNAL OF LEPROSY Volume 72, Number 2Printed in the U.S.A.

(ISSN 0148-916X)

150 International Journal of Leprosy 2004

Leprosy is a chronic infectious diseasecaused by Mycobacterium leprae. After in-fection the response of host immune systemdetermines the course of the disease. On thebasis of cellular immune response of thehost, a spectrum of types of leprosy hasbeen described (27). Some types of leprosypatients suffer from immunological compli-cations known as “lepra reactions” whichinclude both Type 1 (reversal reactions,RR) and Type 2 (erythema nodosum lepro-sum, ENL) reactions (14). Type 1 reaction ischaracterized by episodes of increased in-flammatory activity in skin and in nerves ofpatients with borderline leprosy [Borderlinetuberculoid (BT), Borderline (BB), andBorderline lepromatous (BL)] (5, 31). Type 2reaction or ENL reaction is the most seriousimmunological complication in BL and lep-romatous (LL) patients. Type 1 reaction isknown to be due to changes in (both up anddown regulation) of cell mediated immu-nity (CMI), whereas in Type 2, there is a

rise in immune complexes with their depo-sition in tissues (40). Further, in Type 2 reac-tions a transient rise of CMI with the ex-pression of Th 1 type of cytokines has alsobeen noted (19, 35).

Several stress proteins of M. leprae havebeen cloned (42) and are recognized by bothmurine and human immune cells (22, 24). M.leprae heat shock protein (hsp) 65 kDa, ex-pressed in macrophages transfected withthe mycobacterial gene, is known to be pre-sented to T cells in association with bothMHC class I and class II (32) and also MHCnon-restricted manner (33). Beimnet, et al.(1996) also reported the expression of Hsp60 from a human monocytic cell line in-fected with M. leprae (2). Further, thesestress proteins are known to be major anti-gens of mycobacteria, which induce spe-cific antibody and T cell immune responseduring infections (1, 6, 8,10, 11, 14, 16, 21, 28, 29, 30, 37, 41).Nerve and skin damage in leprosy is re-ported to be associated with increased lev-

tients souffrant de réactions de type 1, en particuliers contre l’antigène 18kDa, accompag-nant une réponse lymphoproliférative élevée contre MLSE, suggère qu’une immunité à mé-diation cellulaire et une immunité à médiation humorale coexistent chez les patients han-séniens pendant les réactions de type 1. A l’opposé, aucun rôle significatif des protéines destress ne fut montré pour les réactions de type 2, à l’exception d’une réponse lymphopro-liférative plus élevée contre l’antigène de 28kDa.

RESUMENEn este estudio se examinó la respuesta inmunitaria contra algunas proteínas de estrés de

Mycobacterium leprae en pacientes con lepra con y sin reacción leprosa. Comparados conlos controles sanos, los pacientes con lepra mostraron altos niveles de anticuerpos contra to-dos los antígenos probados. La respuesta en anticuerpos contra el antígeno de 18 kDa fuesignificativamente mayor en los pacientes con reacción Tipo I que en los pacientes con lepraTT, los pacientes con lepra subpolar sin reacción Tipo 1, o los pacientes con reacción Tipo2. Los pacientes subpolares (BT/BL), los lepromatosos (LL) y aquellos sin reacciones (tipos1 y 2) tuvieron niveles más elevados de anticuerpos contra un extracto soluble de M. leprae(MLSE) y contra la proteína de 65 kDa, que los pacientes del grupo tuberculoide (TT). Lospacientes LL, los subpolares, y los pacientes con reacción Tipo 1 tuvieron mayores nivelesde anticuerpos contra la proteína de 28 kDa que los controles sanos. Sin embargo, no se ob-servaron diferencias significativas en la respuesta en anticuerpos contra los antígenos(MLSE, 65 Kda, y 28 kDa) entre los pacientes con reacción y aquellos sin reacción. Por otrolado, comparados con los pacientes BL/LL, una proporción significativa de los pacientesTT/BT mostraron una respuesta linfoproliferativa positiva contra el MLSE. Además, la re-spuesta linfoproliferativa contra el MLSE fue significativamente mayor en los pacientes conreacción Tipo 1 que en los pacientes sin reacción. En ninguno de los grupos se observó difer-encia en la respuesta proliferativa contra la proteína de 65 kDa. Así, los hallazgos de niveleselevados de anticuerpos contra las proteínas de estrés en los pacientes con reacción Tipo 1,especialmente contra la proteína de 18 kDa, junto con la elevada respuesta proliferativa con-tra el MLSE, sugieren la coexistencia de la inmunidad celular y la inmunidad humoral en lospacientes con lepra durante la reacción Tipo 1. En las reacciones de Tipo 2 no se pudodemostrar un papel significativo de las proteínas de estrés, excepto por la marcada respuestalinfoproliferativa contra el antígeno de 28 kDa.

72, 2 Mohanty, et al.: Immune Response to Stress Proteins in Leprosy Reactions 151

els of intra-lesional hsp (16). The process ofreaction evokes a change in immunologicalstatus of the host leading to stress condi-tions for bacteria, which might result in re-lease of stress proteins. However, the roleof antibodies to stress proteins in Type 1 re-actions has not been elucidated so far ex-cept for the observation of Klatser, et al.(17). To explore the immunological role ofstress proteins of M. leprae in leprosy,analysis of circulating antibodies, and ofthe proliferative response of peripheralblood mononuclear cells (PBMC) to someof the stress proteins were performed inhealthy controls, Tuberculoid (TT), Border-line and LL patients with Type 1 or Type 2reactions, and in patients without reactions.

MATERIALS AND METHODSLeprosy patients attending the out pa-

tient department of the Central JALMA In-stitute for Leprosy (Agra, India) were in-cluded in the study after obtaining theirwritten consent according to the guidelineslaid by the Indian Council of Medical Re-search, India. They were diagnosed clini-cally and bacteriologically and were di-vided into five groups across the diseasespectrum according to the criteria of the In-dian association of Leprologists (12). Allthese patients were clinically active andwere on multi-drug therapy (MDT) duringthe study period.

Serum samples. Serum samples wereobtained from 10 ml of blood drawn by an-tecubital venipuncture from 6 TT, 24 bor-derline (13 BT, 11 BL), 8 LL patients whowere stable in their clinical manifestations,21 BL/LL patients with ENL, and 29BT/BL patients with Type 1 reaction. Nine-teen laboratory volunteers who were hospi-tal contacts served as healthy controls.

Soluble and recombinant antigens ofM. leprae. M. leprae soluble extract(MLSE) (contract No-1-A1-55262) was ob-tained from Dr. P. J. Brennan, ColoradoState University, U.S.A., and the recombi-nant proteins of M. leprae (ML hsp65kDa,ML 28 kDa, ML 18kDa) were gifted by Dr.M. Singh, GBH, Germany.

Enzyme linked immuno sorbent assay(ELISA). Maxisorp (Nunc, Rosklide, Den-mark) plates were coated with 100 mi-crolitre (µl) antigen solutions [MLSE (2µg/ml), 65 kDa (1µg /ml), 28 kDa (2µg/ml)

and 18 kDa (2µg/ml)] in carbonate bicar-bonate buffer (pH 9.2) and kept for 4 hrs at37°C and then overnight at 4°C. The wellswere blocked in phosphate buffered saline(PBS) with 3% Bovine Serum Albumin(BSA) for 1 hr at 37°C. One hundred µl of100-fold diluted sera in PBS containing0.05% Tween 20 and 1% BSA was added induplicate wells. After 2 hrs incubation at37°C, the plates were washed 3 times inPBS Tween (0.05%). One hundred µl of5000-fold diluted horseradish peroxidase-conjugated anti-human IgG antibody (Sigma,St. Louis) was added, and plates were incu-bated for 1 hr 30 minutes at 37°C followedby a final 3 washes. One hundred µl of or-thophenylene diamine hydrochloride solu-tion (0.5 mg/ml substrate in distilled watercontaining 30 µl of 30% H

2O

2) was added

to each well for development of color. Thereaction was stopped after 30 minutes byadding 25 µl of 3N H

2SO

4. The optical den-

sity (OD) was measured in an ELISAreader at 492 nm.

Peripheral blood mononuclear cells(PBMCs). Peripheral venous blood wascollected asceptically in a heparinized tubefrom 2 TT, 22 borderline (10 BT, 12 BL), 7LL patients who were stable in their clinicalmanifestations and 12 BL/LL patients withType 2, 12 BT/BL patients with Type 1, andfrom 12 healthy controls. PBMCs were sep-arated by ficoll hypaque density gradientcentrifugation. The cells were collectedfrom the interface layer and washed threetimes with RPMI 1640 and counted in aNeubaur’s chamber.

Proliferation of peripheral bloodmononuclear cells (Lymphocyte transfor-mation test, LTT). PBMCs (2 × 106 cells/ml)were cultured in quadruplicate wells in RPMI1640 containing 10% human AB serum in96-well plates (Nunc, Rosklide, Denmark)for six days. Optimum concentrations ofMLSE (1 µg/ml) and different recombinantproteins [65kDa (5µg/ml), 28 kDa (10µg/ml)], were added to wells for sensitiza-tion of PBMCs. DNA synthesis was assayedby [3H] labeled thymidine incorporation(Amersham, U.K.). 1 µ Ci of 3H-thymidine(specific activity 5.0 Ci / m mol) was addedto each well on 5th day and after 18 hr cellswere harvested. The lymphocyte stimula-tion index (SI) was calculated using a stan-dard formula (average cpm in the presence

152 International Journal of Leprosy 2004

of antigen/average cpm in the absence ofantigen).

Statistical analysis. Data were analyzedby Student’s t-test by using MS Excel soft-ware program, Chi square test, and Fisherexact test.

RESULTSAntibodies to MLSE, ML 65kDa, ML

28kDa and ML 18kDa. The mean anti-body response (with standard deviations) ofhealthy individuals, all types of leprosy pa-tients is presented in Table 1.

MLSE. It can be noted that all groups ofleprosy patients showed high antibody lev-els to MLSE compared to healthy controls.Antibody level in BT, BL patients, and LLpatients, and in patients during reactions(both Type 1 and Type 2) is significantlyhigher than those of TT group. There wasno difference in antibody level between BTand BL patients and in patients during re-versal reaction. Similarly, there was no dif-

ference in BL/LL patients, and those pa-tients with Type 2 reactions.

65kDa. There was a higher level of anti-body in BT/BL, LL and also in patientswith reactions (ENL and RR) than inhealthy controls and TT patients. No differ-ence could be observed in antibody levelbetween the groups of patients with reac-tions (Type 1 and Type 2) and without reac-tions.

28 kDa. It was also observed that TT,BT/BL and LL patients have higher level ofantibody to this antigen in comparison tohealthy controls. The highest level of anti-body was detected in LL patients, followedby Type 1 patients. The difference betweenantibody levels in BT/BL patients as com-pared to those with Type 1 reactions wasstatistically significant, (p <0.03).

18 kDa. The level of antibody to thisantigen was observed to be at a higher levelin all groups of leprosy patients comparedto those of healthy controls. LL patients had

TABLE 2. The percentage of seropositivity for antibodies against various antigens ofM. leprae.

HC TT Borderline LL ENL RRAntigens N = 19 N = 6 (BT/BL) N = 24 N = 8 N = 21 N = 29

MLSE 0 (0) 0 (0) 10 (41.66) 5 (62.5)♦ 10 (47.61) 15 (51.72)ML65kDa 1 (5.26) 0 (0) 8 (33.33) 4 (50) 6 (28.57) 11 (37.93)ML28kDa 0 (0) 2 (33.33) 8 (33.33) 4 (50) 7 (33.33) 15 (51.72)ML18kDa 0( 0) 0 (0) 7 (29.16) 5 (62.5)♦ 5 (23.80) 16 (55.17)*

Figures in parentheses ( ) show percent positive.Cut off values are mean OD +2 S.D. (MLSE = 0.49), (65kDa = 0.35), (28 kDa = 0.33),

and (28 kDa = 0.33). Individuals having more OD values than cut off points are taken as positive.♦ Significantly more than TT (p <0.03)* Significantly more than borderline group (p <0.06)

TABLE 1. Mean level of antibodies (with standard deviations) to antigens of M. lepraeof healthy controls and leprosy patients.

HC TT Borderline LL ENL RRAntigens N = 19 N = 6 (BT/BL) N = 24 N = 8 N = 21 N = 29

MLSE 0.23 ± 0.13 0.30 ± 0.06 0.51 ± 0.36∇ 0.55 ± 0.19∇ 0.58 ± 0.37∇ 0.51 ± 0.22∇

ML65kDa 0.19 ± 0.08 0.22 ± 0.04 0.29 ± 0.13# 0.37 ± 0.16# 0.30 ± 0.13# 0.35 ± 0.22#

ML28kDa 0.17 ± 0.08 0.30 ± 0.17 0.27 ± 0.13* 0.42 ± 0.25* 0.29 ± 0.13* 0.38 ± 0.19*↑

ML18kDa 0.17 ± 0.08 0.23 ± 0.06♣ 0.28 ± 0.15♣ 0.42 ± 0.19♣ 0.31 ± 0.22♣ 0.46 ± 0.35♣♠

∇ Significantly more than HC & TT (p <0.005)# Significantly more than HC & TT (p <0.02)* Significantly more than HC (p <0.0005)↑ Significantly more than Borderline (BT/BL) and ENL (p <0.03)♣ Significantly more than HC (p <0.05)♠ Significantly more than TT, Borderline (BT/BL) and ENL (p <0.005)

72, 2 Mohanty, et al.: Immune Response to Stress Proteins in Leprosy Reactions 153

significantly higher level of antibody thandid TT patients. The mean antibody level tothis antigen is highest in BT/BL patientsduring Type 1 reactions, which is signifi-cantly different from those of TT patients,BT/BL patients without reaction and pa-tients with Type 2 reaction, (p <0.005).

The percentage of seropositivity of anti-bodies to these antigens is shown in Table2. It was noted that significant proportion ofLL patients showed positivity for MLSEand 18kDa (p <0.03) when compared tothat of TT patients.

There was no significant difference inseropositivity of antibodies to these antigensamongst patients of the BT/BL groups withand without reaction. Nevertheless a signifi-cant proportion of BT/BL patients with Type1 showed higher positivity to 18-kDa anti-gens only when compared to those of bor-derline patients without reactions (p <0.06).

Lymphoproliferative response to MLSEand stress proteins of M. leprae (ML65kDa & ML28kDa). The positivity forlymphoproliferation to these antigens (SI >2)in healthy controls and leprosy patients ispresented in Table 3. The individual lympho-proliferative responses against these antigensare shown in The Figure (a, b, and c).

MLSE. MLSE was found to be the bestinducer for proliferation of lymphocytesamongst all M. leprae proteins tested. Allexcept one of the healthy controls showed apositive response to MLSE, whereas 1 BLpatient, 1 BL patient with Type 2 reactionand none of the LL patients, were positiveto this antigen. 8/12 (66.6%) of Type 1 re-actions, and 6/12 TT/BT (50%) were posi-tive to these antigens. The mean prolifera-tive response of BT/BL patients with Type

1 was significantly greater compared to bor-derline patients without reaction (p <0.02).However, there was no difference in meanproliferative response between BL/LL with-out reactions and Type 2 reaction patients.Further, the mean proliferative responsewas significantly greater in TT/BT patientscompared to BL/LL patients (p <0.03) andType 2 reaction patients (p <0.04). Signifi-cant proportions of TT/BT individualsshowed lymphoproliferation when com-pared to BL/LL patients (p <0.007).

Response to recombinant proteins. ML65kDa. There was no significant difference inlymphoproliferative response to 65kDa pro-tein amongst these groups (Table 3 and TheFigure b). ML 28kDa. While 3/12 patientswith Type 2 showed a positive response,none of the LL patients was responsive to thisantigen (Table 3 and The Figure c).

DISCUSSIONWhen infectious agents enter the host,

they may respond to the host environmentby producing stress proteins. These stressproteins are important in eliciting immuneresponse, which can lead to pathogenesis orprotection in the host. Some recombinantantigens (stress proteins) have been re-ported to be immunologically importantand induce B cell and T cell immune re-sponses in leprosy (6, 8, 16, 21, 24, 30, 37, 38, 41). Al-though these previous studies have beenconducted to analyze the immune responsein leprosy patients, only a few studies werecarried out in leprosy patients with reac-tions. The objective of the present studywas to analyze the level of antibodies andimmunoproliferative response of PBMCs toMLSE and a few stress proteins of M. lep-

TABLE 3. The positivity for lymphoproliferation in healthy controls and leprosy pa-tients towards various antigens of M. leprae.

HC TT/BT♦ BL/LL ENL RRAntigensN = 12 N = 12 N = 19 N = 12 N = 12

MLSE 11 (91.66) 6 (50) 1 (5.2) 1 (8.33) 8 (66.66)*ML65kDa 2 (16.66) 1 (8.33) 1 (5.2) 2 (16.66) 1 (8.33)ML28 kDa 2 (16.66) 0 (0) 0 (0) 3 (25)♣ 2 (16.66)

SI >2 is taken as positive response.Figures in ( ) show the percent positivity.♦ Significantly more than BL/LL (p <0.007)* Significantly more than BT /BL (p <0.04)♣ Significantly more than BL/LL patients (p <0.06) and BT/BL (p <0.02)

154 International Journal of Leprosy 2004

rae in patients associated with reactions,and to compare their levels with patientswho are not associated with reactions. Inaddition, as controls, responses of somehealthy individuals who were exposed toinfection in the hospital were also com-pared with these leprosy patients.

Although the mean antibody level wasfound to be highest in patients with Type 2reactions, this was not significantly differ-ent from other groups. Patients with Type 1reactions also showed almost the same levelof antibody to MLSE as of borderline pa-tients. The mean level of antibodies to

THE FIGURE. Proliferative response of individuals (healthy controls and leprosy patients) to MLSE (a), 65kDa(b), 28kDa(c). PBMCs (2 × 106 cells/ml) were cultured in quadruplicate wells for six days in presence of variousantigens of M. leprae (MLSE, 65kDa, 28kDa) and in absence of antigens. Thymidine [H]3 was added after 5 daysand cells were harvested after 18 hrs. Proliferative response was expressed in stimulation Index (SI). SI was cal-culated by using the formula (Average cpm in antigen pulsed wells/average cpm in control wells).

72, 2 Mohanty, et al.: Immune Response to Stress Proteins in Leprosy Reactions 155

MLSE was found to be significantly higherin all types of leprosy patients except TTpatients when compared to those of healthyindividuals. Among the patient groups, TTpatients showed lowest antibody level. Themean OD value gradually increased fromthe TT end to the LL end of the spectrum. Asimilar finding has been reported earlier byQin-xue, et al. (26). This finding of a gradualincrease of antibody level against MLSEcould possibly be due to the increase inantigenic load from the TT pole to the LLpole.

An increased level of antibodies was seento 65 kDa, 28kDa and 18 kDa stress pro-teins in all groups of leprosy patients com-pared to healthy individuals, similar to pre-vious reports (14, 16). In our study a higherlevel of anti 65kDa antibody was observedin BL/LL and Type 2 patients. However,there was no significant difference in anti-body levels between patients with reactionand patients without reactions. Possibly the65kDa antigen induces an antibody re-sponse in the initial phase of infection andthis does not change during the develop-ment of various stages of disease. This ob-servation would suggest that antibodies to65kDa do not induce any immunopatholog-ical phenomenon in patients associated withreactions. Furthermore, the above finding isconsistent with the observation of Thole, etal. (1995) who did not find any associationwith the 65kDa antigen specific responsesin BT/TT or LL types of leprosy (37). Ofcourse, M. leprae 65 kDa has been noted tobe expressed in skin and nerve of all groupsof leprosy patients (38) and may be pre-sumed to have an important role in Type 1reaction, but it is uncertain whether this ispredominantly related to the initiation ofthe disease or the development of diseaseonce the reaction has started.

We observed a higher positivity for anti-body responses against the 28 kDa antigenin LL patients than TT patients, and this re-sponse was even greater in patients withType 1 reactions. Though other studies haveprovided evidence of the presence of antiM. leprae 28 kDa antibodies in sera of lep-romatous patients (6, 16), this is the first re-port to note such a higher percentage(51.72%) of antibody positivity to thisstress protein of M. leprae in patients dur-ing Type 1 reactions. This antigen has

recently been suggested as a potential can-didate antigen for initiating the Type 1 reac-tion, because it has been demonstrated inmacrophages and Schwann cells of skinand nerve biopsies (20). Hence, our findingof high antibody response may be due toexpression of this antigen by M. leprae dur-ing Type 1 reactions. Interestingly, the M.leprae 28 kDa protein is known to have asequence similarity with human superoxidedismutase (SOD) (67%) and E. coli SOD(55%) (36). The elevated antibody level to28kDa antigen in some of the LL and bor-derline patients with Type 1 reaction maybe attributed to the response against in-creased expression of SOD in response toenvironmental stress during the diseaseprocess or during reaction.

The most interesting finding of our studyis that the antibody level against M. leprae18kDa antigen was much higher in leprosypatients with Type 1 reactions, although thepercent seropositivity was also high in LLpatients without reactions. Our study indi-cates that production of anti18-kDa anti-body is a prominent event in leprosy, as allgroups of leprosy patients except TT pa-tients had a high level of antibodies to thisantigen. Khan, et al. reported a low reactiv-ity to this antigen in multibacillary patients(15), but we observed seropositivity of62.5% in LL patients. Further, Roche, et al.(1991) observed a similar finding of a lowlevel of anti 18kDa antibodies in pau-cibacillary (PB) patients and a high level inmultibacillary (MB) patients (28). Manyother authors have described the 18kDaprotein as one of the important antigenswhich produces significant B cell and T cellimmune response in leprosy (8, 10, 11, 24, 28).However, its association with Type 1 reac-tions has not been described previously.This protein was reported to have strikinglysimilar size and sequence to a family ofheat shock proteins (25) and is expressedduring heat stress (19). So, we postulate thatthe expression of this antigen by M. lepraemight be increasing due to cellular resis-tance by the host, and as a result the host re-sponds by producing antibodies to thisstress protein. This could induce an immuneresponse in the initial phase of infectionsand during Type 1 reactions.

From our observations, we conclude thatcirculating antibodies to some of the stress

156 International Journal of Leprosy 2004

proteins of M. leprae appear to play a rolein Type 1 reactions. We could not observeany significant difference in antibody levelagainst the recombinant proteins in LL pa-tients, nor in patients with Type 2 reaction,though the reactivity was greater to someantigens in patients with Type 2 reactionsthan TT patients or healthy controls. Miller,et al. (1984) have also reported that the oc-currence of Type 2 reaction had no signifi-cant effect on the total level of IgG anti-body against arabinomannan (23).

With regard to the lymphoproliferativeresponses to these antigens, all healthy indi-viduals except one responded to MLSE, butnone of the recombinant proteins induced astrong proliferative response in this group,confirming the earlier report of Wilkinson,et al. (41). Moreover, most of the patientsand all healthy controls were responsive tothe purified protein derivative of M. bovis(data not included). In the present study,TT/BT patients showed stronger lympho-proliferative responses than those of BL/LLpatients only to MLSE and not to recombi-nant proteins of M. leprae, consistent withthe study of Thole, et al. (37) Further, we ob-served a significant lymphoproliferative re-sponse to MLSE in patients with Type 1 re-action. Bjune, et al. (5) have already notedthis with sonicated preparations of M. lep-rae in patients with Type 1 reactions. Thefinding of a significant lymphoproliferativeresponse to MLSE during Type 1 reactions,compared with borderline patients withoutreactions, clearly indicates the upregulationof CMI in such patients.

We did not observe any significant differ-ence in the proliferative response to 65 kDaantigen among patient groups, as reportedby others. Ilangumaran, et al. (13) reportedthat there is an inverse relationship betweencell mediated and humoral immune re-sponses to 65 kDa in leprosy patients. DeLa Barrera, et al. (7) have observed that M.leprae 65 kDa is a poor inducer of cyto-toxic T lymphocyte (CTL) in MB patients,but could induce proliferation and CTL inMB patients with Type 2 reaction.

The finding that the 28 kDa antigen in-duced a poor response both in TT and LLpatients is in agreement with the study ofWilkinson, et al. (41). Lepromatous patientsdid not differ significantly from patientswith reaction in their proliferative responseto all recombinant proteins tested in our

study except that of the 28 kDa antigenwhere a significant proportion of patientswith Type 2 reaction responded to this anti-gen. While a number of studies have re-ported the antigenic potential of 28 kDa inthe humoral immune response, not muchinformation is available regarding the na-ture of cell mediated response against it.Though Wilkinson, et al. (41) have de-scribed this as a moderate stimulator of Tcell responses, they did not investigate theresponse in patients during leprosy reac-tions (Type 1 or Type 2). The significantlygreater positivity in proliferative responsesin Type 2 patients than those of BT/BL/LLmight indicate a response to the expressionof SOD during this reactional stress. Thefinding of a significantly greater number ofType 2 reaction cases responding to the 28kDa antigen compared to BL/LL patientsmight explain their transient boost in CMIas reported earlier by other authors (18).

Although previous workers have demon-strated the presence of antibodies to theseproteins in BT/TT and BL/LL patients, thisappears to be the first study to demonstratea high level of antibodies especially against28 kDa in these patients associated withType 1 reaction. The role of M. leprae anti-gens in Type 1 reactional pathology hasbeen noted others (17, 20, 39). Hence, the highlevel of antibodies observed in patients dur-ing Type 1 reaction may be due to the M.leprae antigens exposed in tissues duringreactions. At this moment, it is not possibleto conclude whether antibodies are induceddue to the development of reactionalpathology, or if it has been initiated due tothe induction of antibodies. The cellular im-mune response associated with Type 1 reac-tion is presumably due to other M. lepraeantigens and not due to the stress proteinsexpressed by M. leprae.

Acknowledgment. Indian Council of Medical Re-search (ICMR), Government of India, supported thisstudy. LEPRA grant was used for purchasing of certainreagents, which were used in the study. We acknowl-edge the help of Mr. V. S. Yadav for statistical analysis(Chi square and Fisher exact test) and Mr. HariomAgarwal for photography. We thank Mr. P. N. Sharma,Mr. M. S. Tomar, Mr. M. Alam, and Mr. K. Kul-shrestha for their technical help.

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1 Received for publication on 22 July 2003. Accepted for publication on 14 January 2004.2 M. Furuta, M.D., Department of Pathology, Izumigaoka Hospital, Turuga-city, Fukui, 914-0770, Japan; K.

Hatano, M.D.; Y. Okano, M.D.; T. Matsuki, M.D., National sanatorium Oku-Komyoen, Okayama, Japan; T.Ikeda, M.D.; K. Nakatani, M.D.; A. Sato, M.D., National sanatorium Minami Kyoto Hospital, Kyoto, Japan; M.Mizushima, M.D., Gifu Prefectural Tajimi Hospital, Tajimi, Japan.

Reprint requests to Dr. Yoshiko Okano, Department of Ophthalmology, National sanatorium Oku-Komyoen,6253 Mushiake, Oku-cho, Oku-gun, Okayama, 701-4593, Japan. E-mail: okano@ nsok.hosp.go. jp

Axonal Spherical Bodies in the Peripheral Nerves of

Leprosy Patients1

Mutsuhiro Furuta, Kentaro Hatano, Yoshiko Okano, Takanobu Matsuki, Takeshi lkeda, Kouichi Nakatani, Atsuo Sato, and Mutsue Mizushima 2

ABSTRACTSpherical bodies, roughly 10 µm in diameter, which have not been reported before, were

found in the peripheral nerve axons of specimens collected during post-mortem examinationof leprosy patients.

These bodies were found in the fascicles of all peripheral nerves of the extremities ex-amined (median, radial, ulnar, peroneal and sciatic nerves). Their incidence was not relatedto the type of leprosy. The area immediately below the thickened perineurium, a feature as-sociated with leprosy, often showed a large number of spherical bodies.

When observed under a transmission electron microscope, the spherical lesions oftenshowed a lamellar structure, although some of them were amorphous. No structure resem-bling organelles was seen within the bodies.

Observation with the merge technique showed a clearly lamellar structure in most of thespherical bodies. These bodies and the surrounding myelin sheaths were partially polarized.

The axonal spherical bodies observed in our study seem to represent lesions graduallyformed due to glycoprotein denaturation over long periods of time and to be associated withleprosy-caused thickening of the perineurium of peripheral nerves.

RÉSUMÉDes corps sphériques, mesurant environ 10 µm de diamètre, qui n’ont pas encore été rap-

portés, furent trouvés dans les axones des nerfs périphériques prélevés à l’autopsie de pa-tients lépreux.

Ces corps furent retrouvés dans les faisceaux de tous les nerfs périphériques des ex-trémités examinées (nerfs médian, radial, ulnaire, péroné et sciatique). Leur incidence n’étaitpas liée au type de lèpre. La zone immédiatement en dessous d’un périneurium épaissi, uncaractère associé à la lèpre, était fréquemment riche de ces corps sphériques.

Lorsque observés au microscope électronique à transmission, ces corps sphériques mon-traient fréquemment une structure lamellaire, bien que certains étaient amorphes. Aucunestructure ressemblant à une organelle ne fut décelée dans ces corps.

L’observation par une technique de concaténation a révélé une structure clairement lamel-laire dans la grande majorité des corps sphériques. Ces corps et les manchons myéliniquesenvironnants n’étaient que partiellement polarisés.

Ces corps sphériques des axones, observés dans notre étude, semblent représenter des lé-sions progressives à long terme de dénaturation des glycoprotéines et être associés auxépaississements du périneurium des nerfs périphériques causés par la lèpre.

RESUMENSe observaron cuerpos esféricos de aproximadamente 10 µm de diámetro en los axones

de especimenes de nervios periféricos colectados durante el examen post-mortem de pa-cientes con lepra. Estos cuerpos esféricos, que no se habían descrito antes, se encontraron enlos fascículos de todos los nervios de las extremidades examinados incluyendo los nerviosmediano, radial, ulnar, peronal y ciático. Su incidencia no estuvo relacionada con el tipo de

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INTERNATIONAL JOURNAL OF LEPROSY Volume 72, Number 2Printed in the U.S.A.

(ISSN 0148-916X)

160 International Journal of Leprosy 2004

During our 40 years of experience of post-mortem examinations of leprosy patients, wehave detected spherical bodies in the periph-eral nerve axons of these patients. Many re-ports on peripheral nerve lesions observed inleprosy patients have been based on electronmicroscopy examinations. These reportshave often mentioned the presence of My-cobacterium leprae in the axons, but noneof them have reported the presence of ax-onal spherical bodies. For the study pre-sented here, axonal spherical bodies wereobserved under light and electron micro-scopes at magnifications up to ×1000.

We used a new method known as mergetechnique (1), which allows for simultane-ous viewing of an object under both a po-larized microscope (PM) and a differentialinterference contrast microscope (DIC)within a common visual field.

MATERIALS AND METHODSSubjects from which specimens were

obtained. Peripheral nerve specimens col-lected from 6 cadavers during post-mortemexamination at the “Oku Komyoen” Na-tional Sanatorium were used for this study.One of the specimens was relatively old(collected in 1983), but the other speci-mens were collected fairly recently (threein 1995 and two in 1996). The specimenswere collected from 6 cases whose historyfollows. In all cases, leprosy-related pe-ripheral nerve disturbance was observed.

Case 1. An 89-year-old male with lepro-matous leprosy, who was diagnosed at age17 with Hansen’s disease, and was admittedto the sanatorium when he was 27. At age89, he was hospitalized because of mildmotor paralysis of the extremities. Duringhis hospitalization, he developed fever andincreased sputum, leading to death fromdyspnea in 1983.

Case 2. A 76-year-old female with tuber-culoid leprosy, who was diagnosed at age26 with Hansen’s disease and admitted tothe sanatorium when she was 27. At age 71,she was diagnosed as having hepatocellularcarcinoma during treatment for hepatitis C,and in 1995, she died of rupture of thecancer-affected liver.

Case 3. An 89-year-old male with lepro-matous leprosy, who was diagnosed withHansen’s disease when he was 25, and wasadmitted to the sanatorium 5 yrs later. At age81, he was hospitalized because of anorexia,and 3 yrs later in 1995, he died of exacerba-tion of pneumonia.

Case 4. A 64-year-old male with lepro-matous leprosy, whose diagnosis ofHansen’s disease was established when hewas 14. The next year he was admitted tothe sanatorium. At age 62, he was diag-nosed with prostate cancer and began hor-mone therapy. When he was 64 years old,the cancer had metastasized to the stomach,and he died of deterioration of his generalcondition in 1995.

Case 5. A 79-year-old male with lepro-matous leprosy, who was diagnosed as hav-ing Hansen’s disease when he was 24, wasadmitted to the sanatorium at age 27. Hewas diagnosed with a urinary bladder tumorwhen he was 77 years old and underwentseveral sessions of transurethral tumor re-section. Two years later, when he was 79,he underwent a total cystectomy, but post-operatively developed metastasis of thecancer to the lumbar vertebrae, liver, subcu-taneous tissue, and other areas. He died ofcancer in the same year in 1996.

Case 6. An 81-year-old male with tuber-culoid leprosy was diagnosed at age 21 ashaving Hansen’s disease and admitted tothe sanatorium at age 26. When he was 81years old, he developed chest pain and hy-

lepra y se observaron con más frecuencia por debajo del perineurio engrosado característicode la lepra.

Observados al microscopio electrónico de transmisión los cuerpos esféricos mostraronuna estructura laminar, aunque algunos de ellos aparecieron amorfos. No se observaron es-tructuras sugerentes de organelos dentro de los cuerpos esféricos. Además de su estructuralaminar, estos cuerpos y las capas envolventes de mielina aparecieron parcialmente polar-izados.

Los cuerpos esféricos axonales observados en nuestro estudio parecen representar le-siones formadas gradualmente por la desnaturalización de glicoproteínas a lo largo de peri-odos prolongados de tiempo y parecen estar asociados con el engrosamiento del perineuriode los nervios periféricos ocasionado por la lepra.

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drothorax associated with thoracic aorticaneurysm, and his condition was compli-cated by DIC, leading to his death in 1996.

Preparation of specimens. All speci-mens of the peripheral nerves were fixed in10% formalin. The specimens collected in1995 and 1996 were 10 cm long were cutinto sections of about 2 cm. They were sub-jected to HE, PAS, Bodian, Luxol fast blue,amyloid and acid-fast bacterium staining.Formalin-fixed specimens that were foundto contain spherical bodies were subjectedto PCR assay to determine the relationshipof the bodies to Mycobacterium leprae. Thespecimens were also subjected to merge ob-servation (allowing simultaneous observa-tion under both PM and DIC in a commonvisual field) and electron microscopy.

Merge technique. Non-stained, deparaf-finized specimens, 5–7 µm thick, weremounted on acryl-based material. The spec-imen can be observed simultaneously in abright visual field under a biomicroscope(BX51, Olympus), a DIC (Olympus) and aPM (Olympus), without the need to movethe stage of any of the microscopes. Micro-scopes capable of magnification up to×1000 were used. Because DIC and PM(two systems with different properties)share the same polarizing filter (polarizer oranalyzer), switching from the DIC to thePM image and vice versa can be done

rapidly, without any distortion in the visualfield. At room temperature and under ordi-nary fluorescent light, polarized imageswere obtained in the FL and differential-interference images in the BF mode with adigital camera (DP-50, Olympus). The im-ages thus taken were subjected to imageanalysis using the Photoshop (Adobe) soft-ware package. During image analysis, po-larizing images were overlapped withdifferential-interference images of the same

FIG. l. Case 1. Several axonal spherical bodies are visible, some of them granular. These bodies were oftendetected immediately below the thickened perineurium (arrow). (×400, PAS).

FIG. 2. Case 1. Two axonal spherical bodies arevisible, one lamellar and the other amorphous. Thesurrounding myelin sheaths are degenerative. (×1000,PAS).

162 International Journal of Leprosy 2004

visual field and with the same number ofpixels.

Polarizing images can be used to checkfor polarizing materials, while differential-interference images provide a view of theentire photographed area. Merged imagesthen make it possible to determine the exactlocation of the polarizing material.

RESULTSSpherical bodies found within periph-

eral nerve axons under a light micro-scope. The diameter of most of the axonalspherical bodies was about 10 µm, with onesection usually containing 1 or 2 sphericalbodies. It was rare for 3 or more bodies tobe observed in one section. The bodieswere visible in HE-stained sections, butmore so in PAS-stained sections (Fig. 1).They were not stained by acid-fast stainingand often had a lamellar structure, althoughsome did not show any specific structure.The bodies were often spherical or oval,and some of them were composed of sev-eral small granules of irregular size.

A narrow area characterized by irregularswelling was often seen around the spheri-cal bodies within the axons (Fig. 2). The le-sions were more frequently seen immedi-ately below the perineurium, and they were

detected in all peripheral nerves examined(median, radial, ulnar, femoral, perinea andsciatic nerves). In Bodian-stained sections,spherical bodies are in the axons and the sil-ver particles are in the center (like a core)(Fig. 3). In Luxol fast blue-stained sections,the spherical bodies did not stain at all (Fig.4). Their incidence did not correlate withthe type of leprosy. These spherical bodiesshowed no chromatic response to amyloidstaining, and, when assayed by PCR, werefound to have no relationship to Mycobac-terium leprae (data not shown).

Findings from transmission electronmicroscopy. The spherical bodies which ap-peared to have no specific structure under thelight microscope were also amorphous whenobserved under the electron microscope. Thebodies with a lamellar structure under thelight microscope, were found under the elec-tron microscope to contain fine powder-likematerials with a high electron density in theircenter (like a core), and show a lamellarstructure composed of rings with differentelectron densities (Fig. 5). The myelinsheaths surrounding the spherical bodieswere of irregular thickness (Fig. 6).

Observation with the merge technique.A few of the spherical bodies showed par-tial polarization, while small areas within

FIG. 3. Case 1. A spherical body is visible in the axon. The silver particles that may be something like a coreare in the center. (×1000, Bodian).

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the myelin sheaths surrounding them weresometimes polarized (Figs. 7, 8). Mostspherical bodies had clearly lamellar struc-ture. A number of minute granular or rod-shaped polarized inclusions were visible inthe histopathology specimens. The polar-ization disappeared after treatment of thesections with an alkaline solution.

DISCUSSIONAlthough a number of reports (3) have

been published concerning peripheral nervelesions associated with Hansen’s disease,none of them have dealt with axonal spher-ical bodies. A search of previous findingsresembling the spherical bodies we detectedin peripheral nerve axons, revealed that the

FIG. 4. Case 1. A spherical body does not stain at all. (×1000, Luxol fast-blue).

FIG. 5. Case 1. Core-like material is visible in thecenter of the axonal spherical body showing a lamellarstructure. (electron micrograph).

FIG. 6. Case 1. An amorphous spherical body isvisible within the axon. The surrounding myelinsheaths vary in thickness. (electron micrograph).

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ballooning and fragmentation of axons (Bo-dian stain) shown in Fig. 3 of the report“Pathology of Peripheral Nerve Lesion inLepromatous Leprosy,” published in 1971by Job (4), resembled our spherical bodies.The spherical body and “spheroid” whichappears pathologically at the time ofamytrophic lateral sclerosis differ fromeach other in the size and the location.

While tissue specimens, sealed in fixationbottles, were available from not only the sixcases included in this study but also fromsome earlier cases, they had been collectedin various ways because post-mortem ex-aminations had been performed by severaldifferent pathologists. These earlier speci-mens were therefore not used for our studybecause it would be difficult to performlight microscopic observation of all nervespecimens under identical conditions. Wedo not think that the spherical bodies resultfrom the post mortem change, because thespherical bodies can be seen in both newand old specimens. The patients’ illnesseshad progressed over more than 10 yrs. Wedid special stains (Bodian and Luxol fast-blue stain) for the relation between thesespherical bodies and the axon or myelinsubstance. But this special stains showed no

relation between the spherical bodies andthe axon or myelin substance.

Under the light microscope, a narrowarea characterized by irregular swelling wasoften observed around the spherical bodywithin the axon. This area seemed to repre-sent a precursor lesion. Among the other ar-eas of the axon, without swelling, the spher-ical body was often seen in the area wherethe peripheral perineurium had thickenedand become abnormally hard. In this con-nection, it is interesting that Kimura, et al.(2) reported that the perineurium of the pe-ripheral nerves appears to be a target of My-cobacterium leprae.

When observed under the transmissionelectron microscope, the spherical lesionsoften showed a lamellar structure, althoughsome lesions had no specific structure. Thelesions contained nothing resembling or-ganelles, nor showed any chromatic re-sponse to amyloid staining, indicating thatthe lesion did not represent amyloid degen-eration. The fine powder-like material witha high electron density observed in the cen-tral area and resembling a core, differed inhardness from the other areas. This materialmade it difficult to slice the specimens intosections suitable for electron microscopy.

FIG. 8. Case 2. (left: DIC, middle: merge technique, right: PM) A group of axonal spherical bodies with po-larization mostly absent, except for some polarized spots.

FIG. 7. Case 2. (left: DIC, middle: merge technique, right: PM) Two partially polarized axonal spherical bod-ies are visible, with the degenerative surrounding myelin sheaths showing some polarized spots. Degenerativeconnective tissue also shows wave-formed polarization. Polarization of distribution is more evident on the imagetaken with the merge technique.

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When observed by the merge technique, thearea around the spherical bodies and themyelin sheaths surrounding them was par-tially polarized in some specimens, althoughinfrequently. Most of the spherical bodieshad a clearly lamellar structure, which isconsistent with the findings from HE andPAS staining and electron microscopy.

The axonal spherical bodies were clearlyPAS-positive, and calcification-like deposi-tions were occasionally seen in their center.These findings, combined with that of thelamellar structure, make it appear likelythat the bodies were gradually formed overlong periods of time. These bodies wereseen in cases of both lepromatous and tu-berculoid leprosy. In conclusion, the axonalspherical bodies observed in the study pre-sented here seem to represent lesions grad-ually formed due to glycoprotein denatura-tion over long periods of time and are asso-ciated with leprosy-caused thickening ofthe perineurium of peripheral nerves.

Acknowledgment. The authors are indebted to Mr.K. Fukuike (technologist) for his help in preparing the

pathology specimens and to Mr. T. Yamada (OlympusPlaza Osaka) for his help with using the merge tech-nique for taking the DIC and PM pictures. The authorsalso wish to thank Prof. C. Ide (Department ofAnatomy, Kyoto University) for the electron micro-graphs he prepared for this study and to Dr. K. Saeki(National Sanatorium Oshima Seishoen) for the PCRassay of formalin-fixed peripheral nerve specimens heconducted for this study.

REFERENCES1. FURUTA, M., HATANO, K., MATSUKI, T., OKANO, Y.,

IKEDA, T., NAKATANI, K., and SATO, A. Observa-tion of acid fast bacilli by merge technique of dif-ferential interference contrast and polarized micro-scopes. Poster presentation at XVI InternationalLeprosy Congress, Salvador, Brazil, 2002.

2. JOB, C. K. Pathology of peripheral nerve lesions inlepromatous leprosy—a light and electron micro-scopic study. Int. J. Lepr. 29 (1971) 251–268.

3. KIMURA, T., INOUE, K., IZUMI, S., HASHIMOTO, K.,and YAHARA, O. Leprous neuropathy morphologi-cal study of biopsied peripheral nerves. Advancesin Neurological Sciences 143 (1999) 929–942. [inJapanese]

4. RIDLEY, D. S., and JOB, C. K. The pathology of lep-rosy. In: Leprosy. 1st edn. New York: ChurchillLivingstone, 1985, pp. 115–117.

Proper classification of a disease is one ofthe fundamental tools of modern medicinein selecting treatment, evaluating prognosis,measuring overall progress, and furtheringthe understanding of that disease. Classifica-tion is an essential tool in our approach to adisease, much as a good map is necessarytool for developing and navigating a coun-try. The development of a comprehensive,practical classification system for leprosymay rate as one of the most important ac-complishments in the extraordinary progressagainst this disease in the 20th century.While the more obvious, essential accom-plishment was the discovery of effectiveanti-microbial agents to treat this infection,the availability of effective treatment doesnot, by itself, guarantee success against adisease. The value of classification is notonly historical; continued application of thebest classification system is essential in theefforts to better understand a disease and toclearly and intelligently develop a strategyto combat it and, ideally, prevent it.

The struggles against leukemia and lym-phoma provide a useful example of the con-tinuing value of classification. Several ef-fective agents were available for these ma-lignancies long before many of the currentsuccesses in treatment. Greater success hasbeen brought about in part by a better un-derstanding of how to use these agents incombination, but also—and very impor-tantly—by better classification systems thatenable physicians to know which types of

leukemia or lymphoma will respond to par-ticular medicines or combinations. Recog-nition of this has stimulated continued, vig-orous research to refine the methods andconcepts of classification of many malig-nancies. Such studies enhance the under-standing of these diseases, with implica-tions not only for treatment but also forearly detection and prevention. Today, nocancer researcher would consider conduct-ing a study, or publishing results of a study,using a primitive or technically outmodedclassification system. And no professionaljournal would accept such a report.

Yet, in current leprosy research, a dis-turbing trend is to do exactly that: to aban-don the best classification system (one thatuses clinical assessment plus histopatho-logic examination of a skin biopsy), andchoose instead to group patients into only 2groups, multibacillary (MB) or paucibacil-lary (PB), according to their bacterial load,or to disregard the bacilli altogether andclassify according to the number of skin le-sions on a patient’s body. This last ap-proach, which disregards the bacteria en-tirely, seems highly ironic for research onan infectious disease. These approaches aretechnically inferior ones that assume, andaccept as satisfactory, a higher degree of in-accuracy than is readily available with stan-dard technology. Such over-simplificationof this complex host-pathogen relationshipis unfortunate and unacceptable. It is as ifsome have grown intellectually weary of

INTERNATIONAL JOURNAL OF LEPROSYand Other Mycobacterial Diseases

OFFICIAL ORGAN OF THE INTERNATIONAL LEPROSY ASSOCIATION

EDITORIALEditorial opinions expressed are those of the writers.

Classification of Leprosy: A Full Color Spectrum, or

Black and White?

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trying to understand the full-color im-munopathologic spectrum, and have de-cided to settle for a black and white outline.

Why is this done? Two related reasons aregenerally given—cost and expertise. TheMB/PB categorization was promulgated bythe World Health Organization in the globalcampaign to eliminate leprosy as a publichealth problem. For some treatment andcontrol programs, where access to expertiseand other resources is severely limited, theuse of a simplified system of classification isreasonable, just as paramedical workers de-liver medical care where there are no doc-tors. It has become too easy, however, to usethis as an excuse to justify a non-critical ac-ceptance of oversimplification that does adisservice to our basic research endeavors.

Research always requires substantial ex-pertise and is inherently costly, and isnearly always conducted with specificallyallocated research funds. Nevertheless, wehave watched with dismay as some investi-gators and collaborative groups apply so-phisticated molecular and immunologictechniques to specimens from patients whoare classified only as MB or PB, or are clas-sified only according to the number of skinlesions. Some of these manuscripts arrive atour office, and some are published in otherjournals. The multiple authorship and ac-knowledgments of support in most of thesepapers clearly indicate that financial re-sources and sophisticated expertise havebeen brought to bear, and funds have beenallocated for expensive instruments andreagents. Experienced clinical leprologistsare virtually always involved in these stud-ies, implying an availability of sufficient re-sources, and it is not acceptable that they donot take the effort also to obtain skin biop-sies and have them examined by an experi-enced professional.

In some instances the pressure to publishquickly appears to play a role. The Ridley-Jopling classification system (1) divides pa-tients into five groups, whereas MB/PBschemes divide into only two groups. It ismuch easier (and faster) to obtain enoughpatients for a 2-group protocol than for onewith 4 or 5 groups. But is this better? Isknowledge really advanced by such a sim-plification? We are very skeptical. No self-respecting academic research advisor willaccept such an excuse from a student, and

the research community should not makean exception and settle for this with respectto research on leprosy.

Any classification system must be ap-plied thoughtfully, and in some circum-stances a simpler system truly will suffice.For example, in epidemiologic or imple-mentation field studies, simplified classifi-cation may be justified.

The diversity inherent in the immuno-logic spectrum of leprosy may not be re-flected in all biological parameters we setout to measure. In some instances, the re-sults may reveal that patients fall into only2 or 3 groups. To discover this is not a fail-ure, nor is it wasted effort. Once such find-ings are established, a 2- or 3-part classifi-cation scheme for that parameter is accept-able. But if the hypothesis is not firstevaluated against the full spectrum, we willnot know if there were more than 2 or 3groups. The burden remains on the investi-gators, however, to explain why better clas-sification was impractical and why a simpli-fied system is actually acceptable in testingtheir hypothesis. If we do not look, we willnot know conditions as they truly exist, andwe may thus overlook important connec-tions and implications.

Researchers in leprosy have before them,at all times, one of the great immunologicalmodels in nature. An essential part of thefoundation of our understanding of leprosyis the recognition that—clinically, histolog-ically and immunologically—polar lepro-matous (LL) differs from borderline lepro-matous (BL), and borderline tuberculoid(BT) differs from polar tuberculoid leprosy(TT). From their first publication in themid-1960’s, the soundness of the theoreti-cal basis for this classification system (2),and the description of practical, straightfor-ward criteria to accomplish such classifica-tion anywhere in the world (1), were hailedas major accomplishments by workerswithin and beyond the field of leprosy. Boththe theory and the practical criteria recog-nize the natural diversity of the immune re-sponse in leprosy that has challenged im-munology for nearly half a century. A morecomplete understanding of the basis for thisdiversity and its underlying mechanismswill most probably be required before thisdisease can be eliminated (i.e., before ahighly effective vaccine can be developed).

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The questions posed by this complex im-munopathologic spectrum have perplexedmore than a few great minds who have at-tempted to tackle them. Although supportfor leprosy research has declined, it seems agrave mistake for those of us who continueto work on leprosy to surrender one of ourbest scientific assets—a practical and theo-retically sound classification system for lep-rosy. Oversimplification fosters the illusionthat this disease is simpler than it appears,and easy to understand (or eliminate). In-fection with Mycobacterium leprae elicitsthe full range of human immunologic re-sponses; this is a natural phenomenon and,like the metastasis of cancer, it will not goaway if ignored, but will be ignored at ourperil.

Today, although the prevalence of lep-rosy has declined worldwide, the number ofnew cases diagnosed annually has not. Thisparadox raises new, important, and interest-ing questions. Answering these and theother still unanswered questions about lep-rosy will require application of the best sci-

entific methods available. It is commonknowledge that funds for leprosy researchare in much shorter supply than they were afew years ago, and that fewer individualsare engaged in leprosy research. This, how-ever, is not an excuse for us to be less rigor-ous. To do so would be a travesty to thehundreds of thousands of patients still diag-nosed every year, to those with lasting dis-abilities from this disease, and to all ofthose who have gone before us, who did notshrink from a rigorous attempt to under-stand the complexity of leprosy eventhough they worked without many of thetechnical advantages we have today.

—DMS

REFERENCES1. RIDLEY, D. S., and JOPLING, W. H. Classification of

leprosy according to immunity, a five-group sys-tem. Int. J. Lepr. 34 (1966) 255–273.

2. SKINSNES, O. K. “The immunological spectrum ofleprosy.” In: Leprosy in Theory and Practice. Balti-more: Williams and Wilkins, 1964, pp. 156–162.

Genomic strategy to identify the chro-mosome 6 leprosy susceptibility gene. Al-though the localization of the leprosy sus-ceptibility locus to chromosome 6q25 wasan enormous breakthrough, the task of iden-tifying the responsible gene still presentedmajor difficulties, since the target regiondelineated by the two markers D6S4155and D6S1277 covered approximately 6.4million nucleotides that encoded 31 knowngenes (2). Since the region was too large toattempt straightforward comparative DNAsequencing, another strategy was needed todissect out the gene. Thus a panel of 64single nucleotide polymorphisms (SNPs)was selected that tagged each of the 31genes in the core interval by at least oneSNP marker (3). The SNP markers used forthe Mira, et al. study were selected eitherdirectly from the human SNP database orobtained by comparative sequencing of un-related Vietnamese individuals. Next, DNAsamples were isolated from all members of197 Vietnamese families composed of twohealthy parents and one leprosy-affectedchild, so called “simplex” families. TheseDNAs were genotyped for the 64 SNPs,and systematic analysis of association withleprosy disease was performed for eachSNP marker (“association scan”). Impor-tantly, the phenotype employed was leprosydisease independent of specific clinical pau-cibacillary or multibacillary forms (PB orMB) of the disease. The association scanshowed that SNPs with strongest associa-tion were located in an 80 kilobase frag-ment that overlapped the promoter (regula-tory region) of the PARK2 (parkin) andPACRG (parkin co-regulated gene) genes.

A higher density screening with a further 81SNPs in the 80 kilobase segment furtherconfirmed the regulatory promoter regionand a total of 17 SNP markers were foundassociated with leprosy disease. By con-ducting a multivariate analysis, it could beshown that only 2 of the 17 SNPs capturedthe entire association between the 80 kilo-base fragment and leprosy disease. Mostimportantly, the findings in the Vietnamesefamilies were confirmed in patients from asecond leprosy endemic country (3). A sub-sequent analysis found that the SNP mark-ers associated with leprosy in Vietnam werealso associated with leprosy susceptibilityin 975 unrelated individuals from Brazil. Inboth populations, the risk alleles are associ-ated with leprosy per se, meaning that per-sons carrying the risk alleles would beequally likely to develop PB as MB. There-fore, the cause of susceptibility is likely to involve an early, common cellular path-way used by the M. leprae bacillus. Whilethe study conclusively implicates PARK2and/or PACRG in leprosy pathogenesis, thequestion if any of the leprosy-associatedSNPs is directly and causally involved inleprosy susceptibility remains unanswered.

PARK2 and PACRG genes reveal thefunction of a novel cellular pathway in sus-ceptibility to leprosy infection. The possibleidentity of a “leprosy per se” pathway wasrevealed through knowledge of the functionof the PARK2 and PACRG genes (1, 5).Mutations in PARK2 are responsible forfamilial early-onset Parkinson disease (PD),which represents approximately 3% of allPD cases. The mutations in PARK2 in PDare not found in leprosy patients. The

COMMENTARYLinkage of Leprosy Susceptibility to

Parkinson’s Disease GenesABSTRACT

In early 2003, an international team of scientists conducted a genome scan in Vietnamesemultiplex leprosy families and found that susceptibility to leprosy was significantly linked to re-gion q25 on the long arm of chromosome 6. Further confirmation of the chromosome 6 locuswas provided by high-resolution linkage mapping in simplex leprosy families. Now, in a con-tinuation of these findings, the team has pinpointed the chromosome 6 susceptibility locus to the5′ regulatory promoter region shared by both the Parkinson’s disease gene PARK2 and its co-regulated gene PACRG. The surprising discovery has important implications for the under-standing of leprosy pathogenesis and for the strategy of genetic analysis of infectious diseases.

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PARK2 (parkin) gene product is an ubiqui-tin-protein ligase, which activates depo-sition of certain proteins such as alpha-synuclein in so-called intracellular Lewybodies. The lack of ubiquitin ligase activityin patients with PARK2 mutations causesprotein accumulation and neurodegenera-tion. PACRG appears to be involved in thetransport of polyubiquitylated proteins tothe proteasome. Overall, PD is a complexdisease with both genetic and environmentalfactors, and it has been suggested that infec-tions may trigger its onset. However, neitherPARK2 or PACRG genes have yet been as-sociated with susceptibility to any infectiousdisease other than leprosy. One importantaspect to determine then is whether thePARK2 or PACRG associations are foundonly in leprosy, or are associated with othermycobacterial diseases, such as tuberculo-sis, and, if the polymorphisms associatedwith leprosy also predict risk of PD.

In vitro experiments to determine thefunction of the leprosy susceptibility gene.The primary focus now should be to establisha connection between the risk alleles for lep-rosy susceptibility, the ubiquitin proteolysispathway and the course of M. leprae infec-tion and growth. There are many unknownsto this next phase of experimentation. Forexample, it is not known whether the lep-rosy susceptibility alleles would up or downregulate the PARK2 or PACRG encodedproteins, or affect the cellular ubiquitinpathway. In the context of further functionalstudies, it is revealing that both PARK2 andPACRG are expressed by Schwann cellsand monocyte-derived macrophages (3).Nevertheless, testing of the risk alleles inpatients will have to await a reliable func-tional assay for biological activity of M.leprae. In parallel, it is also hoped that theanalysis of M. leprae and the parkin geneswill yield information relevant to the neuro-logical aspects of Parkinson’s disease.

CONCLUSIONThe clinical spectrum of leprosy has long

been recognized as an immunologicalmodel in which various aspects of human Tcell subset and cytokine function can becharacterized (4). It is fitting that leprosyhas proven once again to be a model formolecular genomics, by providing the firstinfectious disease locus isolated by posi-tional cloning. Since this success is largely

rooted in the framework provided by theHuman Genome Project, the identificationof this novel and entirely unexpected lep-rosy susceptibility locus also provides agood example that recent advances in ge-nomics can be used for the study of dis-eases primarily prevalent in resource-poorcountries. Taken together, the papers byMira, et al. provide a general framework forthe genetic analysis of complex infectiousdiseases. Above all, it is hoped that thenovel link of leprosy susceptibility to theubiquitin proteolysis pathway will yieldsome insight to the transmission of leprosy,a disease which has so far evaded eradica-tion despite many years of effective drugsand case finding (6).

—Ellen Buschman—Emil Skamene1

McGill Center for the Study of Host Resistance,

Montreal, Canada

REFERENCES1. KITADA, T., ASAKAWA, S., HATTORI, N., MATSUM-

INE, H., YAMAMURA, Y., MINOSHIMA, S., YOKOCHI,M., MIZUNOM Y., and SHIMIZU, N. Mutations in theparkin gene cause autosomal recessive juvenileparkinsonism. Nature 392 (1998) 605–608.

2. MIRA, M. T., ALCAIS, A., NGUYEN, V. T., MORAES,M. O., DI FLUMERI, C., VU, H. T., MAI, C. P.,NGUYEN, T. H., NGUYEN, N. B., PHAM, X. K.,SARNO, E. N., ALTER, A., MONTPETITM A., MORAES,M. E., MORAES, J. R., DORE, C., GALLANT, C. J.,LEPAGE, P., VERNER, A., VAN DE VOSSE, E., HUD-SON, T. J., ABEL, L., and SCHURR, E. Susceptibilityto leprosy is associated with PARK2 and PACRG.Nature 427 (2004) 636–640.

3. MIRA, M. T., ALCAIS, A., VAN THUC, N., THAI, V.H., HUONG, N. T., BA, N. N., VERNER, A., HUDSON,T. J., ABEL, L., and SCHURR, E. Chromosome 6q25is linked to susceptibility to leprosy in a Viet-namese population. Nat. Genet. 33 (2003) 412–415.

4. SALGAME, P., ABRAMS, J. S., CLAYBERGER, C., GOLD-STEIN, H., CONVIT, J., MODLIN, R. L., and BLOOM, B.R. Differing lymphokine profiles of functional sub-sets of human CD4 and CD8 T cell clones. Science254 (1991) 279–282.

5. WEST, A. B., LOCKHART, P. J., O’FARELL, C., andFARRER, M. J. Identification of a novel gene linkedto parkin via a bi-directional promoter. J. Mol. Biol.326 (2003) 11–19.

6. WORLD HEALTH ORGANIZATION. Leprosy. Globalsituation. Weekly. Epidemiol. Rec. 77 (2002) 1–8.

1Reprint requests to: Emil Skamene, Montreal Gen-eral Hospital, Room A6-149, 1650 Cedar Avenue,MONTREAL, Que, H3G 1A4, Canada. Email:emil.skamene@muhc.mcgill.ca

COMMENTARYNeuropathic Pain in Leprosy1

ABSTRACTNeuropathic pain appears to be much more common in leprosy than has been generally

appreciated. Emphasis in leprosy control programs has been on the distribution of multi-drug therapy, on early and better detection, and on the prevention of disability related toanesthetic limbs. Most have thus been inattentive to the problem of neuropathic pain in lep-rosy patients. Neuropathic pain does not respond to the usual analgesics employed for reac-tions, for example, and so it is important that those treating leprosy patients give this prob-lem the special attention it requires, both in diagnosis and in treatment.

RÉSUMÉLes douleurs neurogènes de la lèpre pourraient bien être beaucoup plus fréquentes que ce

qui a été considéré auparavant. L’effort des programmes de contrôle de la lèpre a été orientévers la distribution de la polychimiothérapie, une détection meilleure et plus précoce et dansla prévention des handicaps associés à des membres anesthésiés. La plupart de ces pro-grammes ont ainsi apporté peu d’attention au problème des douleurs neurogènes chez les pa-tients hanséniens. Les douleurs neurogènes ne répondent pas aux analgésiques usuels em-ployés pour traiter par exemple les réactions et il est donc important que les personnes en-gagées dans le traitement des patients hanséniens considèrent particulièrement cet aspect,tant en ce qui concerne le diagnostic que le traitement.

RESUMENEl dolor neuropático en la lepra parece ser mucho más común de lo que usualmente se

considera. Los programas de control de la lepra han hecho mucho énfasis en la distribuciónde drogas para la poliquimioterapia, en la temprana y mejor detección de la enfermedad, yen la prevención de las incapacidades relacionadas con los miembros anestésicos. Muchosprogramas han puesto poca atención al problema del dolor neuropático en los pacientes conlepra. El dolor neuropático no response a los analgésicos usualmente empleados en las reac-ciones de la lepra, por ejemplo. Por esto, es muy importante que los encargados deltratamiento de los pacientes con lepra den a este problema la atención especial que requiere,tanto en el diagnóstico como en el tratamiento.

Stump, et al.’s paper, “Neuropathic painin Leprosy patients,” published in this issueof the JOURNAL, is a timely and importantcontribution to the evaluation and manage-ment of leprosy sufferers. In the widermedical world the management of chronicpain is developing as a specialty in its ownright complete with journals and interna-tional conferences devoted to the subject. Itis an interesting coincidence that a reviewarticle on the same topic has just been pub-lished in the most recent edition of LeprosyReview (1).

In the leprosy world, we have been slowto catch on to the existence of chronic neu-ropathic pain occurring in leprosy patients.Hastings’ textbook Leprosy (1995) does notmention it at all, and there have been re-

1 Received for publication on 23 March 2004. Ac-cepted for publication on 14 April 2004.

markably few papers published on the sub-ject in the world leprosy literature. YetStump and his colleagues report that 56%of the 358 patients assessed for neuropathicpain in his study either had experienced orwere experiencing episodes of pain of suffi-cient intensity to interfere with activities ofdaily life or sleep. The statistics they ad-duce are in line with findings from the fewother studies that have been carried outamongst leprosy sufferers. How could wehave missed it for so long?

There are several overlapping answers tothat question. For several years most pro-grams, NGO and Government alike, havesimply been extremely busy and focussedon case finding and multi-drug therapy(MDT) administration. This has been ex-traordinarily successful in reducing preva-lence and clearing the backlog of patients inthe community. In many places the heat has

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now come out of that approach and perhapsthere is a little more space to reflect on whatour patients—including “cured” patients—are actually experiencing. Another impor-tant focus in leprosy programs and in re-search of the last decade or so has been thedetection and management nerve functionimpairment (NFI) and the prevention of dis-ability. Both of these foci—detection andtreatment, and prevention of disability—have had anaesthesia at the hub, since it isthe absence of sensation that leads both tothe diagnosis of leprosy (and therefore totreatment), and to the development of themost damaging disabilities and consequenthandicap and stigma. We have been so at-tuned to painlessness that we have missedthe fact that a very significant proportion ofour leprosy sufferers experience pain aspart of their dis-ease. Furthermore, theymay continue to suffer long after they havebeen declared “cured” and are lost tofollow-up. That we should have been sodeaf and blind to this most basic of com-plaints—pain—is extraordinary.

As already alluded to, there has been apaucity of studies into neuropathic pain car-ried out amongst leprosy sufferers and veryfew references to it at all in the world liter-ature. There is a clear need for more re-search into this subject and for the findingsto be applied as rapidly as possible. How-ever, much is known already about the di-agnosis and management of neuropathicpain in general that could easily be appliednow. If it is as common as Stump et al. sug-gests—and it probably is—then we shouldget on with it now.

If we are to begin to help leprosy suffer-ers with chronic neuropathic pain then as afirst step we must ask them about it. Itshould not be left to experts in research cen-ters to ask the questions; it should becomepart of the routine history taking of everyparamedical worker. Before that can hap-pen, training institutions must incorporatethis message. Leprosy workers need to un-derstand the difference between nociceptiveand neuropathic pain, and Stump, et al.rightly draw attention to this. Crucially, itshould be understood that neuropathic painwill not respond to simple analgesia, butrather to different drugs such as tricyclicsand anticonvulsants. Then, the treatment ofneuropathic pain must become mainline.

Perhaps a good word to use here is demys-tify. Neuropathic pain is regarded by somedoctors as a little technical, rarefied even.The subject needs to be demystified, itneeds to make the jump to become the reg-ular.

Perhaps the situation we are in is akin tothe situation that existed a decade or so ago,before the widespread use of corticosteroidsat “field level” to treat acute NFI and reac-tions. We knew how to measure NFI, andwe had an effective drug, prednisolone, butit took a paradigm shift in thought and prac-tice for this technology to be widely andsimply applied so that the maximum num-ber of people could benefit. In the sameway, it is known how to diagnose neuro-pathic pain (and it is not difficult), and atleast one very cheap and effective drug isavailable (amitriptyline). A widespread ap-plication of this knowledge down to thegrassroots level could be of considerablebenefit to a large number of people.

The current cut-and-dried WHO recom-mendations for the treatment of leprosy fo-cuses very much on bacteriological curewith discharge after relatively short coursesof treatment. It is well known that thislargely ignores the existence of new nervedamage after release from treatment, but todate there has been very little appreciationof the way that this ignores the presence ofneuropathic pain among “cured” leprosysufferers, as Stump et al. points out in hisconclusions. Indeed, the prevalence of neu-ropathic pain he found is actually higherthan that often quoted for NFI amongst lep-rosy patients.

In summary, Stump’s paper both docu-ments and highlights the existence of a com-mon and significant problem amongst lep-rosy patients, one that has been remarkablyoverlooked. There is a need to demystify thediagnosis and treatment of neuropathic painand to develop simple strategies that will en-able the widespread application of simpleand effective techniques for its manage-ment.

—Richard Croft

REFERENCE1. HAANPÄÄ, M. LOCKWOOD, D. N. J., and HIETA-

HARJU, A. Neuropathic pain in leprosy. Lepr. Rev.75 (2004) 7–18.

172 International Journal of Leprosy 2004

TO THE EDITOR:

According to World Health Organization(WHO) recommendations: “five, or lessernumber of lesions” of leprosy should betreated as paucibacillary (PB) leprosy andshould be given 6 months treatment with ri-fampicin monthly and dapasone daily (5, 6).In this type of simplified classification, thesize of the patches are not considered. How-ever, we feel that size of the patch should beconsidered on deciding whether to treat acase as PB with two drugs for 6 months, oras multibacillary (MB) leprosy with threedrugs for a year. Categorizing leprosy as PBor MB is particularly important in areaswhere treatment is commenced without anybacteriological and histopathological confir-mation. Even in the time honored Ridley-Jopling Classification and its modifications,large patches of leprosy are considered as afeature, more commonly found in border-line, borderline tuberculoid, or subpolar lep-romatous leprosy (2, 4), giving due consider-ation to the size of the lesions.

Histopathologically, in tuberculoid leprosythere are tubercles composed of epithelioidcells. This is due to the process of destructionof lepra bacilli by histiocytes (3). The granu-lomatous reaction thus produced is the resultof a combination of the presence of bacilli

and the host response. Considering that sen-sory impairment and pathological hypopig-mentation in leprosy are due to this host re-sponse by the body in the fight against theleprosy bacilli, it is likely that, the larger thelesions of leprosy, the higher the number ofbacilli that cause the pathology. A granulomawhich originates due to one or more bacilli ina given area can only cause a very limitedspread of its effects, e.g., focal sensory loss inthe affected area. The fact that inoculation ofatypical mycobacteria causes a granuloma inthe immediate vicinity of the inoculation, andthat it spreads very slowly, suggests that pro-liferation of bacteria are necessary to cause alarger lesion. This also means that if there isno proliferation of bacilli in tuberculoid lep-rosy, there can not be evolution of a smallpatch, to become a large patch. This conceptis further supported by the fact that even in aType I leprosy reaction, there is no real lateralspread of a leprosy lesion, though the existinglesions temporarily become inflamed. Thissuggests that a pure immunological responsewithout an increase in bacilli is unlikely tocause a lesion to spread peripherally, to pro-duce a large hypopigmented patch. However,it is known that lowering of one’s cell medi-ated immunity is important in promoting thespread of leprosy lesions. In this situation, thepatient’s ability to destroy the multiplyinglepra bacilli is impaired, allowing the le-sion(s) to enlarge; as in the case of a tubercu-loid leprosy (PB) lesion or lesions in an un-treated patient, evolving towards the “lepro-matous pole” (MB) over several years.

Unless many individual cutaneous nerve

1 Received for publication on 23 October 2003. Ac-cepted for publication on 2 April 2004.

Reprint requests to: S. Prarasd W. Kumarasinghe,Consultant Dermatologist, National Skin Center, Sin-gapore, No. 1, Mandalay Road, Singapore, 308205. E-mail: prasadkumarasinghe@yahoo.com

CORRESPONDENCEThis department is for the publication of informal communications that are of interest

because they are informative and stimulating, and for the discussion of controversialmatters. The mandate of this JOURNAL is to disseminate information relating to leprosy inparticular and also other mycobacterial diseases. Dissident comment or interpretation onpublished research is of course valid, but personality attacks on individuals would seemunnecessary. Political comments, valid or not, also are unwelcome. They might result ininterference with the distribution of the JOURNAL and thus interfere with its prime purpose.

Should Large Lesions of Leprosy Be Considered As

“Multibacillary” for Treatment Purposes Even If the Total

Number of Lesions Is Less Than Five?1

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fibers are affected by separate bacilli, therecan not be sensory impairment in a largearea, involving the total area of the hy-popigmented macule. This is different todistal sensory impairment due to a nervetrunk involvement, for example, sensoryimpairment along ulnar nerve distribution.Furthermore, in monitoring tuberculoid orborderline tuberculoid leprosy, peripheralextension of a lesion is considered to be afeature of failure of treatment or relapse.

Although relapses of leprosy after treat-ment are reportedly uncommon, many au-thorities feel that they may be underesti-mated (1). If an MB case is misdiagnosedand treated with dapsone daily and ri-fampicin monthly as a PB case, that patientreceives only 6 doses of the bactericidal drugrifampicin before stopping the treatment.This would be totally inadequate. Some au-thorities even believe that MB treatmentshould be continued for 24 months ratherthan the WHO recommended 12 months (1).In countries where leprosy is still highlyprevalent, follow-up after discharge from ac-tive treatment is unsatisfactory. Thereforemany relapses or suboptimal treatments maygo unnoticed for many years.

Considering the above facts, we feel thatwhere a large patch (more than 10 cm in di-ameter) of leprosy is present, irrespective ofthe size of the other lesion or lesions, thepatient should be treated as MB and giventreatment at least for 12 months. Just as “5or less leprosy macules are considered asPB” (as recommended by the WHO) is anarbitrary limit, “the dimensions of a lesion”is also an arbitrary measurement, for placeswhere microbiological and histopathologi-cal services are unavailable. It should alsobe emphasized that counting lesions can beerroneous if the whole body is not carefullychecked by the healthcare worker. A personmay have 5 easily visible lesions, but theremay be another small lesion or lesions inunsuspected places such as nasal cleft, atoe, or an elbow posteriorly. In such a situa-tion, a patient would receive only PB treat-ment. However, it is highly unlikely that alarge patch (>10 cm) of leprosy would goundetected by the patient or the clinician orthe health care worker.

In our experience with cases of leprosyin the last two decades (mostly when work-ing in Sri Lanka) we have encountered sev-

eral instances where MB cases had beencategorized and treated as PB, by others,especially by public health workers, due totheir strict categorization according to the“number of patches.” These cases weresubsequently given the MB treatment regi-men. In hospital settings, facilities forbiopsy and smears with microbiologicalevaluation are available and clinicians dono go by the number of patches alone fortreatment.

Long term follow-up of patients withlarge macules of leprosy treated with thestandard WHO treatment regimens wouldbe necessary to ascertain whether relapserate is higher in this group of patients. Per-sonal experience suggests this group hasmore relapses or non responders.

A consensus on the duration and type oftreatment for large macules of leprosywould be desirable for places wherehistopathological and microbiological facil-ities are not available. It appears prudent totreat such cases with MB treatment regimen.

—S. Prasad W. Kumarasinghe, M.D., MBBS, FCCP, FAMS

Consultant DermatologistNational Skin Center, Singapore

—M. P. Kumarasinghe, M.D., MBBS, FRCPA, Dip Cy Path, FCCP, FAMS

Senior Consultant PathologistSingapore General Hospital

REFERENCES1. INTERNATIONAL LEPROSY ASSOCIATION TECHNICAL

FORUM. Report of the International Leprosy Associ-ation Technical Forum. Int. J. Lepr. Other My-cobact. Dis. 70(1)(Suppl) (2002) S3–S60.

2. JOPLING, W. H., and MCDOUGALL, A. C., EDS.Handbook of Leprosy, 4th edn. New Delhi: CBSPublishers, 1992, pp. 22–44.

3. MEHREGAN, A. H., and HASHIMOTO, K. Pinkus’Guide to Dermatohistopathology, 5th edn. EastNorwalk: Appleton and Lange, 1991, pp. 281–290.

4. RIDLEY, D. S., and JOPLING, W. H. Classification ofleprosy according to immunity, a five-group sys-tem. Int. J. Lepr. 34 (1966) 255–273.

5. WHO EXPERT COMMITTEE ON LEPROSY. Seventhreport. Geneva: World Health Organization, 1998.Tech. Rep. Ser. 874.

6. WORLD HEALTH ORGANIZATION. Guide to Elimi-nate Leprosy as a Public Health Problem, 1st ed.Geneva: World Health Organization 2000.

TO THE EDITOR:

It is often difficult to diagnose early lep-rosy by clinical criteria alone. Frequently,the patients’ skin smears are negative, clin-ical findings inconclusive, and history unre-liable and subjective. In many such cases,routine histopathology may also be non-specific as Mycobacterium leprae cannot bedemonstrated in the tissues of many earlylesions. Therefore, the demonstration of M.leprae, or any of its components in a tissuebiopsy, is crucial and important to reach adefinitive diagnosis.

Polymerase chain reaction (PCR) amplifi-cation has been successfully used to detectextremely low numbers of M. leprae in freshunfixed human skin biopsy specimens, pro-viding powerful direct and unequivocal testsfor diagnosis of leprosy (1, 2, 3, 4, 5, 7, 8, 10, 11).However, these studies required the speci-mens to be processed and analyzed promptly,in addition to being properly stored andtransported. Adoption of PCR technologyfor detecting M. leprae and/or its compo-nents in fixed tissues would give cliniciansthe option of examining biopsy specimensfor the presence of M. leprae, which alongwith histology would help in arriving at adefinite diagnosis. This PCR technologywould be of great help to arrive at a quickand conclusive diagnosis, identify and treatearly cases of leprosy, to differentiate lep-rosy from non-leprosy cases and also forepidemiological purposes.

While earlier reports have demonstratedthat buffered formalin was good for bothhistology and PCR detection, formalin fixa-tion of skin biopsy specimens for longerthan 24 hr has been reported to have an in-hibitory effect on PCR amplification (2).

Therefore, we here report the developmentof improved protocol for PCR diagnosis ofM. leprae from formalin-fixed specimensand report the results of a blind study usinga large number of biopsies obtained fromboth paucibacillary and multibacillary lep-rosy patients.

MATERIALS AND METHODSAfter obtaining informed consent and

using aseptic precautions and techniquessuch as cleaning of skin with iodine solu-tion and alcohol, 5 mm punch biopsies weretaken from 78 patients of Indeterminate,polar tuberculoid (TT), borderline tubercu-loid (BT), borderline (BB), borderline-lepromatous (BL), or polar lepromatous(LL) leprosy cases from the Ghatampurfield area of Kanpur district, India. Forcontrols, biopsies from 12 cases of otherdermatological conditions (Pitrrysis alba,Tinea versicolor, and Vitiligo) from thesame population were also taken and ana-lyzed using the same protocol. These biop-sies were fixed and transported in bufferedformalin and processed in a blind mannerafter 5 to 7 days. The scientists performingthe gene amplification assay were unawareof the diagnosis of the case, and that biop-sies of other dermatological cases werealso included in the study. Each biopsy wasdivided into two parts: one part was usedfor histopathology and the other for thegene amplification assay.

Initially, the biopsies were kept in 15 mlof sterile distilled water for 8 hrs, whichhad been determined to be optimal aftertesting with various time periods. The biop-sies were then aseptically homogenized in 1ml of sterile T. E. buffer (Tris 10 mM,EDTA 0.1 mM, pH 8.0) using pestle andmortar in a bio-safety hood.

A technique based on the principle of acombined physiochemical approach, firstfreeze-boiling and then treatment withlysozyme-proteinase K (7) was used for ex-traction of DNA. Briefly, the homogenatesin T. E. buffer were frozen, thawed, and

1 Received for publication on 19 November 2002.Accepted for publication on 13 January 2004.

Reprint requests to: Dr. Kiran Katoch, M.D. DeputyDirector (Sr. Grade) and Head, Medical Unit I, CentralJALMA Institute for Leprosy and Other MycobacterialDiseases (ICMR) Tajganj, Agra-282 001 UP–India. E-mail: jalma@sancharnet.in; rohinik@sancharnet.in

Improved Protocol for PCR Detection of Mycobacterium

leprae in Buffered Formalin-Fixed Skin Biopsies1

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then enzymatically treated at 37°C, firstwith lysozyme (3 mg/ml) for 2 hr followedby proteinase K (250 ug/ml) treatment forone hour at 65°C (9). After de-proteinizationwith chloroform: isoamyl alcohol (24:1),DNA was precipitated with 0.6 volume ofisopropanol. This was dissolved in T. E.buffer and stored at –20°C until further use.Primers and the gene amplification methodas described by Hartskeerl, et al. (3) and asused by de Wit, et al. (1) and Singh, et al. (8)were followed for the amplification of 36kDgene of M. leprae. Primers were obtainedcommercially from Bioserve Biotechnolo-gies (India) Ltd. In all cases, 35 cycles wereused for the amplification and confirmationwas done by the southern blot analysis withrandom digoxygenin (DIG) labeled ampli-fied 36 kDa gene fragment of M. leprae as aprobe. DIG labeling and detection was doneby using a kit from Roche Diagnostics (Cat.No. 1093651).

RESULTSThe percentage positivity of biopsy sam-

ples for M. leprae by gene amplification isshown in The Table. It was observed that48% of Indeterminate, 55% of TT/BT, and83% of BB/BL were positive by gene am-plification. The lone BB patient who wasnegative by this assay was smear-negativeand had taken multi-drug therapy (MDT)for more than a year prior to the biopsy.None of the specimens of non-leprosy caseswere positive by this method, demonstrat-ing the specificity of the method.

For histologic investigations, tissue sam-ples are mostly stored as formalin-fixed,paraffin-embedded blocks. Widening theapplicability of amplification techniques toformalin-fixed blocks could improve theroutine diagnosis of mycobacterial infec-tions. This is particularly important in lep-rosy as M. leprae cannot be grown in any invitro system. Definite diagnosis of early andsuspicious cases of leprosy is required, sothat the patients can be treated at an earlystage to prevent development of deformi-ties, rather than following them up closelyand treating them when definite clinicalsigns appear. While large studies are neces-sary to gain more confidence, published in-formation shows that with PCR technology,identification of M. leprae is sensitive, aswell as and specific (1, 3, 5, 7, 8, 10).

It would be ideal if the PCR could be per-formed on fixed specimens in which boththe detection of M. leprae and/or its compo-nents, and correlation with the histopathol-ogy, can be made. Over the years, differentfixatives have been tried for histology andthe detection of RNA/DNA of the causativeorganisms. Fiallo, et al. (2) and deWit, et al.(1) have observed significant reduction insensitivity of PCR for M. leprae DNA whenZenker’s fluids (mercuric chloride) fixativesare used. Although Carnoy-Lebrun’s fluid isrecommended for fixation of tissues for op-timal staining of glycogen and RNA, unfor-tunately this fixative has the potential forchemical modification, resulting in degrada-tion and excessive depurination of the DNAdue to the acidic nature of fixative. Ethanolwas reported to be a good fixative for PCRanalysis, but this causes excessive shrinkageof the tissues, and is not recommended ifthe same tissue is to be used for histologyand PCR analysis. Neutral buffered forma-lin fixation has been reported to be satisfac-tory for subsequent PCR, but significant re-duction in the level of PCR signals for M.leprae DNA was observed after fixation of4 to 7 days (2). In the case of formalin-fixed,paraffin-embedded specimens of smear-positive tuberculosis, the highest sensitivityrates have been obtained by amplifying thehighly repetitive IS 6110 insertion se-quence, and the different primers testedshowed a sensitivity ranging from 80% to87%, whereas a lower positivity of 47% to80% with primer targeting single copy genewas observed in same study (6).

In the present study, after fixation ofbiopsy specimens and processing thesewithin 5 to 7 days for PCR, 83% positivitywas observed in multibacillary BB/BL pa-tients and 43% to 55% in paucibacillary

THE TABLE. Positivity rates for geneamplification targeting 36 kDa gene inbiopsies collected and fixed in buffered for-malin.

No. No. % Type of leprosytested positive positivity

Indeterminate 25 12 48TT/BT 47 26 55BB/BL 6 5 83Non leprosy cases 12 0 Nil

72, 2 Singh, et al.: M. leprae-PCR on Fixed Biopsies 177

(Indeterminate and BT). This is similar tothe positivity rates of 50% to 70% forsmear-negative and about 100% in smear-positive cases observed with fresh frozenbiopsy specimens in earlier studies (5, 7, 8).Prior to this study it has been reported thatformalin fixation of tissues for longer than24 hrs is detrimental for PCR detection ofM. leprae. It appears that this study pro-vides a major improvement in the ability todetect M. leprae in tissues fixed in bufferedformalin.

This improvement is most likely due tothe soaking of the tissues for 8 hrs in 15 mlof sterile distilled water prior to homoge-nizing tissues, lysing and extraction ofDNA. In the study of Fiallo, et al., tissueswere soaked in HBSS solution for 30 min-utes. In the present study, the tissues werecollected as well as processed in bufferedformalin which did not inhibit the PCR am-plification, and the results are comparableto the earlier reports when PCR amplifica-tion was done in freshly biopsied samplesobtained in the outpatient clinic of the Insti-tute. It appears that the approach of collect-ing the specimens in buffered formalin willbe very suitable for field situations and willhave the added advantage of the same spec-imen being used for histology, probe appli-cation in solution, as well as for in situ ap-plications. This, however, is a pilot studyand trends need to validated by similarstudies on a large number of specimens us-ing the same method by other workers.

Acknowledgment. Authors are thankful to all tech-nical and secretarial colleagues for support in carryingout this study and preparation of this manuscript. Giftof reagents/plasticware by LEPRA (UK) is gratefullyacknowledged. This study has been supported by theDepartment of Biotechnology, Govt. of India (GrantNo. BT/IS/002/95-1998–2002).

—H. B. Singh, Ph.D.Research Assistant

Central JALMA Institute For LeprosyAnd Other Mycobacterial Diseases(ICMR)

—V. M. Katoch, M.D.Director, ICMR

—M. Natrajan, MBBS, DVDDeputy Director, ICMR

—V. D. Sharma, Ph.D.Assistant Director, ICMR

—D. S. Chauhan, Ph.D.Research Officer, ICMR

—Mallika Lavania, M.Sc.Research Assistant, ICMR

—Pragya Sharma, M.Sc.Research Assistant, ICMR

—Mohini Sharma, M.Sc.Research Assistant, ICMR

—K. Katoch M.D.Deputy Director (Sr. Grade), ICMRTajganj, Agra-282001 India

—S. Benara, M.D.Deputy DirectorInstitute for Research in Medical Statistics

(ICMR)

—Padam Singh, Ph.D.Additional Director GeneralIndian Council of Medical Research,Ansari Nagar, New Delhi 11002, India

REFERENCES1. DE WIT, M. Y. L., FABER, W. R., KRIEG, S. R.,

DOUGLAS, J. T., LUCAS, S. B., MONTREEWASUVAT,N., PATTYN, S. R., HUSSAIN, R., PONNIGHAUS, J.M., HARTSKEERL, R. A., and KLASTER, P. R. Ap-plication of polymerase chain reaction for the de-tection of Mycobacterium leprae in skin tissues. J.Clin. Microbiol. 29 (1991) 906–910.

2. FIALLO, P., WILLIAMS, D. L., CHAN, G. P., andGILLIS, T. P. Effects of fixation on polymerasechain reaction detection of Mycobacterium leprae.J. Clin. Microbiol. 30 (1992) 3095–3098.

3. HARTSKEERL, R. A., DE WIT, M. Y. L., andKLASTER. P. R. Polymerase chain reaction for thedetection of Mycobacterium leprae. J. Gen. Mi-crobiol. 135 (1989) 2355–2364.

4. JAMIL, S., WILSON, S. M., HACKEL, M., HUSSAIN,R., and STOKER, N. G. A colorimetric PCRmethod for the detection of Mycobacterium lepraein skin biopsies from leprosy patients. Int. J. Lepr.Other Mycobact. Dis. 62 (1994) 512–526.

5. KATOCH, V. M. New investigative techniques inleprosy. In: Dermatology Update. Mumbai: Bha-lani Publishing House, 1998, pp. 165–174.

6. MARCHETTI, G., GORI, A., CATOZZI, L., VAGO, L.,NEBULONI, M., ROSSI, M. C., ESPOSTI, A. D., BAN-DERA. A., and FRANZETTI, F. Evaluation of PCR indetection of M. tuberculosis from formalin-fixed,

178 International Journal of Leprosy 2004

paraffin-embedded tissues: Comparison of fouramplification assays. J. Clin. Microbiol. 36 (1998)1512–1517.

7. SHARMA, R. K., KATOCH, K., SHIVANNAVAR, C. T.,SHARMA, V. D., NATRAJAN, M., BHATIA, A. S.,SAXENA, N., and KATOCH, V. M. Detection of M.leprae by gene amplification combined ethidium-bromide staining and probe hybridization. Int. J.Lepr. Other Mycobact. Dis. 64 (1996) 409–416.

8. SINGH, H. B., KATOCH, K., NATRAJAN, M.,SHARMA, R. K., GUPTA, U. D., SHARMA, V. D.,SINGH, D., CHAUHAN, D. S., SRIVASTAVA, K., andKATOCH, V. M. Effect of treatment on PCR posi-tivity in multibacillary leprosy patients treatedwith conventional and newer drugs ofloxacin andminocycline. Acta. Leprol. 11 (1999) 179–182.

9. VAN EMBDEN, J. D. A., CAVE, M. D., CRAWFORD, J.

T., DALE, J., EISENACH, K. D., GICQUEL, B., HER-MANS MARTIN, C., MEDAM, R., SHINNICK, T. M.,and SMALL, P. M. Strain identification of Myco-bacterium tuberculosis by DNA fingerprintingrecommendations for standardized methodology.J. Clin. Microbiol. 31 (1993) 406–409.

10. WILLIAMS, D. L., GILLIS, T. P., FIALLO, P., JOB, C.K., GELBER, R. H., HILL, C., and IZUMI, S. Detec-tion of Mycobacterium leprae and the potential ofmonitoring anti-leprosy drug therapy directly fromskin biopsies by polymerase chain reaction. Mol.Cell. Probes 6 (1992) 401–410.

11. WOODS, S. A., and COLE, S. T. A rapid method fordetection of potentially viable Mycobacterium leprae in human biopsies; a novel application of PCR. FEMS Microbiol. Letts. 65 (1989)305–310.

Notice. In recognition of his life longcontribution for the cause of leprosy, Dr. R.Ganapati, Director, Bombay Leprosy Proj-ect (BLP) and the Past President of the In-dian Association of Leprologists (IAL) washonored at the 24th Biennial Conference ofthe IAL in Haldia, West Bengalon 28thFebruary 2004. He received a mementofrom the Member of Parliament, Mr. Lak-shman Seth. Dr. Ganapati had also earlierheld positions of the Honorary Secretaryand Vice President of IAL.

Notice from the ILEP. Three guideswhich have recently been published byILEP on the topics of training in leprosy, in-tegration of leprosy services, and how tocarry out a skin smear. A brief backgroundto each is given below. These publicationscan be ordered through books@ilep.org.uk.

1. ILEP Technical Guide: Training inLeprosy

This guide is aimed at staff who or-ganize, support and run leprosy train-ing activities at national, regional ordistrict level. It offers practical guid-ance on topics such as assessing train-ing needs, effective teaching andlearning methods, online learning, on-the-job training and organizing evalu-ations.

It has been developed in consultationwith a number of practitioners whohave extensive experience in leprosytraining, and this is reflected in themany practical tools and ideas that itcontains. It will be a useful guide fortraining managers, facilitators, trainers,supervisors and other teaching staff.

64 pages, 24 cm × 16 cmISBN 0947543260

2. ILEP Technical Guide: Facilitating theIntegration Process—A guide to the in-

tegration of leprosy services within thegeneral health system

This book offers guidance to publichealth managers and decision-makersat national and regional level facedwith the task of integrating leprosyservices into the general health system.The guide systematically describes allthe steps involved in the integrationprocess, from situation analysis and thedevelopment of a plan of action, to im-plementation and evaluation.

It is founded on the experience ofcountries that have already gonethrough the integration process, andaims to help ensure that the lessonslearned during these experiences areapplied more widely.

36 pages, 24 cm × 16 cmISBN 0947543279

3. How to do a skin smear examinationfor leprosy: ILEP Learning GuideThree (available in English and French)

This guide consists of three laminatedand detachable A4 sheets, and is aclearly presented and durable referenceguide for use in the clinic or laboratory.It describes how to carry out all thesteps involved in taking a skin smear,and is targeted largely at health workersor laboratory staff with responsibilityfor taking and reading skin smears, aswell as laboratory technicians who maybe required to prepare the reagents.

6 A4 pages in 3 detachable lami-nated sheets.

From IDEA. Leprosy and Human Rights.The official discussion of leprosy as a hu-man rights issue continued from May 29through May 31, 2004 during the 60th Ses-sion of the UN Human Rights Commission

179

INTERNATIONAL JOURNAL OF LEPROSY Volume 72, Number 2Printed in the U.S.A.

(ISSN 0148-916X)

NEWS and NOTESThis department furnishes information concerning institutions, organizations, and

individuals engaged in work on leprosy and other mycobacterial diseases, and makes noteof scientific meetings and other matters of interest.

180 International Journal of Leprosy 2004

in Geneva, Switzerland. Representatives ofThe Nippon Foundation, IDEA and theSasasakawa Memorial Health Foundationmet with Acting UN High CommissionerBertrand Ramcharan and also made presen-tations on the denial of human rights expe-rienced by millions of individuals affectedby leprosy and their families. Rights thathave been denied include but are not lim-ited to: the Right to Education, the Right toWork, the Right to Freedom of Movement,the Right to Family; the Right to Freedomfrom Degrading Treatment and the Right toan Existence Worthy of Human Dignity.

For over 3000 years and continuing intothe 21st century, the stigma associated withleprosy remains the most persistent and per-vasive form of social injustice, prejudice,and discrimination that society has forcedupon its fellow human beings. Individuals

Calendar of Meetings and Events

Day mm/yy Location Details Contact E-mail

18–23 Jul-04 Montreal 12th International Congress immuno2004@nrc.ca

of Immunology

26–30 Sep-04 Sydney Joint Meeting of the New Australian Society for Microbiol. admin@theasm.com.au

Zealand and Australian

Societies for Microbiology

29–2 Sep/Oct-04 Tehran 7th International Congress Yahya Dowlati info@iranderm.org

of Dematology

13–8 Sep/Oct-04 Ethiopia “ALERT Training Program ALERT Training Division leprosytb@telecom.net

Tuberculosis, Leprosy and

Research Methodology”

30–4 Sep/Oct-04 Boston Infectious Disease Society Infect. Dis. Soc. of America info@idsociety.org

of America

11–22 Oct-04 Ethiopia “ALERT Training Program ALERT Training Division leprosytb@telecom.net

in Tuberculosis, Leprosy

and Research Methodology”

30–2 Oct/Nov-04 Washington, D.C. 44th Interscience Conference

on Antimicrobial Agents

and Chemotherapy

7–11 Nov-04 Miami 53rd Annual Meeting of the ASTMH

American Society of Tropi-

cal Medicine & Hygiene

17–21 Nov-2004 Florence, Italy 13th Congress of the Euro- EADV 2004 Florence

pean Academy of Derma-

tology and Venerology

1–5 Dec-04 Bangkok 9th Western Pacific Congress Congress Secretariat wpccid2004@idthai.org

on Chemotherapy and

Infectious Diseases

whose lives have been challenged by lep-rosy have had their most basic human rightsdenied by virtually every culture and everymajor religion throughout time. Throughoutthis history individuals with leprosy haveoften been unjustly “blamed” for their dis-ease, with leprosy regarded as punishmentfor supposed wrongdoing. Discussing lep-rosy as a human rights issue shifts the bur-den or responsibility for wrongdoing to so-ciety and thus provides a powerful tool foreliminating the stigma and its associatedprejudice and discrimination that have de-nied millions of individuals their rightfulplace in the world community.

“Our exclusion has been taken forgranted in the cultures, religions and lan-guages of society for generations.”

—Arega Kassa Zelelew, IDEA Ethiopia

Alves Moreira, T. [A panorama of Hansen’sdisease: present status and perspectives].Hist. Cienc. Saude Manguinhos. 10(Suppl.1) (2003) 291–307.

The interviewee speaks about the endemicdisease, which at present contaminates 4.4 outof ten thousand inhabitants in Brazil, thecountry with the second highest number ofpatients. When Tadiana speaks about theBrazilian participation in the programlaunched by the World Health Organization,she explains that WHO’s objective is to ex-tinguish Hansen’s disease on the planet until2005. However, she says that Brazilian maingoal is to reduce the occurrence of the diseaseto less than one case per ten thousand people.In our country, the structure and the organiza-tion of this program, which comprehends theeducation and specialization of professionalsin order to guarantee early diagnoses, as wellas patients’ follow-up during treatment, is de-veloped by Sistema Unico de Saude (SUS)and has been implemented in all the states ofthe federation. The chemotherapy treatmentlasts about a year, when taken seriously. Inmany cases, the disease comes back a whilelater. Tadiana comments on the differences ofthe disease according to the different regionsof the country. It has been extinguished in the

South,whereas the rates in the North and Cen-tral East regions almost reach endemic peaks.Working close to patients and ex-patients as-sociations, first as a nurse and later in the im-plementation of policies for Hansen’s diseaseissues, Tadiana Alves Moreira stresses the im-portance of early diagnoses, which avoid thephysical damages and deformities that takeplace in the advanced stages of the disease, soreducing the stigma over the diseased.—Au-thor’s Abstract

Benchimol, J. L., Sa, M. R., Alves da Cruz,Mde. S., and Magalhaes de Andrade, M.Fight for survival: the life of a Hansen’sdisease sufferer through his correspon-dence with Adolpho Lutz. Hist CiencSaude Manguinhos. 10(Suppl. 1) (2003)361–396. [Article in English, Spanish]

This project presents the complete set ofletters between the family of a Hansen’s dis-ease (leprosy) sufferer in the state of Maran-hao, in the Northeast of Brazil, and the doc-tor and bacteriologist Adolpho Lutz. Formore than twenty years Fabricio Caldas deOliveira and Numa Pires de Oliveira, fatherand son, exchanged a steady flow of letterswith the scientist in pursuit of a cure for the

INTERNATIONAL JOURNAL OF LEPROSY Volume 72, Number 2Printed in the U.S.A.

(ISSN 0148-916X)

CURRENT LITERATURE

This department carries selected abstracts of articles published in current medicaljournals dealing with leprosy and other mycobacterial diseases.

General and Historical 181Chemotherapy 187Clinical Sciences 192Immuno Pathology 198

Leprosy 205Tuberculosis 207

Microbiology 219Leprosy 224Tuberculosis 225

Experimental Infections 230Epidemiology 244Other Mycobacterial Diseases 245Molecular and Genetic Studies 254

General and Historical

181

disease that had assailed Numa since child-hood. The 24 letters compiled here paint aunique portrait of the medical and socialdrama confronted by this family, and the re-sults of the use of chaulmoogra oil and othermedications in their search for alternativetreatments.—Authors’Abstract

Benchimol, J. L., and Romero Sa, M.Adolpho Lutz and controversies over thetransmission of leprosy by mosquitoes.Hist. Cienc. Saude Manguinhos. 10(Suppl.1) (2003) 49–93.

During his years of study in Switzerlandand Germany, Adolpho Lutz published hisfirst articles on zoology, clinical practice,and therapeutics. In Limeira, Sao Paulo, hebegan studies on animal and human diseasescaused by germs and parasites. In 1885–1886,Lutz traveled to Hamburg to study the mor-phology of germs related to skin diseases, inconjunction with Paul Gerson Unna, one ofGermany’s foremost dermatologists. Heproposed the inclusion of Hansen’s andKoch’s bacilli in a new genus. In 1889,Unna nominated his student as physician-in-chief of the Leper Settlement on Molokai Is-land, Hawaii. From then on, Lutz sustainedthe theory that the disease was transmittedby mosquitos. He conducted research toprove this theory when he was head of theInstituto Bacteriologico de Sao Paulo(1893–1908) and, later, after he moved tothe Instituto Oswaldo Cruz (1908–1940).Although this research was not successful,on commissions and at congresses in whichhe participated until his death in October1940, he still held to his conviction that lep-rosy was transmitted by mosquitoes.—Au-thors’Abstract

Cueto, M., and de la Puente, J. C. Vida deleprosa: the testimony of a woman livingwith Hansen’s disease in the PeruvianAmazon, 1947. Hist. Cienc. Saude Man-guinhos. 10(Suppl 1) (2003) 337–360.[Article in English, Spanish]

This is the narrative of a patient made be-fore, during and after being incarcerated inan agricultural colony in the Peruvian Ama-zon. In a vivid style, it narrates the decay of

the body, the stigma and the compulsivesegregation, as well as the hope for a betterlife. It is the perspective of a patient, some-thing that is difficult to find when research-ing the history of health. The original pub-lication was possible thanks to the Germanphysician Maxime H. Kuczynski-Godardwho was a German medical doctor that ar-rived in Peru in the mid 1930s and orga-nized valuable activities in the Peruvianjungle as a part of an effort, which eventu-ally failed, of the Peruvian State to colo-nize, or really to “civilize” the Amazon.—Authors’ Abstract

Levison, J. H. Beyond quarantine: a historyof leprosy in Puerto Rico, 1898–1930s.Hist. Cienc. Saude Manguinhos. 10(Suppl1) (2003) 225–245.

From biblical times to the modern period,leprosy has been a disease associated withstigma. This mark of disgrace, physicallypresent in the sufferers’ sores and disfiguredlimbs, and embodied in the identity of a“leper,” has cast leprosy into the shadows ofsociety. This paper draws on primary sources,written in Spanish, to reconstruct the socialhistory of leprosy in Puerto Rico when theUnited States annexed this island in 1898. Thepublic health policies that developed over theperiod of 1898 to the 1930s were unique toPuerto Rico because of the interplay betweenpolitical events, scientific developments andpopular concerns. Puerto Rico was influencedby the United States’ priorities for publichealth, and the leprosy control policies thatdeveloped were superimposed on vestiges ofthe colonial Spanish public health system.During the United States’ initial occupation,extreme segregation sacrificed the individualrights and liberties of these patients for thebenefit of society. The lives of these leprosysufferers were irrevocably changed as a re-sult.—Author’s Abstract

Manton, J. Global and local contexts: theNorthern Ogoja Leprosy Scheme, Nige-ria, 1945–1960. Hist. Cienc. Saude Man-guinhos. 10(Suppl 1) (2003) 209–23.

Deriving funding from missionary sourcesin Ireland, Britain and the USA, and from

182 International Journal of Leprosy 2004

international leprosy relief organizationssuch as the British Empire Leprosy ReliefAssociation (BELRA) and drawing on de-veloping capacities in international publichealth under the auspices of WHO andUNICEF through the 1950s, the RomanCatholic Mission Ogoja Leprosy Schemeapplied international expertise at a locallevel with ever-increasing success and cov-erage. This paper supplements the presen-tation of a successful leprosy control pro-gram in missionary narratives with anappreciation of how international medicalpolitics shaped the parameters of successand the development of therapeutic under-standing in the late colonial period in Nige-ria.—Author’s Abstract

Monteiro, Y. N. Prophylaxis and exclusion:compulsory isolation of Hansen’s diseasepatients in Sao Paulo. Hist. Cienc. SaudeManguinhos. 10(Suppl 1) (2003) 95–121.

This article aims to retrieve the history ofHansen’s disease in Brazil, analyzing themedical thinking of the time and the shapingof health policies that permitted the imple-mentation, in Sao Paulo, of a prophylacticpolicy of compulsory exclusion for allHansen’s disease patients. It also analyzeshow the structuring and implementation ofthis policy led to a “Sao paulo model” thatstrongly influenced the rest of the country. Itaddresses the creation of the state’s networkof leper colonies, their characteristics andthe emergence of a veritable “parallel state”that endured until 1967, with complete dis-regard of all the changes taking place in bothnational and international prophylactic pol-icymaking.—Author’s Abstract

Mori, S., Kato, S., Yokoyama, H., Tanaka,U., and Kaneda, S. [The history ofYunosawa village and the policy of lep-rosy of Japan. IV]. Nihon HansenbyoGakkai Zasshi. 73(1) (2004) 47–63. [Ar-ticle in Japanese]

There was a village which was calledYunosawa, lots of leprosy patients lived, ex-isted from 1887 to 1941, Kusatu town,Gunnma Prefecture, Japan. It was the onlyplace continued securing self-government

to the last as area was free from the isola-tion policy of State in prewar days there.The aim of this study will make clear thedynamism of “The protection from the ten-sion of the society of leprosy patient cur-rently persecuted” to “The defense of thesociety from the leprosy patient who is asource of infection.” In this study, explainedthe factor of confusion to a National Lep-rosarium Kuryu Rakusen-en during WorldWar II and considered relation between pa-tient movement and residents of Yunosawavillage at the postwar period.—Authors’Abstract

Obregon, D. The anti-leprosy campaign inColombia: the rhetoric of hygiene and sci-ence, 1920–1940. Hist. Cienc. Saude Man-guinhos. 10(Suppl 1) (2003) 179–207.

Since the 1920’s, the medical communityrealized that the strategy of leprosy controlbased on segregation and persecution ofpatients was inefficient and expensive. Inthe 1930’s the new liberal governmentincorporated leprosy within the generalsanitary institutions, by merging the Bu-reau of Lazarettos and the National De-partment of Hygiene. The disease-apartapproach started to be replaced by a moregeneral public health strategy, which in-volved controlling other illnesses. Preven-tion and research played a more influentialrole, and the new sanitary officials sawleprosy in the light of the economic ra-tionality of expenditures, placing moreemphasis on therapies and making themmandatory for all patients. Improvementsin leprosy treatment became widely knownand available. However, the image of lep-rosy as a special condition and the practiceof segregation were deeply entrenchedwithin the Colombian culture and institu-tions. The rhetoric changed, but to breakwith several decades of persecution was adifficult task.—Author’s Abstract

Oliveira, M. L., Mendes, C. M., Tardin,R. T., Cunha, M. D., and Arruda, A.Social representation of Hansen’s diseasethirty years after the term “leprosy” wasreplaced in Brazil. Hist. Cienc. SaudeManguinhos. 10(Suppl 1) (2003) 41–48.

72, 2 Current Literature, General and Historical 183

Based on the theories of social represen-tation (SC) and Central Core (CC), a struc-tural study was undertaken regarding theneologism hanseniase (Hansen’s disease),the term adopted by Brazil’s Ministry ofHealth in the 1970s. Carried out during2001, this study interviewed eight hundredhousewives residing in the Rio de Janeiroand Duque de Caxias municipalities. Itfound that Hansen’s disease is part of aprocess of modernization of common think-ing, anchored in the additional representa-tion of leprosy. This finding is understand-able from the perspective that the centralstructure of a social representation has a his-torical determination, so short- and middle-term changes are not to be expected. Fur-thermore, there has been no ongoinginvestment in social marketing to make thenew terminology more widely known. Theauthors discuss the relation between socialrepresentation and the concept of the historyof mentalities.—Authors’Abstract

Pandya, S. S. The first international leprosyconference, Berlin, 1897: the politics ofsegregation. Hist. Cienc. Saude Man-guinhos. 10(Suppl 1) (2003) 161–177.

The present paper examines the first at-tempts to internationalize the problem of lep-rosy, a subject hitherto overlooked by histo-rians of imperialism and disease. The lastdecade of the nineteenth century saw manyin the “civilized countries” of the imperialistWest gripped by a paranoia about an inva-sion of leprosy via germ-laden immigrantsand returning expatriates who had acquiredthe infection in leprosy-endemic colonialpossessions. Such alarmists clamoured forthe adoption of vigorous leper segregationpolicies in such colonies. But the conta-giousness of leprosy did not go unquestionedby other westerners. The convocation inBerlin of the first international meeting onleprosy revealed the interplay of differingand sometimes incompatible views about thecontainment of leprosy by segregation. Theroles of officials from several countries, aswell as the roles of five protagonists (AlbertAshmead, Jules Goldschmidt, Edvard Ehlers,Armauer Hansen, and Phineas Abraham) inthe shaping of the Berlin Conference are hereexamined.—Author’s Abstract

Porter, J. D., Ogden, J. A., Rao, P. V., Rao,V. P., Rajesh, D., Buskade, R. A., andSoutar, D. Introducing operations re-search into management and policy prac-tices of a non-governmental organization(NGO): a partnership between an Indianleprosy NGO and an international aca-demic institution. Health Policy Plan.19(2) (2004) 80–87.

This paper reports on a partnership be-tween LEPRA, a non-governmental organ-ization (NGO), and the London School ofHygiene and Tropical Medicine (LSHTM) toexplore the feasibility and appropriateness ofincorporating operations research into themanagement and decision-making of a lep-rosy NGO. A pilot study in Orissa was usedto determine the advantages and disadvan-tages of introducing operations research toassist in decision-making and programme im-plementation within the organization. The re-sults highlight the difficulty and complexityof the process, but point to several importantthemes: partnership, changing perspectives,use of time and priority-setting, identificationof gaps in systems, and building institutionaland personal capabilities. The results of thestudy provide support to encourage NGOs tobecome actively involved in research. Be-cause of their work and service to local com-munities, NGOs have the opportunity to col-lect information about the perceptions,resources and constraints of individuals,families and the communities themselves inaccessing appropriate care. Their proximityto communities gives them a feeling of re-sponsibility for ensuring that this informationis translated to the district, national and ulti-mately international level. This will help toensure the creation of appropriate infectiousdisease control policies that support the needsof patients. “Outside” academic institutionscan help NGOs to facilitate this up-streamflow of information from the local to the na-tional and international level, to help to en-sure that international disease control policiesare appropriately serving local communi-ties.—Authors’Abstract

Robertson, J. The papers of StanleyBrowne: leprologist and medical mission-ary (1907–1986). Hist. Cienc. Saude Man-guinhos. 10(Suppl 1) (2003) 427–433.

184 International Journal of Leprosy 2004

This article elaborates a significant archivalacquisition that supplement the collectiondocuments related to the life and work ofStanley George Browne held at the Well-come Library for the History and Under-standing of Medicine in London, specificallyhis work in the Belgian Congo (from 1936 to1959), at Uzuakoli in Nigeria (1959 to 1966),in London with the Leprosy Study Centre(1966–1980), and also in his internationalcapacity as leprosy consultant. It also brieflyrefers to an endangered collection of docu-ments, photographs, files and correspond-ence held in a small museum in CulionSanatorium, The Philippines. This researchis part of the International Leprosy Associ-ation Global Project on the History of Lep-rosy. Its results can be accessed at the sitehttp://www.leprosyhistory.org.—Author’sAbstract

Robertson, J. Leprosy and the elusive M.leprae: colonial and Imperial medical ex-changes in the nineteenth century. Hist.Cienc. Saude Manguinhos. 10(Suppl 1)(2003) 13–40.

In the 1800’s, humoral understandings ofleprosy successively give way to diseasemodels based on morbid anatomy, phys-iopathology, and bacteriology. Linkages be-tween these disease models were reinforcedby the ubiquitous seed/soil metaphor de-ployed both before and after the identifica-tion of M. leprae. While this metaphor pro-vided a continuous link between medicaldescriptions, Henry Vandyke Carter’s OnLeprosy (1874) marks a convergence of dif-ferent models of disease. Simultaneously,this metaphor can be traced in popular med-ical debates in the late nineteenth century,accompanying fears of a resurgence of lep-rosy in Europe. Later the mapping of thegenome ushers in a new model of diseasebut, ironically, while leprosy research drawsits logic from a view of the world in whicha seed and soil metaphor expresses manydifferent aspects of the activity of the dis-ease, the bacillus itself continues to be un-receptive to cultivation.—Author’s Abstract

Rosa Maciel, L., Oliveira, M. L., Gallo,M. E., and Damasco, M. S. Memories

and history of Hansen’s disease in Braziltold by witnesses (1960–2000). Hist.Cienc. Saude Manguinhos. 10(Suppl 1)(2003) 308–335.

This report is a preliminary result of a sur-vey on memories and history of Hansen’sdisease, or ‘hanseniasis,’ prepared by theFundacao Oswaldo Cruz (Fiocruz) and theUniversidade Federal do Rio de Janeiro(UFRJ) using statements from those whohave been afflicted by the disease or thosethat have fought against it. It outlines themethodology used by the authors and givesa succinct history of Hansen’s disease inBrazil, together wish information on thestage of the survey with extracts from ourarchives of statements. The founding andthe role of Movement for the Reintegrationof People Afflicted by Hansen’s Disease(Morhan) are explained in the testimony ofThomas Frist, a social scientist who workedin Brazil in the 1970s and 1980s, when thecountry’s old colonies were being restruc-tured, and Cristiano Torres, a former patientwho spent time in prevention centers andleproseries in Para state and who is now ac-tive in proposing new policy for the controlof Hansen’s disease.—Authors’Abstract

Santos, V. S. [Researching documents on thehistory of hansen’s disease in Brazil]. Hist.Cienc. Saude Manguinhos. 10(Suppl 1)(2003) 415–426. [Article in Spanish]

This article corresponds to part of the re-sults of a research on leprosy-related sourcesdeveloped in several institutions in the cityof Rio de Janeiro. At Real Gabinete Por-tugues de Leitura, Arquivo Nacional andBiblioteca Nacional, banks, indexes, officialdocuments and photos on the administrationof leprosaria and articles on the treatment ofthe disease have been investigated. At Centrode Pesquisa e Documentacao de Historia Con-temporanea do Brazil (CPDOC-FGV), sev-eral files have been researched, mainly thosewith information on the health policies of thefirst Vargas administration (1930–1945). Thisresearch is part of the International LeprosyAssociation Global Project on the History ofLeprosy. Its results can be accessed at the sitehttp://www.leprosyhistory.org—Author’sAbstract

72, 2 Current Literature, General and Historical 185

Smith, T. H. A monument to Lazarus: theleprosy hospital of Rio de Janeiro. Hist.Cienc. Saude Manguinhos 10(Suppl 1)(2003) 143–160.

Soon after the Portuguese made landfallin 1500, Europeans and, later, African slavesintroduced leprosy, and Saint Lazarus, thepatron saint of its victims, into Brazil. Socialand political pressure mounted by the mid-dle of the eighteenth century in the city ofRio de Janeiro to remove those unfortunatesfrom the city’s streets even before the moveof Brazil’s capital in 1763. Frei Antoniomthe bishop of Rio, founded the venerablehospital that year in the neighborhood ofSao Cristovao, He requested that the Irman-dade do Santissimo Sacramento da Cande-laria provide oversight and administration.The brotherhood continues to honor itscovenant of 239 years ago. The history ofthis hospital provides insight into the com-plex relationships that existed between thecitizenry and church and state. Rio’s leprosyhospital, now the Hospital Frei Antonio, hadan important role in the evolution of thehealth care professions, progress in medicalscience, and the genesis of the hygienicmovement in Brazil. This study also con-tributes to the history of a disease that per-sists in 2002 Brazil as a public healthissue.—Author’s Abstract

Staples, J. Delineating disease: self-management of leprosy identities inSouth India. Med. Anthropol. 23(1)(2004) 69–88.

The national and international agenciesworking to eliminate leprosy are also dom-inant in setting the boundaries of official dis-course on the issue. Within these boundariesthe disease is commonly represented as amedical problem with negative social con-sequences, and it is believed that both prob-lem and consequences will be resolved ifleprosy is eliminated and its victims treatedand (if necessary) reintegrated within theirsocial groups. For those affected by leprosythe issues are frequently different, elimina-tion in some respects representing a problemas much as a solution. Against this back-ground, which I describe with reference to agroup of leprosy-affected people in South

India and their position vis-à-vis leprosy or-ganizations, I explore some of the contextsin which leprosy patients actively managetheir own situations, often in defiance ofprevailing development orthodoxies. I con-clude that closer observation and analysis ofthe strategies patients use to manage theirdisease status have important policy impli-cations.—Author’s Abstract

Taylor, G. M., Stewart, G. R., Cooke, M.,Chaplin, S., Ladva, S., Kirkup, J.,Palmer, S., and Young, D. B. Koch’sbacillus—a look at the first isolate of My-cobacterium tuberculosis from a modernperspective. Microbiology 149(Pt 11)(2003) 3213–3220.

Using molecular methods the authorshave studied mycobacterial DNA takenfrom a 19th century victim of tuberculosis.This was the case from which Robert Kochfirst isolated and cultured the organism re-sponsible for tuberculosis. The mycobacte-ria were preserved within five glass culturetubes as abundant bacterial colonies onslopes of a gelatinous culture medium of un-known composition. Originally presented byKoch to surgical laryngologist Walter Job-son Horne in London in 1901, the relic has,since 1983, been in the care of the RoyalCollege of Surgeons of England. Light andelectron microscopy established the pres-ence of acid-fast mycobacteria but showedthat morphological preservation was gener-ally poor. Eleven different genomic lociwere successfully amplified by PCR. Thisseries of experiments confirmed that the or-ganisms were indeed Mycobacterium tuber-culosis and further showed that the originalstrain was in evolutionary terms similar to‘modern’ isolates, having undergone the TBD1 deletion. Attempts to determine thegenotypic group of the isolate were onlypartially successful, due in part to the de-graded nature of the DNA and possibly alsoto a truncation in the katG gene, whichformed part of the classification scheme.Spoligotyping resulted in amplification ofDR spacers consistent with M. tuberculosisbut with discrepancies between independentextracts, stressing the limitations of this typ-ing method when applied to poorly pre-served material.—Authors’Abstract

186 International Journal of Leprosy 2004

Zhang, L. Leprosy in China. Nihon Hansen-byo Gakkai Zasshi. 72(3) (2003) 209–215.

Leprosy has definitely been present inChina for at least 2000 years. Throughpainstaking efforts over the past half century,China has put leprosy under control andreached the WHO’s target at elimination ofleprosy at the national and subnational level.

But difficulties as well as problems like dis-abilities, discrimination, drug-resistance anddismissing of research still remain in thecontrol of leprosy. Highly attention shouldbe continuously paid on to attain the preva-lence rate of less than 0.1/10,000 and the in-cidence rate below 0.5/100,000 in all coun-ties (cities) throughout the country by theyear 2010.—Author’s Abstract

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Chemotherapy

Ashitani, J., Yanagi, S., Arimura, Y., Sano,A., and Mukae, H. Acute respiratory dis-tress syndrome induced by rifampicin withhigh levels of neutrophil and eosinophilproducts in bronchoalveolar lavage fluid.Respiration 70(5) (2003) 541–543.

We reported a case with acute respiratorydistress syndrome (ARDS) caused byrifampicin during therapy for pulmonarytuberculosis. A high level of eosinophilcationic protein in bronchoalveolar lavagefluid (BALF) was detected as well asinterleukin-8 and neutrophil elastase. Basedon these results together with the positive re-sult of the drug lymphocyte-stimulating test,we concluded that rifampicin was thecausative drug leading to ARDS. Cortico-steroid therapy resulted in clinical improve-ment and resolution of the pulmonary infil-trates on the chest radiograph without therecurrence of pulmonary tuberculosis.—Au-thors’Abstract

Brown, C. W., Liu, S., Klucik, J., Berlin,K. D., Brennan, P. J., Kaur, D., andBenbrook, D. M. Novel heteroarotinoidsas potential antagonists of Mycobacte-rium bovis BCG. J. Med. Chem. 47(4)(2004) 1008–1017.

Aseries of 15 heteroarotinoids has been pre-pared and evaluated for activity against Myco-bacterium bovis BCG with the thiourea-containing isoxyl (7) (0.5 microg/mL) as thestandard. 2,2,4-Trimethyl-2H-chromen-7-yl4-(methoxycarbonyl)benzoate (8) displayedthe most significant activity (2.0–4.0 mi-crog/mL) in terms of the lowest concentration(microg/mL) (MIC, minimum inhibitory con-centration) required to produce a 99% reduc-

tion in the number of colonies on a plate ascompared to that system free of the agent atthe same dilution of the culture suspension.Ethyl 4-[[N-(2,2,4,4-tetramethylchroman-6-yl)thiocarbamoyl]amino] benzoate (9) and[[(1E,3Z,5E)-1-aza-4-methyl-6-(1,2,2,4-tetramethyl(1,2-dihydroquinolyl))hexa-1,3,5-trienyl]amino]aminomethane-1-thione(10) exhibited activity at 5.0–10.0 and10.0–20.0 microg/mL, respectively, whilethe other examples had MIC values of 20 mi-crog/mL or greater. The inhibitory ability of8 may occur via the inhibition of mycolicacid synthesis in a like manner as found with7, but this requires further study. The het-eroarotinoids are the first examples to exhibitinhibitory ability against the growth of My-cobacterium bovis BCB.—Authors’Abstract

Bucaretchi, F., Miglioli, L., Baracat, E. C.,Madureira, P. R., Capitani, E. M., andVieira, R. J. [Acute dapsone exposureand methemoglobinemia in children:treatment with multiple doses of acti-vated charcoal with or without the ad-ministration of methylene blue]. J. Pedi-atr. (Rio J). 76(4) (2000) 290–294.

OBJECTIVE: To study the changes inmethemoglobinemia of 17 children admit-ted with acute exposure to dapsone compli-cated by a methemoglobin concentrationgreater than 20% of the total hemoglobin.The children were treated with multipledoses of activated charcoal with or withoutthe administration of methylene blue.PATIENTS AND METHODS: Seventeenpatients (ages 1–13 yrs, median 3 yrs),were admitted 1–72 hr after the ingestionof 100–1200 mg (median 350 mg, 10 pa-tients) or an unknown amount of dapsone

(7 patients). The methemoglobin blood con-centrations upon admission ranged from23.5%–49.7% (median 37.8%), and themain clinical features were cyanosis (17),tachycardia (17), vomiting (11) and tachyp-nea (8). All of the children received multi-ple doses of activated charcoal orally or vianasogastric tube (1g/kg, 10% solution, 4–6times/day, 3–16 doses with a median of 8doses). Twelve of the 14 patients withmethemoglobin levels greater than 30%were also treated with a single dose ofmethylene blue (1–2% solution, 1–2 mg/kg)infused IV over 5 min. RESULTS: Therewas a progressive decrease in the methe-moglobin levels after the beginning of bothtreatments (multiple doses of activatedcharcoal alone or associated with methyleneblue), and only one dose of methylene bluewas necessary. There were no significantstatistical differences between the results ofthe two treatments according to the time-course decrease in methemoglobinemia (p =0.49 Wilcoxon test). CONCLUSIONS: Mul-tiple doses of activated charcoal given whenmethemoglobin levels were greater than20% can be considered as a possible treat-ment for pediatric patients, with or withoutthe administration of methylene blue, afteracute dapsone exposure.—Authors’Abstract

De Carsalade, G. Y., Achirafi, A., andFlageul, B. Pentoxifylline in the treat-ment of Erythema Nodosum Leprosum:Results of an open study. Acta Leprolog-ica 12(3) 117–122.

Erythema nodosum leprosum (ENL) is awell-known immunological serious compli-cation affecting lepromatous multibacillaryleprosy patients. For a long time, ENL hasbeen regarded as an immune complex-mediated disease or Arthus phenomenon. Re-cently, it has been reported that ENL was as-sociated with high serum tumor necrosisfactor-alpha (TNFα) levels, suggesting thatthis cytokine could also play a central role inthe manifestations of ENL. Thalidomide(TH) and systemic steroids (S), both TNFαproduction inhibitors, are the two current ef-fective drugs for the management of ENL.However, TH is rarely available in leprosyendemic countries, and its teratogenicity andneurotoxicity strongly limit its use. More-

over, the morbidity of S and the frequentsteroid-dependence of ENL also create realtherapeutic problems. Recently, the efficacyof pentoxifylline (PTX), which also inhibitsin vitro and in vivo production of TNFα, hasbeen suggested for ENL treatment. We reportour experience on its use for the treatment of15 leprosy patients suffering from a first ENLattack (11 cases), a chronic steroid-dependentENL (3 cases) or chronic steroid- and thalido-mide-dependent ENL (1 case). PTX has beengiven at 800 mg t.i.d. (2 cases), or 400 mgt.i.d. (13 cases) doses. The patients receivedPTX at the initiating dosage until completeclinical cure. At the end of ENL attacks, PTXwas either abruptly stopped or tapered downover the next 4 months. In ten of 11 patientswho developed ENL for the first time, thesystemic symptoms and neuritic pains disap-peared within one week; at three weeks, halfof the patients were cured and the other halfhad striking clinical improvement; completecure was obtained within 7 to 35 days (mean:27 days). A relapse occurred within 2–3months in the 5 patients in which PTX wasabruptly stopped. In contrast, no relapse oc-curred in the patients who benefited from de-creasing doses of PTX. Recurrent ENLepisodes also responded well to PTX. The 3patients who had chronic steroid-dependentENL failed to show any improvement after 3to 6 weeks of PTX. In contrast, steroid ther-apy could be stopped in the steroid- andthalidomide-dependent patient. Our resultsconfirm the action of PTX if it is slowly ta-pered down (4 months seem sufficient) andnot abruptly to avoid relapses. As it is safeuse, PTX could constitute the first line ofENL attack treatment.—Acta Leprologica

Eriksson, T., Hoglund, P., Turesson, I.,Waage, A., Don, B. R., Vu, J., Scheffler,M., and Kaysen, G. A. Pharmacokinet-ics of thalidomide in patients with im-paired renal function and while on and offdialysis. J. Pharm. Pharmacol. 55(12)(2003) 1701–1706.

There is a renewed interest in thalido-mide for use in malignancies and systemicinflammatory diseases. Reduced renal func-tion is not uncommon among patients withthese disease states but the pharmacokinet-ics has not been fully investigated. The aim

188 International Journal of Leprosy 2004

of this study was to investigate the pharma-cokinetics of thalidomide in haemodialysispatients while on and off dialysis and inmyeloma patients with varying degrees ofrenal function. Two studies were per-formed. To establish the pharmacokineticsof thalidomide in patients with mild to mod-erate renal failure, blood samples weretaken over 12 weeks from 40 patients withmultiple myeloma. A second study was per-formed in six patients with end-stage renaldisease both on a non-dialysis day and be-fore and during a haemodialysis session.Thalidomide concentration was determinedby HPLC. A one-compartment open modelwith first-order absorption and eliminationwas used to fit total thalidomide concentra-tion to population pharmacokinetics andstatistical models using the NONMEM pro-gram. Clearance and volumes were slightlybelow 10 L h-1 and 1 L kg-1, respectively,in both patient groups. The inter- and intra-patient variability was low. Clearance wasdoubled during dialysis. There was no cor-relation between thalidomide clearance andrenal function. In conclusion, the pharma-cokinetics of thalidomide in patients withrenal failure are very similar to values re-ported by others for patients with normalrenal function. Although clearance duringdialysis is doubled, thalidomide dose neednot be changed for patients with decreasedkidney function. There is also no need fora supplementary dose due to haemodialy-sis.—Authors’ Abstract

Foroumadi, A., and Soltani, F. Antitubercu-losis agents. IX. In vitro anti-mycobacterialactivity of N-(2-phenyl-2-oxoethyl) and N-[2-(4-fluorophenyl)-2-oxoethyl]-ciprofloxacin derivatives against somedrug-resistant strains of Mycobacteriumtuberculosis and Mycobacterium aviumisolates. Boll. Chim. Farm. 142(6) (2003)248–250.

See Current Literature, Other Mycobac-terial Diseases, p. 247.

Garcia-Garcia, A., Galvez, J., de Julian-Ortiz, J. V., Garcia-Domenech, R.,Munoz, C., Guna, R., and Borras, R.New agents active against Mycobacte-

rium avium complex selected by molec-ular topology: a virtual screening method.J. Antimicrob. Chemother. 53(1) (2004)65–73.

OBJECTIVES: In order to select newdrugs and to predict their in vitro activityagainst Mycobacterium avium complex(MAC), new quantitative structure-activityrelationship (QSAR) models were devel-oped. METHODS: The activities againstMAC of 29 structurally heterogeneousdrugs were examined by means of lineardiscriminant analysis (LDA) and multilinearregression analysis (MLRA) by using topo-logical indices (TI) as structural descriptors.In vitro antimycobacterial activities were de-termined by a broth microdilution methodwith 7H9 medium. RESULTS: The topo-logical model obtained successfully classi-fies over 80% of compounds as active or in-active; consequently, it was applied in thesearch for new molecules active againstMAC. From among the selected candidatesdemonstrating in vitro activity, aflatoxin B1,benzalkonium chloride and pentamidinestand out, with MIC50s between 4 and 32mg/L. CONCLUSION: The method de-scribed in this work is able to select mole-cules active against MAC.—Authors’ Ab-stract

Hansen, J. M., and Harris, C. A novel hy-pothesis for thalidomide-induced limbteratogenesis: redox misregulation of theNF-kappaB pathway. Antioxid. RedoxSignal 6(1) (2004) 1–14.

Several hypotheses have been proposed toexplain the mechanisms of thalidomide ter-atogenesis, although none adequately ac-counts for the observed malformations andexplains the basis for species specificity. Re-cent observations that thalidomide increasesthe production of free radicals and elicits ox-idative stress, coupled with new insightsinto the redox regulation of nuclear tran-scription factors, lead to the suggestion thatthalidomide may act through redox misreg-ulation of the limb outgrowth pathways. Ox-idative stress, as marked by glutathione de-pletion/oxidation and a shift in intracellularredox potential toward the positive, occurspreferentially in limbs of thalidomide-

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sensitive rabbits, but not in resistant rats.DNA binding of nuclear factor kappa-B(NF-kappaB), a redox-sensitive transcrip-tion factor and key regulator of limb out-growth, was shown to be significantly at-tenuated in rabbit limb cells and could berestored following the addition of a free rad-ical spin-trapping agent, phenyl N-tert-butylnitrone. The inability of NF-kappaB to bindto its DNA promoter results in the failure oflimb cells to express fibroblast growth fac-tor (FGF)-10 and twist in the limb progresszone (PZ) mesenchyme, which in turn at-tenuates expression of FGF-8 in the apicalectodermal ridge (AER). Failure to establishan FGF-10/FGF-8 feedback loop betweenthe PZ and AER results in the truncation oflimb outgrowth. We hypothesize that species-selective alterations in redox microenviron-ment caused by free radical production fromthalidomide results in attenuation of the NF-kappaB-mediated gene expression that is re-sponsible for limb outgrowth.—Authors’Abstract

Li Li, and Xue-jun Zhu. Dapsone Hyper-sensitivity Syndrome. J. Clin. Derm.32(Suppl.) (2003) s115–s117.

A 23-year-old woman with linear IgA der-matosis developed dapsone hypersensitivitysyndrome (DHS) after initiation of dapsonetherapy. She had fever, jaundice with hepaticdysfunction, lymphadenopathy, anemia anddermatitis. The symptoms disappeared withmethylprednisolone treatment 40 mg/day.—Journal of Clinical Dermatology

Lu, K. Q., Brenneman, S., Burns, R. Jr.,Vink, A., Gaines, E., Haake, A., andGaspari, A. Thalidomide inhibits UVB-induced mouse keratinocyte apoptosis byboth TNF-alpha-dependent and TNF-alpha-independent pathways. Photoder-matol Photoimmunol Photomed. 19(6)(2003) 272–280.

BACKGROUND: Thalidomide is an anti-inflammatory pharmacologic agent that hasbeen utilized as a therapy for a number ofdermatologic diseases. Its anti-inflammatoryproperties have been attributed to its abilityto antagonize tumor necrosis factor-alpha

(TNF-alpha) production by monocytes.However, its mechanism of action in theskin is not known. PURPOSE: To test ourhypothesis that thalidomide may antago-nize TNF-alpha production in the skin, weused a mouse model for acute ultraviolet-B(UVB) exposure, a known stimulus for in-ducing this cytokine. RESULTS: A singlebolus dose of thalidomide (either 100 or400 mg/kg) given immediately beforeUVB exposure (40–120 mJ/cm2) inhibited,in a dose-dependent manner, sunburn cellformation (i.e., keratinocyte (KC) apopto-sis as defined by histologic appearance andconfirmed by terminal transferase mediatedbiotinylated dUTP nick end labelling stain-ing) in mouse skin biopsy specimens.However, this agent did not affect the for-mation of cyclobutane pyrimidine dimers,a measure of UVB-induced DNA damage,which is an early event associated withapoptosis. RNase protection assays con-firmed that high (400 mg/kg), but not low(100 mg/kg), doses of thalidomide inhib-ited the UVB-induced increase in steady-state TNF-alpha mRNA. Additionally, ourin vitro data using neonatal mouse KCsshowed that thalidomide prevented UVB-induced cell death (JAM assay). The anti-apoptotic effects of thalidomide can be re-versed by the addition of exogenousrecombinant mouse TNF-alpha and hencereconstituting UVB-induced programmedcell death. The inhibition of sunburn cellformation by low-dose thalidomide in theabsence of TNF-alpha inhibition suggeststhat other, unidentified mechanisms ofapoptosis inhibition are active. CONCLU-SIONS: These data suggest that the anti-inflammatory effects of thalidomide can af-fect UVB injury, and may, in part, explainits action in photosensitivity diseases suchas cutaneous lupus erythematosus.—Au-thors’ Abstract

Lu, T., and Drlica, K. In vitro activity of C-8-methoxy fluoroquinolones againstmycobacteria when combined with anti-tuberculosis agents. J. Antimicrob. Chemo-ther. 52(6) (2003) 1025–1028.

OBJECTIVES: To examine the effect offirst-line and second-line anti-tuberculosisagents on the ability of fluoroquinolones to

190 International Journal of Leprosy 2004

kill mycobacteria. METHODS: A clinicalisolate of Mycobacterium tuberculosis and alaboratory strain of Mycobacterium smeg-matis were grown in liquid medium andtreated with a fluoroquinolone in the pres-ence or absence of anti-tuberculosis agents.Bacterial survival was determined by viablecolony counts on agar medium. RESULTS:When moxifloxacin activity was examinedin two-drug combinations containing tradi-tional anti-tuberculosis agents, activity wasgreater than either compound alone withisoniazid, capreomycin and low, but nothigh, concentrations of rifampicin. Cyclo-serine contributed no additional activity, andethambutol interfered with the lethal actionof moxifloxacin and gatifloxacin. Experi-ments with M. smegmatis confirmed thatboth rifampicin and ethambutol reduce fluoro-quinolone lethality. Moreover, ethambutolincreased the recovery of fluoroquinolone-resistant mutants newly created by ethylmethanesulphonate treatment. CONCLU-SIONS: The intrinsic bactericidal activity ofC-8-methoxy fluoroquinolones can be ad-versely affected by some agents currentlyused for treatment of tuberculosis.—Au-thors’Abstract

Pandey, R., Zahoor, A., Sharma, S., andKhuller, G. K. Nanoparticle encapsu-lated antitubercular drugs as a potentialoral drug delivery system against murinetuberculosis. Tuberculosis (Edinb). 83(6)(2003) 373–378.

Patient non-compliance is the major draw-back associated with the long-durationchemotherapy of tuberculosis (TB); hence, re-duction in dosing frequency forms an impor-tant therapeutic strategy. The present study re-ports the formulation of three frontlineantitubercular drugs (ATD), i.e., rifampicin(RIF), isoniazid (INH) and pyrazinamide(PZA) encapsulated in poly (DL-lactide-co-glycolide) (PLG) nanoparticles. Drug en-capsulation efficiencies were 56.9 ± 2.7%for RIF, 66.3 ± 5.8% for INH and 68 ± 5.6%for PZA. Following a single oral adminis-tration of these preparations to mice, thedrugs could be detected in the circulation for6 days (RIF) and 9 days (INH/PZA), whereastherapeutic concentrations in the tissueswere maintained for 9–11 days. Further, on

oral administration of drug-loaded nanopar-ticles to Mycobacterium tuberculosis-infectedmice at every 10th day, no tubercle bacillicould be detected in the tissues after 5 oraldoses of treatment. Therefore, nanoparticle-based ATD therapy forms a sound basis forreduction in dosing frequency for bettermanagement of TB.—Authors’Abstract

Selvaraj, P., Chandra, G., Kurian, S. M.,Reetha, A. M., and Narayanan, P. R.Association of Vitamin D receptor genevariants of BsmI, ApaI, and FokI poly-morphisms with susceptability or resist-ance to pulmonary tuberculosis. Curr.Sci. 84(12) (2003) 1564–1568.

Zhu, X., Giordano, T., Yu, Q. S., Hol-loway, H. W., Perry, T. A., Lahiri, D.K., Brossi, A., and Greig, N. H. Thio-thalidomides: novel isosteric analoguesof thalidomide with enhanced TNF-alphainhibitory activity. J. Med.Chem. 46(24)(2003) 5222–5229.

Thalidomide is being increasingly used inthe clinical management of a wide spectrumof immunologically-mediated and infectiousdiseases, and cancers. However, the mechan-isms underlying its pharmacological actionare still under investigation. In this regard,oral thalidomide is clinically valuable in thetreatment of erythema nodosum leprosum(ENL) and multiple myeloma and effectivelyreduces tumor necrosis factor-alpha (TNF-alpha) levels and angiogenesis in vivo. Thiscontrasts with its relatively weak effects onTNF-alpha and angiogenesis in in vitro stud-ies and implies that active metabolites con-tribute to its in vivo pharmacologic action andthat specific analogues would be endowedwith potent activity. Our focus in the structuralmodification of thalidomide is toward the dis-covery of novel isosteric active analogues. Inthis regard, a series of thiothalidomides andanalogues were synthesized and evaluated fortheir TNF-alpha inhibitory activity againstlipopolysacharide (LPS)-stimulated periph-eral blood mononuclear cells (PBMC), Thiswas combined with a PBMC viability assayto differentiate reductions in TNF-alpha se-cretion from cellular toxicity. Two isosteric

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analogues of thalidomide, compounds 15 and16, that mostly reflect the parent compound,together with the simple structure, dithioglu-tarimide 19, potently inhibited TNF-alpha se-cretion, compared to thalidomide, 1. Themechanism underpinning this most likely isposttranscriptional, as each of these com-pounds decreased TNF-alpha mRNA stabil-

ity via its 3′-UTR. The potency of 19 war-rants further study and suggests that replace-ment of the amide carbonyl with a thiocar-bonyl may be beneficial for increasedTNF-alpha inhibitory action. In addition, anintact phthalimido moiety appeared to be req-uisite for TNF-alpha inhibitory activity.—Authors’Abstract

192 International Journal of Leprosy 2004

Clinical SciencesAlioua, Z., Sbai, M., Elhaouri, M., Boudi,

O., Ghfir, M., Benomar, S., and Se-drati, O. Histoid leprosy with erythemanodosum leprosum. Acta Leprologica12(3) (2004) 107–111.

Histoid leprosy is a particular variant oflepromatous leprosy presenting as cutaneousor subcutaneous nodular and/or plaque-likelesions arising from apparently normal skin.It is characterized histologically by spindle-shaped histiocytes in interlacing bundles andwhorls, containing numerous intact and rod-shaped Mycobacterium leprae. It can occurde novo or secondary in patients treated fora long course by dapsone alone. We describea case of lepromatous leprosy treated ac-cording to the national Moroccan protocolwho developed histoid lesions during histreatment by dapsone. The patient re-sponded well to fluoroquinolone, rifampicinand clofazimine, with however, the occur-rence of erythema nodosum leprosum.

Bartt, R. E. Leprosy (Hansen’s Disease).Curr. Treat Options Neurol. 6(2) (2004)95–103.

Leprosy (Hansen’s disease) causes themost common treatable form of neuropathyin the world. Several endemic countries ac-count for the majority of the world’s casesand most of the cases seen in the US areamongst immigrants. However, endemiccases of leprosy occur in the US. Thepathogen is Mycobacterium leprae, a slow-growing, obligate intracellular pathogen thatconsistently infects skin and peripheralnerves. The clinical appearance of the skinand neurologic deficits develop months toyears after infection and are determined bythe host’s response to the infection. An indi-vidual’s disease classification can change

over time based on the immune status of theindividual. Immune-mediated “reactionalstates” may also occur that require addi-tional recognition and treatment. Varied inits manifestations, a successful treatmentapproach relies on proper recognition andclassification of disease.—Author’s Abstract

Beyene, D., Aseffa, A., Harboe, M., Kidane,D., Macdonald, M., Klatser, P. R., Bjune,G. A., and Smith, W. C. Nasal carriage ofMycobacterium leprae DNA in healthy in-dividuals in Lega Robi village, Ethiopia.Epidemiol. Infect. 131(2) (2003) 841–848.

The number of registered leprosy patientsworld-wide has decreased dramaticallyafter extensive application of WHO recom-mended Multiple Drug Therapy (MDT).The annual number of new cases has, how-ever, been almost unchanged in severalpopulations, indicating that the infection isstill present at community level. Nasal car-riage of Mycobacterium leprae DNA wasstudied in Lega Robi village in Ethiopia.MDT had been applied for more than tenyears, and 718 residents over 5 years oldwere eligible for the study. During the firstsurvey nasal swab samples were collectedfrom 664 (92.5%) individuals. The resultsof a Peptide Nucleic Acid-ELISA test forM. leprae DNA interpreted by stringent sta-tistical criteria were available for 589(88.7%) subjects. Thirty-five (5.9%) indi-viduals without clinical signs of leprosywere positive for M. leprae DNA. SevenPCR positive individuals lived in a house-hold where one or two other members werealso positive for M. leprae DNA. During asecond survey 8 (46%) of 175 interpretablePNA-ELISA tests were positive. Of 137 in-dividuals tested twice, only two were posi-tive on both occasions whereas 10 were

PCR positive only once. The study confirmsthe widespread distribution of M. lepraeDNA in healthy individuals. The feasibilityof curbing possible transmission of subclin-ical infection needs further consideration.

Cortes, S. L., and Rodriguez, G. Leprosyin children: association between clinicaland pathological aspects. J. Trop. Pediatr.50(1) (2004) 12–15.

Leprosy among children is a public healthproblem reflecting the disease’s transmis-sion in the community and the efficiency ofcontrol programmes. To evaluate some clin-ical, epidemiological and histopathologicalcriteria, as well as the level of agreementbetween clinical and histopathological diag-noses, 207 biopsies were studied frompatients less than 15 years old who wereclinically diagnosed with leprosy betweenMarch 1994 and September 2000. Leprosywas confirmed by histopathology in 119cases (57.5 percent). Forty-seven percentof children were 10 years old or more;28.5 percent shared their dwellings with lep-rosy patients; 35 percent had only one le-sion, and 43 percent were multibacillarycases. Agreement between clinical andhistopathological classification was 36 per-cent; hypochromic chronic eczema andpost-inflammatory incontinence of melaninpigment were the clinical lesions most fre-quently mistaken with leprosy. Leprosyamong children represents 7 percent of newleprosy cases in Colombia and the high per-centage of multibacillary cases suggests thatdiagnosis is being made late. The diseasemust be investigated in all children livingwith leprosy patients and skin biopsy is rec-ommended to avoid false-positive diag-noses.—Authors’Abstract

Doffinger, R., Helbert, M. R., Barcenas-Morales, G., Yang, K., Dupuis, S.,Ceron-Gutierrez, L., Espitia-Pinzon, C.,Barnes, N., Bothamley, G., Casanova, J.L., Longhurst, H. J., and Kumararatne,D. S. Autoantibodies to interferon-gammain a patient with selective susceptibility tomycobacterial infection and organ-specificautoimmunity. Clin. Infect. Dis. 38(1)(2004) e10–4.

We evaluated a patient with disseminatedMycobacterium tuberculosis and Mycobac-terium chelonae infection, of which he died.He also developed autoimmune (type I)diabetes and primary hypothyroidism. Hisserum contained a high titer of immuno-globulin G autoantibody to interferon-gamma (IFN-gamma) capable of blockingin vitro responses to this cytokine by pe-ripheral blood mononuclear cells from nor-mal donors. These results suggest thatautoantibodies to IFN-gamma can inducesusceptibility to disseminated mycobacterialinfection, which may be refractory tochemotherapy.—Authors’Abstract

Fenniche, S., Ben Jennet, S., Marrak, H.,Khayat, O., Zghal, M., Ben Ayed, M.,and Mokhtar, I. [Cutaneous tuberculo-sis: anatomoclinical features and clinicalcourse (26 cases)]. Ann. Dermatol.Venereol. 130(11) (2003) 1021–1024.[Article in French]

INTRODUCTION: Despite preventionprograms, tuberculosis is still progressingendemically in developing countries. Theprevalence of cutaneous tuberculosis is esti-mated as 2.1 p. 100 and represents a rare lo-calization among the extra-pulmonaryforms. In order to study the epidemiology,the most frequent anatomoclinical formsand the progressive features of cutaneous tu-berculosis, we conducted a study in the areaof Tunis over a 20-year period. PATIENTSAND METHODS: All cases of cutaneoustuberculosis observed between 1981 and2000 in the dermatology department of theHabib Thameur hospital were included in aretrospective study. Diagnosis of cutaneoustuberculosis was challenging and requiredthe correlation of clinical, biological andprogressive features. RESULTS: Twenty-sixpatients were observed in the study. Therewere 12 men and 14 women with a meanage of 30.4 years (range: 6 to 74) and 20 p.100 of infantile cases. Of the various pat-terns of cutaneous tuberculosis seen, 11 (42p. 100) had lupus tuberculosis, 10 (38 p.100) had scrofuloderma, 4 (15 p. 100) hadtuberculosis verrucosa cutis and 1 child hada perianal tubercular ulcer. The Mantoux testwas positive in 20/24 patients. Histologicaltuberculoid granuloma was seen in 25 cases

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(96 p. 100) associated with caseatingnecrosis in 10 cases (38 p. 100). All pa-tients were treated successfully with tripleor quadruple anti-tubercular drugs for 6 to10 months. One patient exhibited a squa-mous cell carcinoma on a lupus tuberculo-sis scar four years later. DISCUSSION:The progression of cutaneous tuberculosisremains stable, ranging from 1.4 cases/yearbetween 1981 and 1990 to 1.2 cases/yearbetween 1991 and 2000. In our study, fe-males were slightly more affected than menwith a M/F sex ration of 0.86. Before 1984,scrofuloderma was the most frequent formamong the cutaneous tuberculoses. Nowthe frequency of lupus tuberculosis hasreached that of scrofuloderma, demonstrat-ing the increase in the incidence of clinicalpattern of cutaneous tuberculosis withstrong immunity probably related to theimprovement in health conditions and gen-eralization of vaccination programs.—Au-thors’ Abstract

Flageul, B. Prise en charge médicale actu-uelle de la maladie de Hansen. Bull. Soc.Pathol. Exot. 96(5) (2003) 357–360. [Ar-ticle in French]

During the last 20 years, the global lep-rosy situation has strikingly changed with adecrease of cases from 12 millions esti-mated cases in 1982 to 600,000 registeredcases in the year 2000. However, during thepast 15 years, about 700,000 new cases arestill detected annually. The systematic use ofmultidrug therapy (MDT), as recommendedby a WHO Study Group in 1982, has provenits efficacy as assessed by the low reportedrelapse rate (less than 1% per year). Theinitial PCT schedule has been modified sev-eral times, but this PCT remains the recom-mended chemotherapy for the great major-ity of patients. New potent antibacillarydrugs (ofloxacin, minocycline, clarithromy-cine) have been discovered; however, theircurrent use is limited and should remain lim-ited until under way trials could confirmtheir efficacy. With the use of PCT, the fre-quency of immunologically mediated reac-tional states have changed. The occurrenceof reversal reaction (type 1 reaction), hassignificantly increased while that of ery-thema nodosum leprosum (ENL, type 2) ap-

peared less common. Because of the highrisk of neurological permanent damage, re-versal reaction needs to be diagnosed andtreated as soon as possible. Herein, the cur-rent antibacillary and antireactional treat-ments are being reviewed.—Bulletin de laSociété de Pathologie Exotique

Jardim, M. R., Antunes, S. L. G., Santos,A. R., Nascimento, O. J. M., Nery, J. A.C., Sales, A. M., Illarramendi, X., Dup-pre, N., Chimelli, L., Sampio, E. P.,Sarno, E. P. N. Criteria for diagnosis ofpure neural leprosy. J. Neurol. 250(7)(2003) 806–809.—Tropical Disease Bul-letin

The clinical diagnosis of pure neural lep-rosy (PNL) remains a public health careproblem mainly because skin lesions—thecardinal features of leprosy—are always ab-sent. Moreover, the identification of the lep-rosy bacillus is not easily achieved evenwhen a nerve biopsy can be performed. Inan attempt to reach a reliable PNL diagnosisin patients referred to our Leprosy Outpa-tient Clinic, this study employed a variety ofcriteria. The nerve biopsies performed onthe 67 individuals whose clinical, neurolog-ical, and electrophysiological examinationfindings strongly suggested peripheral neu-ropathy were submitted to M. leprae identi-fication via a polymerase chain reaction(PCR). Mononeuropathy multiplex was themost frequent clinical and electrophysiolog-ical pattern of nerve dysfunction, while sen-sory impairment occurred in 89% of allcases and motor dysfunction in 81%. Axonalneuropathy was the most prominent electro-physiological finding, while the histopatho-logical nerve study showed epithelioid gran-uloma in 14% of the patients, acid fastbacilli in 16%, and nonspecific inflamma-tory infiltrate and/or fibrosis in 39%. PCRfor M. leprae was positive in 47% of thenerve biopsy samples (n =23). PCR, in con-junction with clinical and neurological ex-amination results, can be a powerful tool inattempting to identify and confirm a PNL di-agnosis.—Tropical Disease Bulletin

Matsuo, E. [The sequelae of Hansen’s dis-ease. (Pathologic viewpoint of etiologies,

194 International Journal of Leprosy 2004

morphologies and countermeasures)].Nihon Hansenbyo Gakkai Zasshi 72(3)(2003) 251–257. [Article in Japanese]

The proportion of glomerulonephritis,often a sequence of arteriolitis, among thesequelae of Hansen’s disease after the intro-duction of chemotherapy increasedmarkedly in Japan and nullified that of onceprevalent tuberculosis after 1960s. How-ever, most significant aftermath of the dis-ease for numbers of years in the past havebeen peripheral nerve injuries worldwide forwhich effective countermeasures are yet tobe developed. In this brief autopsy casesstudy from 1960s to 1990s, we confirmedthe presence of cases in which arteriolitisand resulted infarction of peripheral nervesand not M. leprae itself were shown to bethe major cause of axonal damages. Therewere also cases in which the accumulationof the bacilli without vascular changes didnot damage the axons. The cases as thesecould not be solitary but should be rathercommon in this time of chemotherapy. If so,the methods to reconstruct nerves and bloodvessels by promoting those regenerationshould be developed to cope with the situa-tion for surgeon, assisted by pathologists.—Authors’Abstract

Mert, A., Ozaras, R., Tabak, F., and Oz-turk, R. Primary tuberculosis cases pre-senting with erythema nodosum. J. Der-matol. 31(1) (2004) 66–68.

Erythema nodosum (EN) is seen only inthe primary tuberculosis (TB) form of tu-berculous diseases. Among the etiologiesof EN, TB is the most frequent disorder indeveloping countries. We aimed to assessour patients with EN in reference to pri-mary TB. We evaluated 335 patients withthe diagnosis of TB during last 20 years;retrospectively 61 (18%) of these caseshad pulmonary and 274 (82%) had extra-pulmonary TB. Ten (16%) of the pul-monary TB cases were primary. All 10 pa-tients with primary TB presented with EN.Among 50 patients with EN diagnosed andfollowed during the last 10 years, the eti-ology was determined in 56%, and primaryTB was the most frequent: 20%.—Au-thors’ Abstract

Modi, K., Mancini, M., and Joyce, M. P.Lepromatous leprosy in a heart transplantrecipient. Am. J. Transplant 3(12) (2003)1600–1603.

Northern Louisiana is not an area for in-digenous cases of leprosy. Limited data areavailable on the occurrence of leprosy inorgan transplant recipients. No cases havebeen reported in heart transplant recipients.Mr J.R. is a 68-year-old man from Shreve-port, Louisiana. He underwent orthotopicheart transplantation in March 1996. Hepresented in March 2000 with a macu-lopapular skin rash and intermittent handswelling for 5 months. He also complainedof intermittent burning of his feet for a year.The skin lesions were of two types—a finered migratory, intermittent maculopapularrash over the upper torso and a raised,larger, violaceaous lesion on his hands.Neurological examination revealed com-plete loss of protective sensation in the rightfoot by filamentous test and some loss in theleft foot. Punch skin biopsies from his rightarm and right chest lesion revealed abun-dant acid-fast bacilli (AFB). Histopatho-logic examination revealed perivascular, in-terstitial and perineural granulomatousinflammation and a large number of AFBorganisms within histiocytes. Culture of theskin biopsy specimen was negative for My-cobacterium tuberculosis or atypical myco-bacterium. Polymerase chain reaction(PCR) performed for Mycobacterium lep-rae was positive. The patient was treatedwith a modified regimen consisting of dap-sone 100 mg qd, ethionamide 250 mg qd,and minocycline 100 mg qd. His skin rashand neurological symptoms have re-solved.—Authors’ Abstract

Moses, A. E., Adelowo, K. A., and Ajayi,B. B. Prevalence of HIV-1 infectionamong patients with leprosy and pul-monary tuberculosis in a semi-arid re-gion, Nigeria. J. R. Soc. Promotion Health123(2) (2003) 117–119.

Much evidence exists on pulmonary tu-berculosis (PTB) as a presenting feature ofHIV infection or AIDS-related complex,while few reports exist of a direct associa-tion between HIV infection and leprosy.

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This study was carried out to see whether ornot an association between leprosy and HIVinfection existed, similar to that of PTB inthe region of Maiduguri, Nigeria. Of 105 pa-tients with leprosy, 11 (10.5%) were positivefor HIV antibody. Of 58 patients with sus-pected PTB, 11 (19%) were positive forHIV antibody. Twenty-seven (47%) of the58 had active PTB, with results of sputumsmear and culture positive for mycobacte-rium, and six of these (22.2%) were alsopositive for HIV antibody. Odds ratios (OR)obtained by conditional logistic regression(matched) analysis were 3.52 (95%, CI1.03–12.07) and 2.53 (95%, CI 1.04–6.15)for association between HIV-1 and PTB andleprosy, respectively. HIV infection wasmore prevalent among leprosy patients agedunder 30 years, OR = 4.25 (95%, CI1.25–14.42). The prevalence of HIV-1 in-fection was at borderline significance,higher in PTB and leprosy patients than inblood donors, Fisher’s exact test (two-tailed)p = 0.07 and p = 0.05, respectively.

Pimentel, M. I. F., Borges, E., da CostaNery, J. A., and Gonçalves, R. R. Initialneurological exam of multibacillary lep-rosy: correlation between the presence ofaffected nerves and the disability presentat diagnosis and with the occurrence ofovert neuritis. An. bras. Dermatol., Rio deJaneiro, 78(5) (2003) 561–568.

BACKGROUND: Disabilities constitute themain problem of leprosy. It is important toidentify risk factors involved, so it can bepossible the prone patients be followed-upmore carefully.

OBJECTIVES: To determine if the presenceof thick and/or painful peripheral nerves atdiagnosis correlates with disabilities al-ready present at the initial examination, aswell as with subsequent development ofneuritis, during and after multidrug therapy.

METHODS: One hundred and three patientswith multibacillary forms of leprosy werestudied and we noted the presence of com-promised peripheral nerves at diagnosis, thedisability grade before treatment (DGBT),and the occurrence of neuritis episodes dur-ing and after multibacillary multidrug ther-apy.

RESULTS: The detection of affected pe-

ripheral nerves at diagnosis, correlated sta-tistically (p <0.005) with the occurrence ofdisabilities (DGBT >0). It also correlatedsigificantly with the development of neuri-tis in the follow-up (average of 64.6 monthsfrom diagnosis, during and after multidrugtherapy).

CONCLUSIONS: We emphasize the need ofa good examination of peripheral nervetrunks in multibacillary patients at the diag-nosis, in order to improve the detection ofdiabilities already present, and specially toprevent further disabilities. Healthy profes-sionals who deal with leprosy patients mustbe aware to the initial neurological impair-ments because those patients are more sus-ceptible to the occurrence of neuritis andneurological sequelae.—Anais Braseileirosde Dermatologia

Rodriguez, G. [Generalized adenopathy asa manifestation of type 2 reactional lep-rosy]. Biomedica 23(4) (2003) 373–387.[Article in Spanish]

Generalized adenopathy as a manifesta-tion of type 2 reactional leprosy Leprosy pa-tient’s reactions are severe clinical manifes-tations of acute inflammation of chroniclesions, capable of producing irreversibleand invalidating damage. We studied a 46year-old man with a type 2 leprosy reaction,who presented fever, cutaneous nodules,nasal obstruction and generalized adenopa-thy. The hemogram showed leucocytosiswith neutrophilia. None of the initial diag-noses included leprosy. A lymph nodebiopsy revealed extensive necrotic areas in-filtrated with polymorphonuclear lympho-cytes, and foamy macrophages. Eosino-phylic necrosis and thrombosis of venuleswith lymphoid nodule depletion was also inevidence. Ziehl Neelsen stain was not done,but the Gomori stain clearly showedHansen’s bacilli. These were were not de-tected by the pathologist and therefore afinal diagnosis was not provided. Twentymonths later, the patient presented similarsymptoms, but with more generalized lym-phadenopathy and presence of cutaneousnodules. Nodule biopsy showed leproma-tous leprosy with erythema nodusum lepro-sum or type 2 reaction. Polychemotherapytreatment and anti-reaction treatment with

196 International Journal of Leprosy 2004

thalidomide cured the patient. No sequelaewere noted in 3 years following the treat-ment. A literature review of the type 2 reac-tion in leprosy is provided, including dis-cussion of risk factors, histopathology,differential diagnosis for leprosy adenopa-thy, pathogenesis, prognosis, and treatment.Type 2 leprosy must be treated immediatelyupon diagnosis as it can cause serious andpermanent tissue damage. As had occurredin the above patient, the disease can proceedwith generalized and symptomatic lymph-adenopathy.—Authors’Abstract

Thompson, A. M., Lynn, A. A., Robson,K., Joyce. M, P., Fivenson, D. P., andScollard, D. Lepromatous phlebitis ofthe external jugular vein. J. Am. Acad.Dermatol. 49(6) (2003) 1180–1182.

Mycobacterium leprae (M. leprae), thecausative agent of Hansen’s disease, is en-demic in many areas of Asia, sub-SaharanAfrica, South and Central America, the Pa-cific Islands, and the Philippines. The spec-trum of clinical disease is dependent on thepatient’s cell-mediated immunity and mightrange from localized anesthetic patches orplaques to disseminated disease. If undiag-nosed, progression with damage to the in-volved sensory and motor nerves mightoccur. Lepromatous vasculitis occurs mostcommonly in patients with severe dissemi-nated disease. Vascular disease, as the initialpresenting sign of tuberculoid leprosy, is,however, rare. We present one patient inwhom the development of Hansen’s diseasewas associated with involvement of the ex-ternal jugular vein and was initially seen asexternal jugular vein fibrosis.—Authors’Abstract

Warren, R. M., Victor, T. C., Streicher, E.M., Richardson, M., Beyers, N., vanPittius, N. C., and van Helden, P. D. Pa-tients with active tuberculosis often havedifferent strains in the same sputum spec-imen. Am. J. Respir. Crit. Care Med.169(5) (2004) 610–614.

It is generally accepted that tuberculosisresults from a single infection with a singleMycobacterium tuberculosis strain. Such in-

fections are thought to confer protective im-munity against exogenous reinfection. Inthis study, a novel polymerase chain reac-tion method was developed to specificallyidentify M. tuberculosis strains belonging tothe Beijing and non-Beijing evolutionarylineages in sputum specimens collectedfrom tuberculosis patients resident in an epi-demiologic field site in Cape Town, SouthAfrica. The sensitivity and specificity of thepolymerase chain reaction-based strain clas-sification method were 100% (95% confi-dence interval, 85–100%) when comparedwith DNA fingerprinting and spacer oligo-typing (spoligotyping). Application of thismethod showed that 19% of all patientswere simultaneously infected with Beijingand non-Beijing strains, and 57% of patientsinfected with a Beijing strain were also in-fected with a non-Beijing strain. Multipleinfections were more frequent in retreatmentcases (23%) as compared with new cases(17%), but were not associated with sex,age, or smear grading. These results suggestthat multiple infections are frequent, imply-ing high reinfection rates and the absence ofefficient protective immunity conferred bythe initial infection. This finding could in-fluence our understanding of the epidemiol-ogy of disease in high-incidence regions andour understanding for vaccine develop-ment.—Authors’Abstract

Yan, L., Zhang, G., Zheng, Z., Li, W.,Zheng, T., Watson, J. M., and Piefer, A.Comprehensive treatment of complicatedplantar ulcers in leprosy. Chin. Med. J.(Engl). 116(12) (2003) 1946–1948.

OBJECTIVE: To investigate feasible treat-ment methods for plantar ulcers in leprosypatients according to the agreement betweenthe Ministry of Health (MOH) of China andthe Leprosy Mission International (LMI).METHODS: A total of 2599 complicated footulcers in 1804 leprosy cases underwent surgictreatment. Plastic fixation and supports wereused, dressings were changed regularly, andprotective footwear and modified insoles wereprovided. RESULTS: Of the 2599 foot ulcers1446 (55.64%) healed. The cure rate of thepatients treated in leprosy hospitals was71.31%, with 219 (15.15%) recurrences offoot ulcers. The recurrence rate of those who

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lived at home was 18.35%. CONCLUSIONS:Comprehensive treatment of foot ulcers hasa high cure rate and a low recurrence rate. Re-duction of workload, avoidance of long dis-

tance walking, intensification of education onfoot self-care and provision of financial sup-port are the main measures for preventing arecurrence of foot ulcers.—Authors’Abstract

198 International Journal of Leprosy 2004

Immuno PathologyAlvarez, G. R., Zwilling, B. S., and Lafuse,

W. P. Mycobacterium avium inhibition ofIFN-gamma signaling in mouse macro-phages: Toll-like receptor 2 stimulationincreases expression of dominant-nega-tive STAT1 beta by mRNA stabilization.J. Immunol. 171(12) 2003 6766–6773.

Mycobacterial infections of macrophageshave been shown to inhibit the ability of themacrophage to respond to IFN-gamma. Wepreviously reported that Mycobacteriumavium infection of mouse macrophages de-creases IFN-gamma-induced STAT1 tyrosinephosphorylation and STAT1 DNA binding.Because macrophages respond to M. aviumthrough Toll-like receptor 2 (TLR2), we de-termined whether TLR2 stimulation inhibitsthe response to IFN-gamma. Treatment ofmouse RAW264.7 macrophages with TLR2agonists inhibited the induction of IFN-gamma-inducible genes by IFN-gamma. Incontrast to M. avium infection, TLR2 agonistsdid not inhibit the IFN-gamma induction ofDNA-binding activity of STAT1 and thetyrosine phosphorylation of STAT1alpha.Instead, IFN-gamma induction of RAW264.7cells treated with TLR2 agonists resulted inan increase in the tyrosine phosphorylationof the dominant-negative STAT1beta. TLR2stimulation of RAW264.7 cells increasedboth STAT1beta protein and mRNAexpression, suggesting that the increasedSTAT1beta phosphoylation results from in-creased STAT1beta expression. BecauseSTAT1alpha and STAT1beta mRNA havedifferent 3′ untranslated regions, and 3′ un-translated regions can regulate mRNA stabil-ity, we examined the effects of TLR2 stimu-lation on mRNA stability. TLR2 stimulationof RAW264.7 cells increased the stability ofSTAT1beta mRNA, while not affecting thestability of STAT1alpha mRNA. The abilityof STAT1beta to function as a dominant neg-ative was confirmed by overexpression ofSTAT1beta in RAW264.7 macrophages bytransient transfection, which inhibited IFN-

gamma-induced gene expression. These find-ings suggest that M. avium infection of mousemacrophages inhibits IFN-gamma signal-ing through a TLR2-dependent increase inSTAT1beta expression by mRNAstablizationand a TLR2-independent inhibition of STAT1tyrosine phosphorylation.—Authors’Abstract

Bisen, P. S., Garg, S. K., Tiwari, R. P.,Tagore, P. R., Chandra, R., Karnik, R.,Thaker, N., Desai, N., Ghosh, P. K.,Fraziano, M., and Colizzi, V. Analysisof the shotgun expression library of theMycobacterium tuberculosis genome forimmunodominant polypeptides: potentialuse in serodiagnosis. Clin. Diagn. Lab.Immunol. 10(6) (2003) 1051–1058.

See Current Literature, Molecular andGenetic Studies, p. 255.

Chen, K., Lu, J., Wang, L., and Gan, Y. H.Mycobacterial heat shock protein 65 en-hances antigen cross-presentation in den-dritic cells independent of Toll-like re-ceptor 4 signaling. J. Leukoc. Biol. 75(2)(2004) 260–266.

Heat shock proteins (HSP) have beenshown to enhance antigen processing andpresentation through their association withantigenic peptides and delivery of these moi-eties into major histocompatibility complexclass I pathways. In this study, mycobacterialHsp65 is demonstrated to have the ability tohelp cross-present an exogenous protein bydendritic cells (DC) to CD8 T cells withoutthe need for complex formation betweenHsp65 and the protein. This ability of Hsp65to enhance cross-presentation is independentof its weak stimulatory effect on DC, the lat-ter seen only after prolonged incubation.When the effect of lipopolysaccharide con-tamination is abrogated, Hsp65 is unable toactivate Toll-like receptor (TLR)4 in the

presence of CD14 and MD2. This accountsfor the inability of Hsp65 to drive maturationof DC and shows that Hsp65 is not a potentstimulator of DC. Thus, Hsp65 enhances thecross-presentation of a soluble, free antigenby DC, independent of TLR4 signaling andup-regulation of costimulatory molecules.—Authors’Abstract

Curto, M., Reali, C., Palmieri, G., Scintu,F., Schivo, M. L., Sogos, V., Marcialis,M. A., Ennas, M. G., Schwarz, H.,Pozzi, G., and Gremo, F. Inhibition ofcytokines expression in human microgliainfected by virulent and non-virulent my-cobacteria. Neurochem. Int. 44(6) (2004)381–392.

The pathogenesis of tuberculosis (TBC)meningitis is still unknown. As shown byprevious studies, human microglia can bethe target of mycobacteria, but no data areavailable about their cellular response to in-fection. Consequently, we studied the ex-pression of tumor necrosis factor-alpha(TNF-alpha), interleukin-1 (IL-1) and IL-10in human microglia pure cultures infectedwith the two variants of Mycobacteriumavium (domed-opaque (SmD) and transpar-ent (SmT)) and with Mycobacterium tuber-culosis. Results showed that microglia wasproductively infected by mycobacteriawhich could grow inside the cells. Myco-bacteria internalization was more rapid forM. avium, but M. tuberculosis infectionturned out to be more efficient due to the in-corporation of densely packed bacteria.TNF-alpha expression was not affected byM. avium, whereas an increase followed bya decrease was observed in M. tuberculosis.Both IL-1 and IL-10 cytokine expressionwas rapidly inhibited by infection with themore virulent bacteria, whereas the non-pathogenic one had almost no effect. Also,the expression of the co-stimulatory mole-cule CD137, a member of tumor necrosisfactor receptor family, was affected byinfection with virulent mycobacteria. Ourresults show that microglia response to my-cobacterial infection is modulated in cor-relation with virulence, mainly toward inhi-bition of inflammatory response. Thisobservation might be one of the mechanismsby which non-pathogenic mycobacteria are

quickly eliminated, explaining one of thebases of virulence.—Authors’Abstract

Deng, L., Ding, W., and Granstein, R. D.Thalidomide inhibits tumor necrosisfactor-alpha production and antigen pre-sentation by Langerhans cells. J. Invest.Dermatol. 121(5) (2003) 1060–1065.

Thalidomide is an effective treatmentfor several inflammatory and autoimmunedisorders including erythema nodosumleprosum, Behcet’s syndrome, discoidlupus erythematosus, and Crohn’s disease.Thalidomide is believed to exert its anti-inflammatory effects, at least in part, byinhibiting tumor necrosis factor-alpha(TNF-alpha) production by monocytes. Westudied the effects of thalidomide on epi-dermal Langerhans cells (LC). LCs areepidermal antigen-presenting dendriticcells that play important roles in skin im-mune responses. Using the murineepidermis-derived dendritic cell lines,XS106A from A/J mice and XS52 fromBALB/c mice as surrogates for LC, wefound that thalidomide inhibited TNF-alpha production in a concentration-dependent manner. Northern blot analysisrevealed that thalidomide significantly de-creased the peak-induced mRNA level ofTNF-alpha in XS106A cells and XS52cells. We then examined the effect ofthalidomide on fresh LC enriched to ap-proximately 98% using positive selectionof Ia+ cells with antibodies conjugated tomagnetic microspheres. TNF-alpha pro-duction was reduced by 67.7% at a thalido-mide concentration of 200 microg per mL.Thalidomide also had a profound in-hibitory effect on the ability of LC to pre-sent antigen to a responsive TH1 clone.Thalidomide inhibits TNF-alpha produc-tion and the antigen-presenting ability ofepidermal LCs. These mechanisms maycontribute to the therapeutic effects ob-served with this agent.—Authors’Abstract

Dugue, C., Perraut, R., Youinou, P., andRenaudineau, Y. Effects of anti-endothelial cell antibodies in leprosy andmalaria. Infect. Immun. 72(1) (2004)301–309.

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As a result of damaging endothelial cells(ECs), Mycobacterium leprae triggers theproduction of antibodies (Abs). These anti-EC Abs (AECAs) can be divided into twotypes. The first type nonspecifically reactswith components of the cytosol (CY) andcan be detected by enzyme-linked im-munosorbent assay (ELISA). The secondspecifically reacts with the EC membrane(MB) and requires fluorescence-activatedcell sorter (FACS) analysis to be detected.The presence of both types of AECAs wasdetermined in 68 leprosy patients. TheELISA was positive for 35 of them but alsofor 30 of 34 malaria patients and 17 of 50healthy African controls. However, whereasFACS analysis showed MB reactivity inonly three malaria patients and four con-trols, this reactivity was found in 27 leprosypatients, more of those having the leproma-tous than the tuberculoid form. Specificityfor MB, which we failed to absorb by incu-bation with CY lysates, predominated overthat for CY in leprosy, unlike malaria, wherethe EC reactivity was restricted to the CY.Western blot analysis and two-dimensionalelectrophoresis revealed that calreticulin, vi-mentin, tubulin, and heat shock protein 70were targeted by AECAs from leprosy pa-tients, but other proteins remained unidenti-fied. These auto-Abs, but not those frommalaria patients, did activate ECs, as indi-cated by the E-selectin and intercellular ad-hesion molecule 1 upregulation, and/or in-duced them into apoptosis, as documentedby four different methods. Our findings sug-gest that, in some but not all leprosy pa-tients, AECAs may play a role in pathogen-esis.—Authors’Abstract

Gaede, K. I., Mamat, U., and Muller-Quernheim, J. Differential gene expres-sion pattern in alveolar macrophages ofpatients with sarcoidosis and tuberculo-sis. J. Mol. Med. 82(3) (2004) 206–210.

Sarcoidosis is a multisystem granuloma-tous disorder of unknown origin character-ized by the presence of epithelioid granulo-mata in the affected organs. Histological andclinical similarities between sarcoidosis andtuberculosis caused by M. tuberculosis sug-gest a shared underlying pathophysiology.However, specific markers are needed. This

study examined the differential gene ex-pression pattern in alveolar macrophages ofpatients with granulomatous disorders. Thedifferential mRNA regulation pattern ofalveolar macrophages in the bronchoalveo-lar lavage of healthy controls was comparedto that of patients with sarcoidosis and tu-berculosis by means of differential displayreverse transcription PCR. Comparativeanalysis of 2498 PCR products in controls,sarcoidosis, and tuberculosis revealed a dif-ferential regulation of expressed sequencetags in only 6.5%. 1.8% showed a shared ex-pression pattern between sarcoidosis and tu-berculosis in contrast to the control. It canbe assumed that these alterations are associ-ated with common granulomatous features.In contrast, 3.0% of the amplified sequencetags showed specific up- or downregulationin sarcoidosis and 1.6% in tuberculosis.These data indicate a significant proportionof common granuloma-associated features,independent of the origin of the granuloma-tous disorder.—Authors’Abstract

Geluk, A., van Meijgaarden, K. E.,Franken, K. L., Wieles, B., Arend, S.M., Faber, W. R., Naafs, B., and Otten-hoff, T. H. Immunological crossreactiv-ity of the Mycobacterium leprae CFP-10with its homologue in Mycobacterium tu-berculosis. Scand. J. Immunol. 59(1)(2004) 66–70.

Mycobacterium tuberculosis culture fil-trate protein-10 (CFP-10) (Rv3874) is con-sidered a promising antigen for the immuno-diagnosis of tuberculosis (TB) together withearly secreted antigens of M. tuberculosis(ESAT-6). Both ESAT-6 and CFP-10 are en-coded by the RD1 region that is deletedfrom all tested M. bovis bacille Calmette-Guerin (BCG) strains but present in M. lep-rae, M. tuberculosis, M. bovis, M. kansasii,M. africanum and M. marinum. In thisstudy, the homologue of CFP-10 in M. lep-rae (ML0050) is identified and character-ized. Interferon-gamma production in re-sponse to this homologue by T cells fromleprosy patients, TB patients and unexposedcontrols shows that CFP-10 of M. leprae isa potent antigen that crossreacts with CFP-10 of M. tuberculosis at the T-cell level. Thiscrossreactivity has implications for the use

200 International Journal of Leprosy 2004

of CFP-10 of these mycobacterial species asdiagnostic tool in areas endemic for both thediseases.—Authors’Abstract

Imai, K., Kurita-Ochiai, T., and Ochiai, K.Mycobacterium bovis bacillus Calmette-Guerin infection promotes SOCS induc-tion and inhibits IFN-gamma-stimulatedJAK/STAT signaling in J774 macro-phages. FEMS Immunol. Med. Micro-biol. 39(2) (2003) 173–180.

The resurgence in mycobacterial infec-tion worldwide has led to renewed attentionto the pathogenesis of Mycobacteriumspecies. Although interferon-gamma (IFN-gamma) is a principal mediator of macro-phage activation, macrophages infectedwith Mycobacterium are poor in response atthe cytokine. However, the molecularmechanisms underlying mycobacterial in-fection remain unclear. The purpose of thisstudy was to elucidate the mechanism of thepoor response to IFN-gamma in mycobac-terial infection. Our data clearly demon-strate that this is due to induction of sup-pressor of cytokine signal (SOCS) negativeregulators of IFN-gamma signal transduc-tion that closely correlates with the inhibi-tion of JAK/STAT signaling and gene ex-pression stimulated by IFN-gamma.Mycobacterium bovis bacillus Calmette-Guerin infection induces the production ofSOCS-1 and SOCS-3 in murine J774macrophages. The level of SOCS-1 mRNAincreased 1 h and reached a maximum 3 hafter the addition of the bacteria. SOCS-3mRNA expression appeared as early as 1 hafter the infection. We also observed thattrehalose 6,6′-dimycolate/cord factor, amajor component of the Mycobacterium tu-berculosis cell wall, induces expression ofSOCS and inhibits IFN-gamma-stimulatedphosphorylation of STAT1 extensively inthe cells. The results in this study suggestthat a molecular mechanism of mycobacte-rial infection affects the unresponsivenessto IFN-gamma in the subsequent growthand spread of macrophages.—Authors’Ab-stract

Kang, S.-J., and Cresswell, P. Saposins fa-cilitate CD1d-restricted presentation of

an exogenous lipid antigen to T cells. Na-ture Immunol. 5(2) (2004) 175–181.

Members of the CD1 family present anti-genic lipids to T lymphocytes. CD1 mole-cules survey endocytic compartments forlipid antigens that are sorted into these vesi-cles after incorporation into the membranebilayer, and extraction from the bilayer islikely to be a critical step for lipid associa-tion. We hypothesized that lysosomalsaposins, which are cofactors required forsphingolipid degradation, might be involvedin this process. Here we show that saposins,although not required for the autoreactiverecognition of CD1d by natural killer Tcells, are indispensable for the binding of anexogenous lipid antigen, α-galactosylce-ramide, to CD1d in the endocytic pathway.We suggest that saposins mobilize mono-meric lipids from lysosomal membranes andfacilitate their association with CD1d.—Na-ture Immunology

Mendez-Samperio, P., Ayala, H., Trejo, A.,and Ramirez, F. A. Differential inductionof TNF-alpha and NOS2 by mitogen-activated protein kinase signaling path-ways during Mycobacterium bovis infec-tion. J. Infect. 48(1) (2004) 66–73.

The role of mitogen-activated protein ki-nase (MAPK) signaling pathways in the reg-ulation of TNF-alpha and NOS2 productionby human monocytes infected with Myco-bacterium bovis BCG was examined. Inhi-bition studies showed that ERK1/2 and p38MAPK activation were necessary for themonocyte response to M. bovis infection.Analysis of MAPK activation showed rapidphosphorylation of ERK1/2 and p38 in re-sponse to M. bovis BCG. Phosphorylationwas not due to an autocrine effect of TNF-alpha secretion, since an anti-TNF-alpha an-tibody had no significant effect on the levelsof p38 phosphorylation. The inhibitorPD98059 significantly reduced M. bovisBCG-induced TNF-alpha production and al-most completely abrogated phosphorylationof ERK1/2; in addition the potent MEK in-hibitor U0126 also abrogated phosphoryla-tion. In contrast, studies using inhibitors se-lective for ERK1/2 and p38 showed that p38

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plays an essential role in the induction ofNOS2, whereas the role of ERK1/2 wasminor. These results suggest that ERK1/2and p38 kinases differentially regulate theM. bovis BCG-mediated induction of TNF-alpha and NOS2 in human monocytes.—Authors’Abstract

Penido, C., Vieira-de-Abreu, A., Bozza,M. T., Castro-Faria-Neto, H. C., andBozza, P. T. Role of monocyte chemo-tactic protein-1/CC chemokine ligand 2on gamma delta T lymphocyte traffickingduring inflammation induced by lipo-polysaccharide or Mycobacterium bovisbacille Calmette-Guerin. J. Immunol.171(12) (2003) 6788–6794

Gammadelta T lymphocytes are involvedin a great variety of inflammatory and infec-tious responses. However, the mechanismsby which gammadelta T lymphocytes mi-grate to inflamed sites are poorly understood.In this study we investigate the role of mono-cyte chemotactic protein (MCP)-1 in regu-lating gammadelta T cell migration after LPSor Mycobacterium bovis bacille Calmette-Guerin (BCG) challenge. LPS-induced gam-madelta T cell influx was significantlyinhibited by either pretreatment with dexa-methasone or vaccinia virus Lister 35-kDachemokine binding protein, vCKBP, a CCchemokine neutralizing protein, suggestinga role for CC chemokines in this phenome-non. LPS stimulation increased the expres-sion of MCP-1 mRNA and protein at the in-flammation site within 6 hr. It is noteworthythat LPS was unable to increase MCP-1 pro-duction or gammadelta T cell recruitment inC3H/HeJ, indicative of the involvement ofToll-like receptor 4. Gammadelta T cells ex-press MCP-1 receptor CCR2. Pretreatmentwith anti-MCP-1 mAb drastically inhibitedLPS-induced in vivo gammadelta T cell mo-bilization. Indeed, MCP-1 knockout micewere unable to recruit gammadelta T cells tothe pleural cavity after LPS stimulation, ef-fect that could be restored by coadministra-tion of MCP-1. In addition, BCG-inducedgammadelta lymphocyte accumulation wassignificantly reduced in MCP-1 knockoutmice when compared with wild-type mice.In conclusion, our results indicate that LPS-induced gammadelta T lymphocyte migra-

tion is dependent on Toll-like receptor 4 andsensitive to both dexamethasone and CCchemokine-binding protein inhibition. More-over, by using MCP-1 neutralizing Abs andgenetically deficient mice we show that LPS-and BCG-induced gammadelta T lympho-cyte influx to the pleural cavity of mice ismainly orchestrated by the CC chemokineMCP-1.—Authors’Abstract

Ranjbar, S., Ly, N., Thim, S., Reynes, J.M., and Goldfeld, A. E. Mycobacteriumtuberculosis recall antigens suppressHIV-1 replication in anergic donor cellsvia CD8+ T cell expansion and increasedIL-10 levels. J. Immunol. 172(3) (2004)1953–1959.

Mycobacterium tuberculosis (MTb) is theleading cause of death in the setting ofAIDS. MTb enhances the pathogenicity andaccelerates the course of HIV disease and,furthermore, infection with HIV-1 increasesthe risk of reactivation or reinfection withMTb. In this study, we show that host-specific recall responses to one pathogen,MTb, has a direct effect upon the regulationof a second pathogen, HIV-1. Using cellsfrom immunocompetent former tuberculo-sis (TB) patients who displayed either a per-sistently positive (responsive) or negative(anergic), delayed-type hypersensitivity(DTH) reaction to intradermal injection ofpurified protein derivative (PPD), we inves-tigated the effect of recall Ags to MTb uponthe replication of HIV-1 primary isolates invitro. We show that HIV-1 replication of a Tcell-tropic isolate was significantly impairedin MTb-stimulated PBMC from PPD-anergicdonors. Furthermore, these donors displayeda significant increase in CD8(+) T cells andIL-10 levels and lower levels of IL-2 andTNF-alpha relative to PPD-responsivedonors in response to PPD stimulation.Strikingly, CD8(+) T cell depletion andblocking of IL-10 significantly increasedHIV-1 replication in these PPD-anergicdonors, indicating that an immunosuppres-sive response to MTb recall Ags inhibitsHIV-1 replication in PPD-anergic individuals.Therefore, immunotherapeutic approachesaimed at recapitulating Ag-specific MTb an-ergy in vivo could result in novel and effec-tive approaches to inhibit HIV-1 disease

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progression in MTb/HIV-1 coinfection.—Authors’Abstract

Reddy, V. M., and Suleman, F. G. Myco-bacterium avium-superoxide dismutasebinds to epithelial cell aldolase, glycer-aldehyde-3-phosphate dehydrogenaseand cyclophilin A. Microb. Pathog. 36(2)(2004) 67–74.

Mycobacterium avium complex (MAC)adheres, invades and multiplies inside epi-thelial cells. Earlier, we demonstrated twoMAC protein adhesins, 25 and 31 kDa,binding with HEp-2 cells. The 25 kDa MACadhesin was found to be superoxide dismu-tase (SOD). In this study, epithelial cell(HEp-2 and A549) ligands for MAC-SODwere identified by probing two-dimensionalwestern blots of epithelial extracts withMAC proteins followed by monoclonal anti-MAC-SOD antibodies. Three epithelial cellproteins with molecular masses 43, 40 and18 kDa, present in both membrane and cy-tosolic fractions, were found to bind withMAC-SOD. Based on the N-terminal aminoacid sequences, the 43, 40 and 18 kDa epi-thelial proteins were identified as aldolase,glyceraldehyde-3-phosphate dehydrogenase(GAPDH) and cyclophilin A (CypA), re-spectively. Furthermore, MAC-SOD wasfound to bind to purified rabbit muscle al-dolase, GAPDH and recombinant CypA inwestern blotting.—Authors’Abstract

Remus, N., Alcais, A., and Abel, L. Humangenetics of common mycobacterial in-fections. Immunol. Res. 28(2) (2003)109–129.

See Current Literature, Molecular andGenetic Studies, p. 260.

Saunders, B. M., Fernando, S. L., Sluyter,R., Britton, W. J., and Wiley, J. S. Aloss-of-function polymorphism in thehuman P2X7 receptor abolishes ATP-mediated killing of mycobacteria. J. Im-munol. 171(10) (2003) 5442–5446.

Protective immunity to mycobacterial in-fections requires activation of the antibacte-

rial mechanisms of infected macrophages. Ithas previously been reported that ATP treat-ment of mycobacteria-infected macrophagesinduces apoptosis mediated via the P2X(7)pathway and that this leads to the death ofboth the host cell and the internalized bacilli.We have recently identified a single nu-cleotide polymorphism in the P2X7 gene(1513A→C), with 1–2% prevalence in thehomozygous state, which codes for a non-functional receptor. IFN-gamma-primed,mycobacteria-infected macrophages fromwild-type individuals were incubated withATP and this induced apoptosis and reducedmycobacterial viability by 90%. Similartreatment of macrophages from individualshomozygous for the 1513C polymorphismfailed to induce apoptosis and did not leadto mycobacterial killing via the P2X(7)-mediated pathway. These data demonstratethat a single nucleotide polymorphism in theP2X7 gene can allow survival of mycobac-teria within infected host cells.—Authors’Abstract

Sen Gupta, R., Hillemann, D., Kubica, T.,Zissel, G., Muller-Quernheim, J.,Galle, J., Vollmer, E., and Goldmann,T. HOPE-fixation enables improvedPCR-based detection and differentiationof Mycobacterium tuberculosis complexin paraffin-embedded tissues. Pathol.Res. Pract. 199(9) (2003) 619–623.

Standard PCR-based detection of myco-bacterial DNA in paraffin-embedded speci-mens may lack sufficient sensitivity becauseof the degradation of nucleic acids causedby routinely used formalin fixation. There-fore, we set up an approach that aimed atimproving the results by applying the novelHOPE-fixative in PCR-detection of myco-bacteria in paraffin-embedded tissues. Com-parison of PCR-results using DNA extractedfrom either HOPE- or formalin-fixed speci-mens in BCG-infected SCID-mice revealeda more than 100fold enhanced sensitivity forthe HOPE-fixed material. Owing to thepreservation of DNA from degradation inHOPE-fixed tissues, even differentiationwithin the M. tuberculosis complex waspossible by spoligotyping. We thereforeconclude that the HOPE-fixative is a usefultool for molecular pathology that enhances

72, 2 Current Literature, Immuno Pathology 203

the sensitivity of PCR-based methods forthe detection of pathogens in paraffin-embedded tissues compared to formalin-fixation. Owing to the better preservedDNA, improved differentiation of myco-bacteria from archived materials is possible.These results promise new and a substan-tially wider range of possibilities in the fieldof molecular pathology.—Authors’Abstract

Siemion, I. Z., Gawlowska, M., Krajew-ski, K., Strug, I., and Wieczorek, Z.Analogs of RGDVY and GRGD peptidesinhibit Mycobacterium kansasii phagocy-tosis. Peptides 24(8) (2003) 1109–1115.

Continuing our research on Mycobacteriakansasii phagocytosis inhibition, we have ex-amined in that context three series of peptidesderived from the RGDVY and GRGD se-quences. It was found that the levels of the in-hibitory activity depend on the amino acidcomposition as well as on the particular pep-tide sequence. Distinct inhibitory activity wasfound in the case of thymopentin (RKDVY),the active fragment of thymopoietin. In thiscase the Mycobacterium phagocytosis inhi-bition should be combined with general im-munostimulatory activity of RKDVY pep-tide. Our examination of a series of GRGDVanalogs with a successively prolonged oligo-Gly linker inserted into the peptide chainshowed that the distance between the Arg andAsp residues required for such an activityshould be about 9A.—Authors’Abstract

Stamm, L. M., Morisaki, J. H., Gao, L. Y.,Jeng, R. L., McDonald, K. L., Roth, R.,Takeshita, S., Heuser, J., Welch, M. D.,and Brown, E. J. Mycobacterium mar-inum escapes from phagosomes and ispropelled by actin-based motility. J. Exp.Med. 198(9) (2003) 1361–1368.

Mycobacteria are responsible for a num-ber of human and animal diseases and areclassical intracellular pathogens, living in-side macrophages rather than as free-livingorganisms during infection. Numerous in-tracellular pathogens, including Listeriamonocytogenes, Shigella flexneri, and Rick-ettsia rickettsii, exploit the host cytoskele-ton by using actin-based motility for cell to

cell spread during infection. Here we showthat Mycobacterium marinum, a naturalpathogen of fish and frogs and an occasionalpathogen of humans, is capable of activelyinducing actin polymerization within mac-rophages. M. marinum that polymerizedactin were free in the cytoplasm and pro-pelled by actin-based motility into adjacentcells. Immunofluorescence demonstrated thepresence of host cytoskeletal proteins, in-cluding the Arp2/3 complex and vasodilator-stimulated phosphoprotein, throughout theactin tails. In contrast, Wiskott-Aldrich syn-drome protein localized exclusively at theactin-polymerizing pole of M. marinum.These findings show that M. marinum canescape into the cytoplasm of infectedmacrophages, where it can recruit host cellcytoskeletal factors to induce actin poly-merization leading to direct cell to cellspread.—Authors’Abstract

Weir, R. E., Black, G. F., Dockrell, H. M.,Floyd, S., Fine, P. E., Chaguluka, S. D.,Stenson, S., King, E., Nazareth, B.,Warndorff, D. K., Ngwira, B., Crampin,A. C., Mwaungulu, L., Sichali, L., Jar-man, E., Donovan, L., and Blackwell, J.M. Mycobacterial purified protein deriv-atives stimulate innate immunity: Malaw-ians show enhanced tumor necrosis fac-tor alpha, interleukin-1beta (IL-1beta),and IL-10 responses compared to those ofadolescents in the United Kingdom. In-fect. Immun. 72(3) (2004) 1807–1811.

To investigate the role of innate immunityin variable efficacy of Mycobacterium bovisBCG vaccination in Malawi and the UnitedKingdom, we examined 24-hr tumor necro-sis factor alpha, interleukin-1beta (IL-1beta), and IL-10 responses to mycobacte-rial purified protein derivatives (PPDs). Therank order in stimulatory potency for differ-ent PPDs was the same for all three cy-tokines. Before vaccination Malawiansmade higher pro- and anti-inflammatory re-sponses than did United Kingdom subjects.Fewer than 5% of United Kingdom subjectsmade IL-10 in response to any PPD, com-pared to 19 to 57% responders amongMalawians. Priming for regulatory IL-10may contribute to the smaller increase ingamma interferon responses in Malawians

204 International Journal of Leprosy 2004

compared to United Kingdom subjects fol-lowing BCG vaccination.—Authors’ Ab-stract

Zhong, J., Gilbertson, B., and Cheers, C.Apoptosis of CD4+ and CD8+ T cellsduring experimental infection with My-cobacterium avium is controlled byFas/FasL and Bcl-2-sensitive pathways,respectively. Immunol. Cell. Biol. 81(6)(2003) 480–486.

Both CD4+ and CD8+ T cells from miceinfected with Mycobacterium avium suf-fered a high rate of apoptosis, beginningwith the onset of the immune response andculminating in the loss of T cells from thetissues and loss of IFN-gamma production.

Fas expression increased over the course ofinfection on both T cell populations, as didtheir susceptibility to the induction of apop-tosis in vitro by anti-Fas mAb. Nevertheless,although the rate of apoptosis among CD4+T cells from infected mice was reduced tonormal levels in lpr mice with a defectiveFas, CD8+ T cells were unaffected, imply-ing that Fas/FasL interaction was not im-portant in these cells in vivo. Conversely,over-expression of B-cell lymphoma-2 (Bcl-2), which is known to protect T cells fromapoptosis signalled through the TNF recep-tor or due to the withdrawal of cytokines, to-tally protected CD8+ T cells from infectedmice but had no effect on CD4+. It is of in-terest that these two contrasting pathways ofT-cell apoptosis operate at the same timeduring a single infection.—Authors’Abstract

72, 2 Current Literature, Immuno Pathology (Leprosy) 205

Immuno Pathology (Leprosy) de la Barrera, S., Finiasz, M., Fink, S.,

Ilarregui, J., Aleman, M., Olivares, L.,Franco, M. C., Pizzariello, G., and delCarmen Sasiain, M. NK cells modulatethe cytotoxic activity generated by My-cobacterium leprae-hsp65 in leprosy pa-tients: role of IL-18 and IL-13. Clin. Exp.Immunol. 135(1) (2004) 105–113.

Protection against intracellular pathogenssuch as Mycobacterium leprae is criticallydependent on the function of NK cells atearly stages of the immune response and onTh1 cells at later stages. In the present reportwe evaluated the role of IL-18 and IL-13, twocytokines that can influence NK cell activity,in the generation of M. leprae-derived hsp65-cytotoxic T lymphocytes (CTL) from periph-eral blood mononuclear cells (PBMC) of lep-rosy patients. We demonstrated that IL-18modulates hsp65-induced CTL generationand collaborates with IL-12 for this effect. Inpaucibacillary (PB) patients and normal con-trols (N) depletion of NK cells reduces thecytolytic activity. Under these conditions, IL-12 cannot up-regulate this CTL generation,while, in contrast, IL-18 increases the cyto-toxic activity both in the presence or absenceof NK cells. IL-13 down-regulates the hsp65-induced CTL generation and counteracts thepositive effect of IL-18. The negative effectof IL-13 is observed in the early stages of the

response, suggesting that this cytokine affectsIFNgamma production by NK cells. mRNAcoding for IFNgamma is induced by IL-18and reduced in the presence of IL-13, whenPBMC from N or PB patients are stimulatedwith hsp65. Neutralization of IL-13 in PBMCfrom multibacillary (MB) leprosy patients in-duces the production of IFNgamma proteinby lymphocytes. A modulatory role on thegeneration of hsp65 induced CTL is demon-strated for IL-18 and IL-13 and this effecttakes place through the production ofIFNgamma.—Authors’Abstract

Job, C. K. Nine-banded armadillo and lep-rosy research. Indian J. Pathol. Micro-biol. 46(4) (2003) 541–550.—IndianJournal of Pathology and Microbiology

In this presentation an attempt has beenmade to describe the nine-banded armadilloas an animal model, probably the only one inwhich lepromatous leprosy similar to thatfound in humans can be experimentally pro-duced. Some unique features of the physiol-ogy of the animal are mentioned. The pathol-ogy and the microbiology of leprosy in thearmadillo are described in detail. The discov-ery of lepromatous leprosy in the wild ar-madillos in the southern parts of the UnitedStates, the transmission of disease among

them through trauma and thorn pricks and thepathogenesis of the disease are presented. Theimpact of leprosy in the wild animals mayhave on human leprosy is discussed.—IndianJournal of Pathology and Microbiology

Johansen, P., Raynaud, C., Yang, M., Col-ston, M. J., Tascon, R. E., and Lowrie,D. B. Anti-mycobacterial immunity in-duced by a single injection of M. lepraeHsp65-encoding plasmid DNA in bio-degradable microparticles. Immunol. Lett.90(2–3) (2003) 81–85.

See Current Literature, Experimental In-fections, p. 235.

Kirkaldy, A. A., Musonda, A. C.,Khanolkhar-Young, S., Suneetha, S.,and Lockwood, D. N. Expression of CCand CXC chemokines and chemokinereceptors in human leprosy skin lesions.Clin. Exp. Immunol. 134(3) (2003)447–453.

We have investigated the expression ofchemokines and their receptors in leprosy skinlesions using immunohistochemistry. Skinbiopsies from 25 leprosy patients across theleprosy spectrum, 11 patients undergoing typeI reversal reactions and four normal donorswere immunostained by ABC peroxidasemethod using antibodies against CC and CXCchemokines and their receptors. Using an insitu hybridization technique we have alsostudied the expression of monocyte chemo-attractant protein 1 (MCP-1), RANTES andinterleukin (IL)-8 chemokines mRNA in lep-rosy skin lesions. Chemokines and receptorexpression was detected in all leprosy skinbiopsies. Expression of CC chemokinesMCP-1 (p <0.01) and RANTES (p <0.01)were elevated significantly in borderline tu-berculoid leprosy in reversal reaction com-pared to non-reactional borderline tuberculoidleprosy, but there was no difference in the ex-pression of IL-8 chemokine. Surprisingly,there was no significant difference in the ex-pression of CC (CCR2 and CCR5) and CXC(CXCR2) chemokine receptors across the lep-rosy spectrum. Similarly, there was no signif-icant difference in the expression of mRNA

for MCP-1, regulated upon activation normalT cell expressed and secreted (RANTES) andIL-8 chemokines. Here, the presence of a neu-trophil chemoattractant IL-8 in leprosy le-sions, which do not contain neutrophils, sug-gests strongly a role of IL-8 as a monocyteand lymphocyte recruiter in leprosy lesions.These results suggest that the chemokines andtheir receptors, which are known to chemo-attract T lymphocytes and macrophages, areinvolved in assembling the cellular infiltratefound in lesions across the leprosy spec-trum.—Authors’Abstract

Leal, A. M., Magalhaes, P. K., Souza, C.S., and Foss, N. T. Adrenocortical hor-mones and interleukin patterns in leprosy.Parasite Immunol. 25(8–9) (2003)457–461.

The functional status of adrenocorticalhormones and their relationship to the pat-tern of inflammatory cytokines in the lepro-matous and tuberculoid poles of leprosywere investigated. Interleukin (IL)-1beta,IL-6 and tumour necrosis factor (TNF)-alpha plasma levels, C-reactive protein(CRP) concentrations and erythrocyte sedi-mentation rates (ESR) were significantlyhigher in LL/BL (lepromatous) leprosy pa-tients than in control subjects. There was asignificant positive correlation between IL-6and TNF-alpha plasma levels and ESR andCRP concentrations. IL-1beta was posi-tively correlated with ESR but not withCRP. Both baseline and stimulated adreno-corticotropic hormone and cortisol plasmalevels were not different between patientsand control subjects. In contrast, adrenal an-drogen dehydroepiandrosterone sulphate(DHEA-S) plasma levels were significantlylower in leprosy patients than in sex-matched control subjects. There was a sig-nificant inverse correlation between DHEA-Sand IL-6, TNF-alpha, and CRP concentra-tions. This finding may be of pathogeneticsignificance in this disease and in other in-flammatory states.—Authors’Abstract

Sridevi, K., Neena, K., Chitralekha, K. T.,Arif, A. K., Tomar, D., and Rao, D. N.Expression of costimulatory molecules(CD80, CD86, CD28, CD152), accessory

206 International Journal of Leprosy 2004

molecules (TCR alphabeta, TCR gam-madelta) and T cell lineage molecules(CD4+, CD8+) in PBMC of leprosy pa-tients using Mycobacterium leprae anti-gen (MLCWA) with murabutide and Tcell peptide of Trat protein. Int. Im-munopharmacol. 4(1) (2004) 1–14.

In leprosy, cell-mediated immunity (CMI)is more significant than humoral response toeliminate intracellular pathogen. T cell defectis a common feature in lepromatous leprosy(LL) patients as compared to tuberculoid type(TT) patients. For efficient initiation of CD4+,T cell response requires T cell receptor (TCR)activation and costimulation provided by mol-ecules on antigen-presenting cells (APC) andtheir counter receptors on T cells. In our pre-vious study, the defective T cell function inLL patients was restored to a proliferatingstate with the release of TH1 type cytokinesusing mycobacterial antigen(s) with two im-munomodulators (Murabutide (MDP-BE)and T cell epitope of Trat protein of Escheri-chia coli) by presenting the antigen in partic-ulate form in vitro to PBMC derived from lep-rosy patients. This observation prompted us tostudy the expression of the costimulatory mol-ecules (CD80, CD86, CD28, CD152), otheraccessory molecules (TCR alphabeta/gam-madelta) and T cell lineage molecules (CD4+and CD8+) during constitutive and activatedstate of peripheral blood mononuclear cells(PBMC) derived from normal and leprosy in-dividuals using different formulations of My-cobacterium leprae total cell wall antigen(MLCWA), Trat and MDP-BE using flow cy-

tometric analysis. An increased surface ex-pression of CD80, CD86 and CD28 but de-creased CD152 expression was observedwhen PBMC of normal, BT/TT (tubercu-loid) and BL/LL (lepromatous) patientswere stimulated in vitro with MLCWA+MDP-BE+Trat peptide using liposomalmode of antigen delivery, while opposite re-sults were obtained with the antigen alone.Antibody inhibition study using antihumanCD80 or CD86 completely abolished the Tcell lymphoproliferation, thereby recon-firming the importance of these costim-ulatory molecules during T cell activa-tion/differentiation. Though the liposome-entrapped antigen formulation has no effecton expression of alphabeta/gammadelta Tcell receptor, the constitutive levels of TCRgammadelta were high in lepromatous pa-tients. Thus, TCR bearing gammadelta ap-pears to have a negligible regulatory role inperipheral blood of leprosy patients. Thepercentage of cells positive for CD4+ areincreased in inducible state in all the threegroups, while CD8+-positive cells weredecreased in LL patients, thereby recon-firming the fact that priming of CD4+ cellsare necessary for producing final effectorfunctions. Lastly, intracellular cytokinestaining experiment indicated that CD4+cells are the major producers of IFN-gammabut not NK cells. The study highlights thereversal of T cell anergy especially in lepro-matous patients through the modulation ofcostimulatory molecule expression underthe influence of Th1 cytokines, i.e., IL-2 andIFNgamma.—Authors’Abstract

72, 2 Current Literature, Immuno Pathology (Tuberculosis) 207

Immuno Pathology (Tuberculosis)

Aagaard, C., Brock, I., Olsen, A., Otten-hoff, T. H., Weldingh, K., and Ander-sen, P. Mapping immune reactivity to-ward Rv2653 and Rv2654: two novellow-molecular-mass antigens foundspecifically in the Mycobacterium tuber-culosis complex. J. Infect. Dis. 189(5)2004 812–819.

New tools are urgently needed for the de-tection of latent tuberculosis (TB). We eval-uated the diagnostic potential of 2 novel My-cobacterium tuberculosis complex-specific

candidate antigens (Rv2653 and Rv2654)and investigated T cell recognition duringnatural infection in humans and experimen-tal infection in guinea pigs. Peripheral bloodmononuclear cells stimulated with peptidepools covering the full length of Rv2654 in-duced interferon-gamma release in 10 of 19patients with TB. Neither Rv2654 single pep-tides nor Rv2654 pools were recognized bybacille Calmette-Guerin-vaccinated donors.However, peptides from Rv2653 were rec-ognized by both patients group. The cross-reactive epitope(s) in Rv2653 were located in

a 36-amino acid stretch in the center of themolecule. Rv2654 also induced M. tubercu-losis-specific skin-test responses in 3 of 4aerosol-infected guinea pigs. Rv2654 is astrongly recognized T cell antigen that ishighly specific for TB and has potential as anovel cell-mediated immunity-based TB di-agnostic agent.—Authors’Abstract

Agger, E. M., Brock, I., Okkels, L. M.,Arend, S. M., Aagaard, C. S., Weldingh,K. N., and Andersen, P. Human T-cell re-sponses to the RD1-encoded proteinTB27.4 (Rv3878) from Mycobacteriumtuberculosis. Immunology 110(4) 2003507–512.

In recent years, there has been considerablefocus on the discovery and characterization ofproteins derived from Mycobacterium tuber-culosis leading to the identification of a num-ber of candidate antigens for use in vaccinedevelopment or for diagnostic purposes. Pre-vious experiments have demonstrated an im-portant immunological role for proteins en-coded by the RD1 region, which is absent fromall strains of bacillus Calmette-Guerin (BCG)but present in the genomes of virulent M.bovis and M. tuberculosis. Herein, we havestudied human T-cell responses to the antigenencoded by the putative open reading frame(rv3878) of the RD1 region. Immunoblotanalysis revealed that rv3878 was expressedand the native protein was designated TB27.4.Immunological evaluations demonstrate thatTB27.4 elicits a prominent immune responsein human tuberculosis patients with a domi-nant region in the C-terminal part of the mol-ecule. In contrast, very limited responses wereseen in M. bovis BCG-vaccinated donors.This study therefore emphasizes the diagnos-tic potential of proteins encoded by the RD1region.—Authors’Abstract

Allen, S. S., Cassone, L., Lasco, T. M., andMcMurray, D. N. Effect of neutralizingtransforming growth factor beta1 on theimmune response against Mycobacteriumtuberculosis in guinea pigs. Infect.Immun. 72(3) 2004 1358–1363.

Transforming growth factor beta (TGF-beta) is a cytokine which has been shown

to suppress the antimycobacterial immuneresponses of humans and experimental an-imals. In this study, the contributions ofTGF-beta to cytokine production in vivowere investigated by using the establishedguinea pig model of tuberculous pleurisy.Mycobacterium bovis BCG-vaccinatedguinea pigs were injected intrapleurallywith heat-killed virulent Mycobacteriumtuberculosis. Eight days following induc-tion of an antigen-specific pleural effusion,guinea pigs were injected intrapleurallywith anti-TGF-beta1 or isotype control an-tibody. The following day, pleural exudateswere removed, and the fluid volume andcharacteristics of the infiltrating cells weredetermined. Pleural fluid was analyzed fortotal interferon (IFN) and tumor necrosisfactor (TNF) protein levels by using ap-propriate bioassays. RNA from pleural ef-fusion cells was examined to determineTGF-beta1, TNF-alpha, IFN-gamma, andinterleukin-8 mRNA levels by using real-time PCR. Proliferative responses of pleu-ral effusion lymphocytes were examined inresponse to concanavalin A and purifiedprotein derivative (PPD) in vitro. Treat-ment with anti-TGF-beta1 resulted in de-creased pleural fluid volume and decreasedcell numbers in the pleural space alongwith an increased percentage of lympho-cytes and a decreased percentage of neu-trophils. The bioactive TNF protein levelsin pleural fluid were increased in guineapigs treated with anti-TGF-beta1, while thebioactive IFN protein concentrations werenot altered. Expression of TGF-beta1 andTNF-alpha mRNA was significantly in-creased following TGF-beta1 neutraliza-tion. Finally, PPD-induced proliferative re-sponses of pleural cells from anti-TGF-beta1-treated animals were significantlyenhanced. Thus, TGF-beta1 may be in-volved in the resolution of this local, my-cobacterial antigen-specific inflammatoryresponse.—Authors’ Abstract

Ando, M., Yoshimatsu, T., Ko, C., Con-verse, P. J., and Bishai, W. R. Deletionof Mycobacterium tuberculosis sigmafactor E results in delayed time to deathwith bacterial persistence in the lungs ofaerosol-infected mice. Infect. Immun.71(12) (2003) 7170–7172.

208 International Journal of Leprosy 2004

See Current Literature, Experimental In-fections, p. 230.

Brookes, R. H., Pathan, A. A., McShane,H., Hensmann, M., Price, D. A., andHill, A. V. CD8+ T cell-mediated sup-pression of intracellular Mycobacteriumtuberculosis growth in activated humanmacrophages. Eur. J. Immunol. 33(12)(2003) 3293–3302.

Animal models of tuberculosis point to aprotective role for MHC class I-restrictedCD8(+) T cells, yet it is unclear how thesecells protect or whether such findings ex-tend to humans. Here we report that macro-phages infected with Mycobacterium tu-berculosis, rapidly process and present anearly secreted antigenic target (ESAT-6)-specific HLA class I-restricted CD8(+) Tcell epitope. When cocultured with CD8(+)T cells restricted through classical HLAclass I molecules the growth of bacilliwithin macrophages is significantly im-paired after 7 days. This slow antimy-cobacterial activity did not correlate withmacrophage lysis but required cell contact.We also found that inhibitors of apoptosiseither had no effect or augmented the CD8-mediated suppressive activity, suggestingthat an activation signal might be involved.Indeed we show that CD8(+) T cells wereable to activate macrophages through re-ceptors that include CD95 (Fas). Consistentwith these findings the CD8-mediated sup-pression of mycobacterial growth was par-tially reversed by Fas blockade. These dataidentify a previously unrecognized CD8(+)T cell-mediated mechanism used to controlan intracellular infection of macrophages.—Authors’ Abstract

Drennan, M. B., Nicolle, D., Quesniaux,V. J., Jacobs, M., Allie, N., Mpagi, J.,Fremond, C., Wagner, H., Kirschning,C., and Ryffel, B. Toll-like receptor 2-deficient mice succumb to Mycobacte-rium tuberculosis infection. Am. J. Pathol.164(1) (2004) 49–57.

Recognition of Mycobacterium tubercu-losis by the innate immune system is essen-tial in the development of an adaptive im-

mune response. Mycobacterial cell wallcomponents activate macrophages throughToll-like receptor (TLR) 2, suggesting thatthis innate immune receptor plays a role inthe host response to M. tuberculosis infec-tion. After aerosol infection with either 100or 500 live mycobacteria, TLR2-deficientmice display reduced bacterial clearance, adefective granulomatous response, anddevelop chronic pneumonia. Analysis ofpulmonary immune responses in TLR2-deficient mice after 500 mycobacterialaerosol challenge showed increased levelsof interferon-gamma, tumor necrosis factor-alpha, and interleukin-12p40 as well as in-creased numbers of CD4(+) and CD8(+)cells. Furthermore, TLR2-deficient micemounted elevated Ag-specific type 1 T-cellresponses that were not protective becauseall deficient mice succumb to infectionwithin 5 months. Taken together, the datasuggests that TLR2 may function as a regu-lator of inflammation, and in its absence anexaggerated immune inflammatory responsedevelops.—Authors’Abstract

Geiman, D. E., Kaushal, D., Ko, C., Tyagi,S., Manabe, Y. C., Schroeder, B. G.,Fleischmann, R. D., Morrison, N. E.,Converse, P. J., Chen, P., and Bishai,W. R. Attenuation of late-stage disease inmice infected by the Mycobacterium tu-berculosis mutant lacking the SigF alter-nate sigma factor and identification ofSigF-dependent genes by microarrayanalysis. Infect. Immun. 72(3) (2004)1733–1745.

The Mycobacterium tuberculosis alternatesigma factor, SigF, is expressed during sta-tionary growth phase and under stress con-ditions in vitro. To better understand thefunction of SigF we studied the phenotypeof the M. tuberculosis DeltasigF mutant invivo during mouse infection, tested the mu-tant as a vaccine in rabbits, and evaluatedthe mutant’s microarray expression profilein comparison with the wild type. In micethe growth rates of the DeltasigF mutant andwild-type strains were nearly identical dur-ing the first 8 weeks after infection. At 8weeks, the DeltasigF mutant persisted in thelung, while the wild type continued growingthrough 20 weeks. Histopathological analy-

72, 2 Current Literature, Immuno Pathology (Tuberculosis) 209

sis showed that both wild-type and mutantstrains had similar degrees of interstitial andgranulomatous inflammation during the first12 weeks of infection. However, from 12 to20 weeks the mutant strain showed smallerand fewer lesions and less inflammation inthe lungs and spleen. Intradermal vaccina-tion of rabbits with the M. tuberculosisDeltasigF strain, followed by aerosol chal-lenge, resulted in fewer tubercles than didintradermal M. bovis BCG vaccination.Complete genomic microarray analysis re-vealed that 187 genes were relatively un-derexpressed in the absence of SigF in earlystationary phase, 277 in late stationaryphase, and only 38 genes in exponentialgrowth phase. Numerous regulatory genesand those involved in cell envelope synthe-sis were down-regulated in the absence ofSigF; moreover, the DeltasigF mutant strainlacked neutral red staining, suggesting areduction in the expression of envelope-associated sulfolipids. Examination of 5′-untranslated sequences among the down-regulated genes revealed multiple instancesof a putative SigF consensus recognition se-quence: GGTTTCX(18)GGGTAT. These re-sults indicate that in the mouse the M. tu-berculosis DeltasigF mutant strain persistsin the lung but at lower bacterial burdensthan wild type and is attenuated byhistopathologic assessment. Microarrayanalysis has identified SigF-dependentgenes and a putative SigF consensus recog-nition site.

Gilleron, M., Stenger, S., Mazorra, Z.,Wittke, F., Mariotti, S., Bohmer, G.,Prandi, J., Mori, L., Puzo, G., and DeLibero, G. Diacylated SulfoglycolipidsAre Novel Mycobacterial Antigens Stim-ulating CD1-restricted T Cells during In-fection with Mycobacterium tuberculosis.J. Exp. Med. 199(5) (2004) 649–659.

Mycobacterial lipids comprise a heteroge-neous group of molecules capable of inducingT cell responses in humans. To identify novelantigenic lipids and increase our understand-ing of lipid-mediated immune responses, weestablished a panel of T cell clones with dif-ferent lipid specificities. Using this approachwe characterized a novel lipid antigen be-longing to the group of diacylated sulfogly-

colipids purified from Mycobacterium tuber-culosis. The structure of this sulfoglycolipidwas identified as 2-palmitoyl or 2-stearoyl-3-hydroxyphthioceranoyl-2′-sulfate-alpha-alpha′-d-trehalose (Ac(2)SGL). Its immuno-genicity is dependent on the presence of thesulfate group and of the two fatty acids.Ac(2)SGL is mainly presented by CD1b mol-ecules after internalization in a cellular com-partment with low pH. Ac(2)SGL-specific Tcells release interferon gamma, efficientlyrecognize M. tuberculosis-infected cells, andkill intracellular bacteria. The presence ofAc(2)SGL-responsive T cells in vivo isstrictly dependent on previous contact withM. tuberculosis, but independent from thedevelopment of clinically overt disease.These properties identify Ac(2)SGL as apromising candidate to be tested in novelvaccines against tuberculosis.—Authors’Abstract

Gold, J. A., Hoshino, Y., Tanaka, N., Rom,W. N., Raju, B., Condos, R., and Wei-den, M. D. Surfactant protein A modu-lates the inflammatory response inmacrophages during tuberculosis. Infect.Immun. 72(2) (2004) 645–650.

Tuberculosis leads to immune activationand increased human immunodeficiencyvirus type 1 (HIV-1) replication in the lung.However, in vitro models of mycobacterialinfection of human macrophages do notfully reproduce these in vivo observations,suggesting that there are additional host fac-tors. Surfactant protein A (SP-A) is an im-portant mediator of innate immunity in thelung. SP-A levels were assayed in thehuman lung by using bronchoalveolarlavage (BAL). There was a threefold reduc-tion in SP-A levels during tuberculosis onlyin the radiographically involved lung seg-ments, and the levels returned to normalafter 1 month of treatment. The SP-A levelswere inversely correlated with the percent-age of neutrophils in BAL fluid, suggestingthat low SP-A levels were associated withincreased inflammation in the lung. Differ-entiated THP-1 macrophages were used totest the effect of decreasing SP-A levels onimmune function. In the absence of infec-tion with Mycobacterium tuberculosis, SP-Aat doses ranging from 5 to 0.01 micro g/ml

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inhibited both interleukin-6 (IL-6) produc-tion and HIV-1 long terminal repeat (LTR)activity. In macrophages infected with M.tuberculosis, SP-A augmented both IL-6production and HIV-1 LTR activity. To bet-ter understand the effect of SP-A, we mea-sured expression of CAAT/enhancer bindingprotein beta (C/EBPbeta), a transcriptionfactor central to the regulation of IL-6 andthe HIV-1 LTR. In macrophages infectedwith M. tuberculosis, SP-A reduced expres-sion of a dominant negative isoform ofC/EBPbeta. These data suggest that SP-Ahas pleiotropic effects even at the low con-centrations found in tuberculosis patients.This protein augments inflammation in thepresence of infection and inhibits inflam-mation in uninfected macrophages, protect-ing uninvolved lung segments from thedeleterious effects of inflammation.—Au-thors’Abstract

Goletti, D., Carrara, S., Vincenti, D., Gia-comini, E., Fattorini, L., Garbuglia, A.R., Capobianchi, M. R., Alonzi, T.,Fimia, G. M., Federico, M., Poli, G.,and Coccia, E. Inhibition of HIV-1 repli-cation in monocyte-derived macrophagesby Mycobacterium tuberculosis. J. Infect.Dis. 189(4) (2004) 624–633.

Controversial results have been obtainedin studies of the effect of Mycobacterium tu-berculosis on human immunodeficiencyvirus type 1 (HIV-1) replication in cells ofthe macrophage lineage. In the present study,monocyte-derived macrophages (MDMs),previously incubated for 2 days with heat-inactivated M. tuberculosis, were infectedwith HIV-1. M. tuberculosis consistently in-hibited viral replication, and a similar resultalso was observed in the presence of super-natants from M. tuberculosis-stimulatedMDMs, which indicates that this effect wasmediated by soluble factors. AlthoughCCR5-binding chemokines were induced byM. tuberculosis stimulation, the results ofneutralization experiments indicated that itis unlikely that they were responsible forviral suppression. Inhibition occurredmainly after viral entry (demonstrated byuse of a vesicular stomatitis virus G-pseudo-typed HIV-1 and by analysis of HIV-1 earlyand late reverse-transcription products).

Therefore, M. tuberculosis-induced factorsmay inhibit in vitro HIV-1 replication inmacrophages by affecting an early postentrystep in the HIV-1 cycle.—Authors’Abstract

Guinn, K. M., Hickey, M. J., Mathur, S.K., Zakel, K. L., Grotzke, J. E., Lewin-sohn, D. M., Smith, S., and Sherman,D. R. Individual RD1-region genes arerequired for export of ESAT-6/CFP-10and for virulence of Mycobacterium tu-berculosis. Mol. Microbiol. 51(2) (2004)359–370.

The RD1 genomic region is present in vir-ulent strains of Mycobacterium tuberculosis(MTB), missing from the vaccine strain M.bovis BCG, and its importance to virulencehas been established experimentally. Basedon in silico analysis, it has been suggestedthat RD1 may encode a novel secretion sys-tem, but the mechanism by which this re-gion affects virulence is unknown. Here weexamined mutants disrupted in five indi-vidual RD1 genes. Both in vitro and in vivo,each mutant displayed an attenuated pheno-type very similar to a mutant missing the en-tire RD1 region. Genetic complementationof individual genes restored virulence. At-tenuated mutants could multiply withinTHP-1 cells, but they were unable to spreadto uninfected macrophages. We also exam-ined export of two immunodominant RD1proteins, CFP-10 and ESAT-6. Export ofthese proteins was greatly reduced or abol-ished in each attenuated mutant. Again, ge-netic complementation restored a wild-typephenotype. Our results indicate that RD1genes work together to form a single viru-lence determinant, and argue that RD1 en-codes a novel specialized secretion systemthat is required for pathogenesis of MTB.—Authors’Abstract

Harboe, M., Das, A. K., Mitra, D., Ul-vund, G., Ahmad, S., Harkness, R. E.,Das, D., Mustafa, A. S., and Wiker, H.G. Immunodominant B-cell epitope inthe Mce1A mammalian cell entry proteinof Mycobacterium tuberculosis cross-reacting with glutathione S-transferase.Scand. J. Immunol. 59(2) (2004) 190–197.

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The TB1-5 76C monoclonal antibodyraised against a synthetic 60-mer peptidein the N-terminal part of the Mce1A mam-malian cell entry protein of Mycobacte-rium tuberculosis has previously beenshown to react with a linear epitope in theKRRITPKD region, residues 131–138 inMce1A, and to cross-react with Mce1F. Sixadditional monoclonal antibodies raisedagainst the same peptide were also shown tocross-react with Mce1F. Four of them re-acted with a linear epitope in the same area,indicating that this area is immunodominantbut showed distinct differrences in finespecificity. Two monoclonal antibodies didnot react with synthetic peptides from thisregion on the solid phase in enzyme-linkedimmunosorbent assay, indicating greaterinfluence of conformation on reactivity.None of the monoclonal antibodies reactedwith 14-mer synthetic peptides from thecorresponding area in Mce2A, Mce3A,Mce4A, M. avium, M. smegmatis or M.leprae. The reaction pattern of the mono-clonal antibodies was analysed in relation toour model of the Mce1A molecule (AK Das,et al. Biochem Biophys Res Commun2003;302:442–7). The epitope is located onthe surface of Mce1A, at the distal beta-strand-loop region in the beta-domain sup-porting its potential role in promoting up-take of M. tuberculosis in host cells.Monoclonal antibody TB1-5 19C cross-reacted with glutathione S-transferase ofSchistosoma japonicum containing a PKEtriplet. Monoclonal antibody TB1-5 76Cgave a major band at about 44 kDa in West-ern blotting of M. tuberculosis sonicate,whereas polyclonal rabbit anti-Mce1A pep-tide antibodies reacting with the extendedTTPKNPTKRRITPKDVI area of Mce1Ashowed a distinct band above the 160 kDamolecular mass standard.—Authors’ Ab-stract

Higuchi, K., Sekiya, Y., and Harada, N.Characterization of M. Tuberculosis-derived IL-12-inducing material by al-veolar macrophages. Vaccine 22(5–6)(2004) 724–734.

We have investigated the substance de-rived from Mycobacterium tuberculosis(Mtb) that induces interleukin (IL)-12 pro-

duction by alveolar macrophages (AMs) invitro. The cytosol fraction of live MtbH37Rv induced IL-12 production by AMs ina dose-dependent manner. The addition ofinterferon-gamma (IFN-gamma) augmentedIL-12 production. IL-12-inducing activityby AMs (termed as surely active keepingrescue antigen, SAKRA) was purified by gelfiltration and ion exchange column chro-matography, and the molecular weight ofSAKRA was estimated by gel filtration to bemore than 700 kDa. SDS-polyacrylamidegel electrophoresis (PAGE) and Westernblotting of SAKRA using rabbit anti-SAKRA antibody suggested that SAKRA iscomposed with several low molecularweight proteins. Amino acids sequenceanalysis of several bands after SDS-PAGEsuggested that SAKRA is a part of ribo-somes. RT-PCR showed that SAKRA in-duced not only expression of IL-12 p40mRNA, but expression of tumor necrosisfactor (TNF)-alpha and inducible nitricoxide synthase (iNOS) mRNA at least 6 hrafter stimulation, suggesting that SAKRAactivates the bactericidal activity of macro-phages. To investigate the potential use ofSAKRA as a vaccine against tuberculosis,SAKRA was administered to BALB/cmouse that had been immunized with BCGfor 18 months, and mouse were infectedwith Mtb H37Rv via a respiratory route.Replication of Mtb in lungs and spleens wasexamined 6 weeks after infection. Admini-stration of SAKRA to BCG-vaccinated micesignificantly reduced the numbers of Mtb inlungs and spleens as compared with BCG-vaccinated control mice. Taken together,these results suggest that SAKRA is one ofthe Mtb-derived immunomodulatory sub-stances which induce IL-12 production dur-ing infection and also increases mycobacte-ricidal activities of macrophages, and thatSAKRA may be a promising new vaccinecandidate against tuberculosis.

Junqueira-Kipnis, A. P., Kipnis, A.,Jamieson, A., Juarrero, M. G., Diefen-bach, A., Raulet, D. H., Turner, J., andOrme, I. M. NK cells respond to pul-monary infection with Mycobacteriumtuberculosis, but play a minimal role inprotection. J. Immunol. 171(11) (2003)6039–6045.

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Both innate and adaptive immune systemscontribute to host defense against infectionwith Mycobacterium tuberculosis. NK cellshave been associated with early resistanceagainst intracellular pathogens and areknown to be potent producers of the cy-tokine IFN-gamma. In C57BL/6 mice in-fected by aerosol exposure with M. tubercu-losis, NK cells increased in the lungs overthe first 21 days of infection. Expansion ofthe NK cell subset was associated with in-creased expression of activation and matu-ration markers. In addition, NK cells iso-lated from the infected lungs were capableof producing IFN-gamma and became pos-itive for perforin. In vivo depletion of NKcells using a lytic Ab had no influence onbacterial load within the lungs. These find-ings indicate that NK cells can become acti-vated during the early response to pul-monary tuberculosis in the mouse modeland are a source of IFN-gamma, but their re-moval does not substantially alter the ex-pression of host resistance.—Authors’ Ab-stract

Kanaujia, G. V., Motzel, S., Garcia, M. A.,Andersen, P., and Gennaro, M. L.Recognition of ESAT-6 sequences by an-tibodies in sera of tuberculous nonhumanprimates. Clin. Diagn. Lab. Immunol.11(1) (2004) 222–226.

See Current Literature, Experimental In-fections, p. 235.

Kanaujia, G. V., Garcia, M. A., Bouley, D.M., Peters, R., and Gennaro, M. L.Detection of early secretory antigenictarget-6 antibody for diagnosis of tuber-culosis in non-human primates. Comp.Med. 53(6) (2003) 602–606.

See Current Literature, Experimental In-fections, p. 235.

Kaplan, G., Post, F. A., Moreira, A. L.,Wainwright, H., Kreiswirth, B. N.,Tanverdi, M., Mathema, B., Ra-maswamy, S. V., Walther, G., Steyn, L.M., Barry, C. E. 3rd, and Bekker, L. G.Mycobacterium tuberculosis growth at

the cavity surface: a microenvironmentwith failed immunity. Infect. Immun.71(12) (2003) 7099–7108.

Protective immunity against pulmonarytuberculosis (TB) is characterized by theformation in the lungs of granulomas con-sisting of macrophages and activated T cellsproducing tumor necrosis factor alpha andgamma interferon, both required for the ac-tivation of the phagocytes. In 90% of im-munocompetent humans, this response con-trols the infection. To understand whyimmunity fails in the other 10%, we studiedthe lungs of six patients who underwent sur-gery for incurable TB. Histologic examina-tion of different lung lesions revealed het-erogeneous morphology and distribution ofacid-fast bacilli; only at the surface of cavi-ties, i.e., in granulomas with a patent con-nection to the airways, were there numerousbacilli. The mutation profile of the isolatessuggested that a single founder strain of My-cobacterium tuberculosis may undergo ge-netic changes during treatment, leading toacquisition of additional drug resistance in-dependently in discrete physical locales. Ad-ditional drug resistance was preferentiallyobserved at the cavity surface. Cytokinegene expression revealed that failure to con-trol the bacilli was not associated with ageneralized suppression of cellular immu-nity, since cytokine mRNA was up regu-lated in all lesions tested. Rather, a selectiveabsence of CD4(+) and CD8(+) T cells wasnoted at the luminal surface of the cavity,preventing direct T-cell-macrophage inter-actions at this site, probably allowing lumi-nal phagocytes to remain permissive forbacillary growth. In contrast, in the peri-necrotic zone of the granulomas, the twocell types colocalized and bacillary num-bers were substantially lower, suggestingthat in this microenvironment an efficientbacteriostatic or bactericidal phagocytepopulation was generated.—Authors’ Ab-stract

Lazarevic, V., Myers, A. J., Scanga, C. A.,and Flynn, J. L. CD40, but not CD40L,is required for the optimal priming of Tcells and control of aerosol M. tuberculo-sis infection. Immunity. 19(6) (2003)823–835.

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CD40(–/–) mice succumbed to low-doseaerosol infection with M. tuberculosis due todeficient IL-12 production leading to im-paired priming of IFN-gamma T cell re-sponses. In contrast, CD40L(–/–) mice wereresistant to M. tuberculosis. This asymmetryin outcome of infection between the twoknockout strains is likely due to the exis-tence of an alternative ligand for CD40.Both in vitro M. tuberculosis infection andrecombinant M. tuberculosis Hsp70 elicitedIL-12 production from WT dendritic cells.This response was absent in both CD40(–/–)dendritic cells and CD40(–/–) mice, sug-gesting that M. tuberculosis Hsp70 serves asan alternative ligand for CD40 in vivo.—Authors’Abstract

Majlessi, L., Rojas, M. J., Brodin, P., andLeclerc, C. CD8+-T-cell responses ofMycobacterium-infected mice to a newlyidentified major histocompatibility com-plex class I-restricted epitope shared byproteins of the ESAT-6 family. Infect.Immun. 71(12) (2003) 7173–7177.

Here we describe the identification of a newCD8(+)-T-cell epitope, the GYAGTLQSLnonamer, shared by the TB10.3 and TB10.4proteins of the Mycobacterium tuberculosisESAT-6 family. Cytotoxic T cells frommycobacterium-infected mice efficientlyrecognized this epitope. GYAGTLQSL-specific T-cell hybridomas, which were ableto recognize Mycobacterium bovis BCG-infected macrophages, were generated andnow allow investigation of mycobacterial-antigen processing through the major histo-compatibility complex class I pathway.—Authors’Abstract

Malhotra, V., Sharma, D., Ramanathan,V. D., Shakila, H., Saini, D. K.,Chakravorty, S., Das, T. K., Li, Q., Sil-ver, R. F., Narayanan, P. R., Tyagi, J. S.Disruption of response regulator gene,devR, leads to attenuation in virulence ofMycobacterium tuberculosis. FEMS Mi-crobiol Lett. 20231(2) (2004) 237–245.

The devR-devS two-component system ofMycobacterium tuberculosis was identifiedearlier and partially characterized in our lab-

oratory. A devR::kan mutant of M. tubercu-losis was constructed by allelic exchange.The devR mutant strain showed reducedcell-to-cell adherence in comparison to theparental strain in laboratory culture media.This phenotype was reversed on comple-mentation with a wild-type copy of devR.The devR mutant and parental strains grewat equivalent rates within human monocyteseither in the absence or in the presence oflymphocytic cells. The expression of DevRwas not modulated upon entry of M. tuber-culosis into human monocytes. However,guinea pigs infected with the mutant strainshowed a significant decrease in gross le-sions in lung, liver and spleen; only mildpathological changes in liver and lung; anda nearly 3 log lower bacterial burden inspleen compared to guinea pigs infectedwith the parental strain. Our results suggestthat DevR is required for virulence in guineapigs but is not essential for entry, survivaland multiplication of M. tuberculosis withinhuman monocytes in vitro. The attenuationin virulence of the devR mutant in guineapigs together with DevR-DevS being a bonafide signal transduction system indicates thatDevR plays a critical and regulatory role inthe adaptation and survival of M. tuberculo-sis within tissues.—Authors’Abstract

Mattow, J., Schaible, U. E., Schmidt, F.,Hagens, K., Siejak, F., Brestrich, G.,Haeselbarth, G., Muller, E. C., Jung-blut, P. R., and Kaufmann, S. H.Comparative proteome analysis of culturesupernatant proteins from virulent Myco-bacterium tuberculosis H37Rv and atten-uated M. bovis BCG Copenhagen. Elec-trophoresis 24(19–20) (2004) 3405–3420.

A comprehensive analysis of culturesupernatant (CSN) proteins of Mycobacte-rium tuberculosis H37Rv was accomplishedby combination of two-dimensional elec-trophoresis (2-DE), mass spectrometry, andN-terminal sequencing by Edman degrada-tion. Analytical 2-DE gels resolved approx-imately 1250 protein spots from CSN of M.tuberculosis H37Rv, 381 of which wereidentified by mass spectrometry and/orEdman degradation. This study revealed 137different proteins, 42 of which had previ-ously been described as secreted. Compar-

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ative proteome analysis of CSN from viru-lent M. tuberculosis H37Rv and attenuatedMycobacterium bovis BCG Copenhagenidentified 39 M. tuberculosis-specific spotscontaining 27 different proteins, represent-ing candidate antigens for novel vaccinesand diagnostics in tuberculosis. These in-cluded five proteins encoded by open read-ing frames absent from M. bovis BCG, e.g.,early secretory antigen target (Esat6), aswell as 22 novel differential proteins, suchas acetyl-CoA C-acetyltransferase (Rv0243)and two putative Esat6-like proteins(Rv1198, Rv1793).—Authors’Abstract

McCarthy, A. A., Knijff, R., Peterson, N.A., and Baker, E. N. Crystallization andpreliminary X-ray analysis of N-acetyl-1-D-myo-inosityl-2-deoxy-alpha-D-glucopyranoside deacetylase (MshB)from Mycobacterium tuberculosis. Acta.Crystallogr. D. Biol. Crystallogr. 59(Pt12) (2003) 2316–2318.

Mycobacteria synthesize mycothiol (MSH)as a low-molecular-weight thiol that protectsagainst oxidative stress in a similar role tothat of glutathione in many other species.The absence of MSH in mammals suggeststhat enzymes from its biosynthetic path-way in Mycobacterium tuberculosis couldbe useful targets for drug design. The genefor MshB (Rv1170), the enzyme that catal-yses the second step in MSH biosynthesisin M. tuberculosis, has been cloned and theprotein has been expressed in Escherichiacoli both in native and SeMet-substitutedforms and crystallized in two crystalforms. One of these, prepared in the pres-ence of beta-octylglucoside as a key addi-tive, is suitable for high-resolution X-raystructural analysis. The crystals are or-thorhombic, with unit-cell parameters a =71.69, b = 83.74, c = 95.65 A, space groupP2(1)2(1)2(1) and two molecules in theasymmetric unit. X-ray diffraction data to1.9 A resolution have been collected.—Authors’ Abstract

Pasquinelli, V., Quiroga, M. F., Martinez,G. J., Zorrilla, L. C., Musella, R. M.,Bracco, M. M., Belmonte, L., Malbran,A., Fainboim, L., Sieling, P. A., and Gar-

cia, V. E. Expression of signaling lympho-cytic activation molecule-associated pro-tein interrupts IFN-gamma production inhuman tuberculosis. J. Immunol. 172(2)(2004) 1177–1185.

Production of the Th1 cytokine IFN-gamma by T cells is considered crucial forimmunity against Mycobacterium tubercu-losis infection. We evaluated IFN-gammaproduction in tuberculosis in the context ofsignaling molecules known to regulate Th1cytokines. Two populations of patients whohave active tuberculosis were identified,based on their T cell responses to the bacte-rium. High responder tuberculosis patientsdisplayed significant M. tuberculosis-dependent T cell proliferation and IFN-gamma production, whereas low respondertuberculosis patients displayed weak or no Tcell responses to M. tuberculosis. The ex-pression of the signaling lymphocytic acti-vation molecule (SLAM)-associated protein(SAP) on cells from tuberculosis patientswas inversely correlated with IFN-gammaproduction in those individuals. Moreover,patients with a nonfunctional SAP gene dis-played immune responses to M. tuberculo-sis similar to those of high responder tuber-culosis patients. In contrast to SAP, T cellexpression of SLAM was directly correlatedwith responsiveness to M. tuberculosis Ag.Our data suggest that expression of SAP in-terferes with Th1 responses whereas SLAMexpression contributes to Th1 cytokine re-sponses in tuberculosis. The study furthersuggests that SAP and SLAM might befocal points for therapeutic modulation of Tcell cytokine responses in tuberculosis.—Authors’Abstract

Pereira, C. B., Palaci, M., Leite, O. H.,Duarte, A. J., and Benard, G. Mono-cyte cytokine secretion in patients withpulmonary tuberculosis differs from thatof healthy infected subjects and corre-lates with clinical manifestations. Mi-crobes Infect. 6(1) (2004) 25–33.

Cell-mediated immunity, leading to My-cobacterium tuberculosis (Mtb)-constraininggranuloma formation, is the major compo-nent of host defense against tuberculosis andis regulated by the balance of cytokines se-

72, 2 Current Literature, Immuno Pathology (Tuberculosis) 215

creted mostly by mononuclear phagocytesand lymphocytes. To better understand therole of monocytes in the regulation of theimmune response against pulmonary tuber-culosis, we examined IL-10, IL-12 andTNF-alpha release by monocytes fromhealthy purified protein derivative (PPD) re-actors and pulmonary tuberculosis patientswith or without systemic reactions (e.g.,fever, weight loss, asthenia). Our studyshows that, probably as a result of in vivopriming by circulating antigens, monocytesfrom patients, especially those with sys-temic manifestations, have a biased ex vivocytokine secretion, with high IL-10 andTNF-alpha but low IL-12, in contrast withPPD reactors. Higher spontaneous IL-10and TNF-alpha release persisted whenmonocytes were co-cultured with autolo-gous lymphocytes. Challenge of patients’monocytes with a virulent Mtb strain led toa further enhancement of IL-10 and TNF-alpha, but not of IL-12. When lymphocyteswere added to these cultures, IL-10 andTNF-alpha elevation persisted and, in thepatients with a systemic reaction, both IL-12and IFN-gamma were significantly reducedcompared to PPD reactors. Intragroup com-parisons revealed that in the patients with sys-temic reactions, the lymphocyte-monocyteinteraction resulted in a positive feedbackfor IL-10 secretion, while in the patientswithout systemic reaction and PPD reactors,the feedback was positive for IL-12 secre-tion. Thus, in tuberculosis, there appears toexist a relationship between the immuno-logical findings and the distinct clinicalmanifestations.—Authors’Abstract

Qiao, Y., Prabhakar, S., Canova, A.,Hoshino, Y., Weiden, M., and Pine, R.Posttranscriptional inhibition of gene ex-pression by Mycobacterium tuberculosisoffsets transcriptional synergism withIFN-gamma and posttranscriptional up-regulation by IFN-gamma. J. Immunol.172(5) (2004) 2935–2943.

Host defense against Mycobacterium tu-berculosis requires the cytokine IFN-gamma and IFN regulatory factor 1 (IRF-1), a transcription factor that is induced tohigh levels by IFN-gamma. Therefore, wechose to study regulation of IRF-1 expres-

sion as a model for effects of M. tuberculo-sis on response to IFN-gamma. We foundthat IRF-1 mRNA abundance increased farmore than transcription rate in humanmonocytic THP-1 cells stimulated by IFN-gamma, but less than transcription rate incells infected by M. tuberculosis. IFN-gamma stimulation of infected cells causeda synergistic increase in IRF-1 transcrip-tion, yet IRF-1 mRNA abundance was sim-ilar in uninfected and infected cells stimu-lated by IFN-gamma, as was the IRF-1protein level. Comparable infection byMycobacterium bovis bacillus Calmette-Guerin failed to induce IRF-1 expressionand had no effect on the response to IFN-gamma. We also examined the kinetics oftranscription, the mRNA t(1/2), and the dis-tribution of IRF-1 transcripts among totalnuclear RNA, poly(A) nuclear RNA, andpoly(A) cytoplasmic RNA pools in cellsthat were infected by M. tuberculosis and/orstimulated by IFN-gamma. Our data sug-gest that infection by M. tuberculosis in-hibits RNA export from the nucleus. More-over, the results indicate that regulatedentry of nascent transcripts into the pool oftotal nuclear RNA affects IRF-1 expressionand that this process is stimulated by IFN-gamma and inhibited by M. tuberculosis.The ability of infection by M. tuberculosisto limit the increase in IRF-1 mRNA ex-pression that typically follows transcrip-tional synergism may contribute to thepathogenicity of M. tuberculosis.—Au-thors’ Abstract

Rousseau, C., Winter, N., Pivert, E., Bor-dat, Y., Neyrolles, O., Ave, P., Huerre,M., Gicquel, B., and Jackson, M. Pro-duction of phthiocerol dimycocerosatesprotects Mycobacterium tuberculosisfrom the cidal activity of reactive nitro-gen intermediates produced by macro-phages and modulates the early immuneresponse to infection. Cell Microbiol.6(3) (2004) 277–287.

The growth of Mycobacterium tubercu-losis mutants unable to synthesize phthio-cerol dimycocerosates (DIMs) was recentlyshown to be impaired in mouse lungs.However, the precise role of these mole-cules in the course of infection remained to

216 International Journal of Leprosy 2004

be determined. Here, we provide evidencethat the attenuation of a DIM-deficientstrain takes place during the acute phase ofinfection in both lungs and spleen of mice,and that this attenuation results in part fromthe increased sensitivity of the mutant tothe cidal activity of reactive nitrogen inter-mediates released by activated mac-rophages. We also show that the DIM-deficient mutant, the growth and survivalof which were not impaired within restingmacrophages and dendritic cells, inducedthese cells to secrete more tumour necrosisfactor (TNF)-alpha and interleukin (IL)-6than the wild-type strain. Although purifiedDIM molecules by themselves had no ef-fect on the activation of macrophages anddendritic cells in vitro, we found that theproper localization of DIMs in the cell en-velope of M. tuberculosis is critical to theirbiological effects. Thus, our findings sug-gest that DIM production contributes to theinitial growth of M. tuberculosis by pro-tecting it from the nitric oxide-dependentkilling of macrophages and modulating theearly immune response to infection.—Au-thors’ Abstract

Shanmugalakshmi, S., Dheenadhayalan,V., Muthuveeralakshmi, P., Arivarig-nan, G., and Pitchappan, R. M. Myco-bacterium bovis BCG scar status andHLA class II alleles influence purifiedprotein derivative-specific T-cell receptorV? expression in pulmonary tuberculosispatients from Southern India. Infect. Im-munity 71(8) (2003) 4544–4553.

Purified protein derivative (PPD) RT23-recalled T-cell receptor (TCR) Vβ expres-sion was studied in the peripheral blood of42 pulmonary tuberculosis patients and 44healthy controls from southern India, a re-gion where tuberculosis is endemic. Forty-eight-hour whole-blood cultures in the pres-ence or absence of PPD-RT23 were set up,and at the end of the culture period totalRNA was extracted and cDNA was synthe-sized. Expression of various TCR Vβ fami-lies was assessed by using family-specificprimers. PPD-specific expression (usage) ofTCR Vβ families 4, 6, 8 to 12, and 14 wasfound in more controls than patients. Amongthe responders (individuals who showed

PPD-specific expression), endemic controlshad significantly higher responses than thepatients had for TCR Vβ families 2, 3, 7, 13,and 17. The majority of the patients did notshow usage of most of the TCR Vβ families,and this was attributed to T-cell downregu-lation. A four-way nested classificationanalysis revealed that TCR Vβ family 1, 5,9, 12, and 13 usage in the context of HLAclass II high-risk alleles) (DRB1*1501,DRB1*08, and DQB1*0601) and Mycobac-terium bovis BCG scar status were the de-termining factors in susceptibility and re-sistance to tuberculosis. The healthier statusof controls was attributed to the wider usageof many TCR Vβ families readily recalledby PPD, while the disease status of the pa-tients was attributed to TCR Vβ downreg-ulation and the resultant T-cell (memorycell?) unresponsiveness. Host genetics(HLA status) and BCG vaccination (scarstatus) seem to play important roles in skew-ing the immune response in adult suscepti-bility to pulmonary tuberculosis throughTCR Vβ usage.—Tropical Disease Bulletin

Stanton, L. A., Fenhalls, G., Lucas, A.,Gough, P., Greaves, D. R., Mahoney, J.A., Helden, P., and Gordon, S. Im-munophenotyping of macrophages inhuman pulmonary tuberculosis and sar-coidosis. Int. J. Exp. Pathol. 84(6) (2003)289–304.

Classic studies of tuberculosis (TB) re-vealed morphologic evidence of consider-able heterogeneity of macrophages (MOs),but the functional significance of this het-erogeneity remains unknown. We have usednewly available specific antibodies for se-lected membrane and secretory molecules toexamine the phenotype of MOs in situ in arange of South African patients with TB,compared with sarcoidosis. Patients werehuman immunodeficiency virus-negativeadults and children, and the examinedbiopsy specimens included lung and lymphnodes. Mature pulmonary MOs (alveolar,interstitial, epithelioid and multinucleatedgiant cells) selectively expressed scavengerreceptor type Aand a novel carboxypeptidase-like antigen called carboxypeptidase-relatedvitellogenin-like MO molecule (CPVL).CPVL did not display enhanced expression

72, 2 Current Literature, Immuno Pathology (Tuberculosis) 217

in sarcoidosis, vs. TB patients, as observedwith angiotensin-converting enzyme (ACE),a related molecule. Immunocytochemicalstudies with surfactant proteins (SP)-A and-D showed that type II alveolar cells ex-pressed these collectins, as did MOs, possi-bly after binding of secreted proteins. Stud-ies with an antibody specific for theC-terminus of fractalkine, a tethered CX3Cchemokine, confirmed synthesis of this mol-ecule by bronchiolar epithelial cells and oc-casional endothelial cells. These studies pro-vide new marker antigens and extendprevious studies on MO differentiation, ac-tivation and local interactions in chronichuman granulomatous inflammation in thelung.—Authors’Abstract

Turner, J., and Orme, I. M. The expressionof early resistance to an infection withMycobacterium tuberculosis by old miceis dependent on IFN type II (IFN-gamma) but not IFN type I. Mech. Age-ing Dev. 125(1) (2004) 1–9.

Old mice can express a transient early re-sistance to infection with M. tuberculosisthat requires the presence of CD8 T cellswithin the lungs. Further characterization ofthose CD8 T cells within the aged lung es-tablished that the majority of CD8 T cellsfrom old mice expressed the IL-15 receptor(CD122) in combination with bright ex-pression of CD44 (CD44(hi)), and were ca-pable of producing IFN-gamma after T cellreceptor cross-linking. It has been previ-ously described that CD8 CD44(hi) T cellsproliferate in response to IFN-I, acting viaIL-15, and therefore we determined whetherIFN-I signaling could be a participant in theresponse of CD8 T cells within the lungs ofold mice infected with M. tuberculosis. Wedemonstrate here that IFN-I signaling wasrequired for the expansion of CD8 T cellswithin the aging lung in response to infec-tion with M. tuberculosis, but that IFN-I sig-naling had no influence on the capacity ofold mice to express early resistance to an in-fection with M. tuberculosis. Resident CD8T cells were still however capable of pro-ducing IFN-gamma, which we demonstratehere to be critical in the expression of earlyresistance, suggesting that the expression ofearly resistance requires the participation,

but not expansion, of the CD8 T cell poolwithin the aging lung.—Authors’Abstract

Vergne, I., Fratti, R. A., Hill, P. J., Chua,J., Belisle, J., and Deretic, V. Mycobac-terium tuberculosis phagosome maturationarrest: mycobacterial phosphatidylinositolanalog phosphatidylinositol mannosidestimulates early endosomal fusion. Mol.Biol. Cell. 15(2) (2004) 751–760.

Mycobacterium tuberculosis is a faculta-tive intracellular pathogen that parasitizesmacrophages by modulating properties ofthe Mycobacterium-containing phagosome.Mycobacterial phagosomes do not fuse withlate endosomal/lysosomal organelles but re-tain access to early endosomal contents byan unknown mechanism. We have previ-ously reported that mycobacterial phos-phatidylinositol analog lipoarabinomannan(LAM) blocks a trans-Golgi network-to-phagosome phosphatidylinositol 3-kinase-dependent pathway. In this work, we extendour investigations of the effects of myco-bacterial phosphoinositides on host mem-brane trafficking. We present data demon-strating that phosphatidylinositol mannoside(PIM) specifically stimulated homotypic fu-sion of early endosomes in an ATP-, cytosol-,and N-ethylmaleimide sensitive factor-dependent manner. The fusion showedabsolute requirement for small Rab GT-Pases, and the stimulatory effect of PIM in-creased upon partial depletion of membraneRabs with RabGDI. We found that stimula-tion of early endosomal fusion by PIM washigher when phosphatidylinositol 3-kinasewas inhibited by wortmannin. PIM alsostimulated in vitro fusion between modelphagosomes and early endosomes. Finally,PIM displayed in vivo effects in macro-phages by increasing accumulation ofplasma membrane-endosomal syntaxin 4and transferrin receptor on PIM-coated latexbead phagosomes. In addition, inhibition ofphagosomal acidification was detected withPIM-coated beads. The effects of PIM,along with the previously reported action ofLAM, suggest that M. tuberculosis hasevolved a two-prong strategy to modify itsintracellular niche: its products block acqui-sition of late endosomal/lysosomal con-stituents, while facilitating fusion with early

218 International Journal of Leprosy 2004

endosomal compartments.—Authors’ Ab-stract

Wang, J. P., Rought, S. E., Corbeil, J.,and Guiney, D. G. Gene expression pro-filing detects patterns of human macro-

phage responses following Mycobacte-rium tuberculosis infection. FEMS Im-munol. Med. Microbiol. 39(2) (2003)163–172.

See Current Literature, Molecular andGenetic Studies, p. 262.

72, 2 Current Literature, Microbiology 219

MicrobiologyAdekambi, T., Colson, P., and Drancourt,

M. rpoB-based identification of nonpig-mented and late-pigmenting rapidlygrowing mycobacteria. J. Clin. Micro-biol. 41(12) 2003 5699–5708.

Nonpigmented and late-pigmenting rap-idly growing mycobacteria (RGM) are in-creasingly isolated in clinical microbiologylaboratories. Their accurate identification re-mains problematic because classification islabor intensive work and because new taxaare not often incorporated into classificationdatabases. Also, 16S rRNA gene sequenceanalysis underestimates RGM diversity anddoes not distinguish between all taxa. We de-termined the complete nucleotide sequenceof the rpoB gene, which encodes the bacterialbeta subunit of the RNA polymerase, for 20RGM type strains. After using in-house soft-ware which analyzes and graphically repre-sents variability stretches of 60 bp along thenucleotide sequence, our analysis focused ona 723-bp variable region exhibiting 83.9 to97% interspecies similarity and 0 to 1.7% in-traspecific divergence. Primer pair Myco-F-Myco-R was designed as a tool for both PCRamplification and sequencing of this regionfor molecular identification of RGM. Thistool was used for identification of 63 RGMclinical isolates previously identified at thespecies level on the basis of phenotypic char-acteristics and by 16S rRNA gene sequenceanalysis. Of 63 clinical isolates, 59 (94%) ex-hibited <2% partial rpoB gene sequence di-vergence from 1 of 20 species under studyand were regarded as correctly identified atthe species level. Mycobacterium abscessusand Mycobacterium mucogenicum isolateswere clearly distinguished from Mycobacte-rium chelonae; Mycobacterium mageritenseisolates were clearly distinguished from “My-cobacterium houstonense.” Four isolateswere not identified at the species level be-cause they exhibited >3% partial rpoB gene

sequence divergence from the correspondingtype strain; they belonged to three taxa re-lated to M. mucogenicum, Mycobacteriumsmegmatis, and Mycobacterium porcinum.For M. abscessus and M. mucogenicum, thispartial sequence yielded a high genetic het-erogeneity within the clinical isolates. Weconclude that molecular identification byanalysis of the 723-bp rpoB sequence is arapid and accurate tool for identification ofRGM.—Authors’Abstract

Chattopadhyay, C., Sau, S., and Mandal,N. C. Cloning and characterization of thepromoters of temperate mycobacterio-phage L1. J. Biochem. Mol. Biol. 36(6)(2003) 586–592.

See Current Literature, Molecular andGenetic Studies, p. 256.

Chui, L. W., King, R., Lu, P., Manninen,K., and Sim, J. Evaluation of four DNAextraction methods for the detection of My-cobacterium avium subsp. paratuberculo-sis by polymerase chain reaction. Diagn.Microbiol. Infect. Dis. 48(1) (2004) 39–45.

Polymerase chain reaction (PCR) hasbeen widely used due to its high specificity,sensitivity, and rapid turn-around time.However, inhibitory factors may be co-extracted with the target nucleic acid thatwill hinder the performance of PCR. In thisstudy, DNA extraction methods for Myco-bacterium avium subsp. paratuberculosiswere evaluated including rapid lysis, or-ganic extraction, silica-based and magneticparticle-based (MagaZorb) technologies onbacterial cells, and spiked bovine feces. Ef-ficiency of the extraction was determined byPCR end point titration with primers target-ing the insertion sequence, IS900. Results of

the end point titrations are identical for bac-terial cells and spiked feces. Inhibition wasobserved in PCR with DNA isolated fromspiked feces, and a 1/100 dilution was ableto alleviate this problem with DNA ex-tracted by MagaZorb. A 1/1000 dilution wasrequired for the other three methods. Mag-aZorb proved to be more efficient at remov-ing inhibitory factors and required the leastlabor and completion time. Further evalua-tion is required for its utilization in otherclinical specimens.—Authors’Abstract

Cociorva, O. M., and Lowary, T. L.Synthesis of oligosaccharides as potentialinhibitors of mycobacterial arabinosyl-transferases. Di- and trisaccharides con-taining C-5 modified arabinofuranosylresidues. Carbohydr. Res. 339(4) (2004)853–865.

The synthesis of a panel of oligosac-charides containing C-5 arabinofuranosylresidues (9–20) is described. These com-pounds are of interest as potential inhibitorsof the alpha-(1→5)-arabinosyltransferaseinvolved in the assembly of mycobacterialcell-wall arabinan. In the series of com-pounds prepared, the 5-OH group on thenonreducing residue(s) is replaced, inde-pendently, with an amino, azido, fluoro, ormethoxy functionality. The synthesis of thetarget compounds involved the preparationof a series of C-5 modified arabinofuranosylthioglycosides (24–26) and their subsequentcoupling to the appropriate acceptor species(21–23). Deprotection of the glycosylationproducts afforded the azido, fluoro, ormethoxy analogs directly. The amino deriv-atives were obtained in one additional stepby reduction of the azido compounds.—Au-thors’Abstract

Daniel, A. K., Lee, R. E., Portaels, F., andSmall, P. L. Analysis of Mycobacteriumspecies for the presence of a macrolidetoxin, mycolactone. Infect. Immun. 72(1)(2004) 123–132.

Mycobacterium ulcerans is an environ-mental organism which is responsible forthe disease Buruli ulcer, a necrotizing skindisease emerging in west Africa. M. ul-

cerans produces the polyketide-derivedmacrolide mycolactone, which is requiredfor the immunosuppression and tissue dam-age which characterizes Buruli ulcer. Wehave extracted lipids from the cell envelopeand culture filtrate from 52 isolates of My-cobacterium species, analyzed them withthin-layer chromatography, and tested themin a murine fibroblast cell line (L929) cyto-toxicity assay to investigate whether thesemycobacterial species produce mycolac-tone. For these studies chloroform-methanol(2:1, vol/vol) extracts were prepared fromrepresentative fast- and slow-growing my-cobacterial species. Isolates tested included16 uncharacterized, slow-growing, environ-mental mycobacterial species isolated fromareas in which M. ulcerans infection is en-demic. Although several strains of myco-bacteria studied produced cytopathic lipids,none of these produced a phenotype on cul-tured cells consistent with that produced bymycolactone. Two mycobacterial species,M. scrofulaceum and M. kansasii, and eightof the environmental mycobacterial isolatescontained cell-associated lipids cytopathicto fibroblasts at concentrations of 33 to 1000microg/ml. In contrast, mycolactone pro-duces cytotoxicity at less than 2 ng/ml.Analysis of 16S rRNA sequences from theeight environmental isolates suggests thatthese are novel mycobacterial species. Re-sults from these studies suggest that, al-though production of cytopathic lipids isrelatively common among mycobacterialspecies, the production of mycolactone as acell-associated or secreted molecule appearsso far to be restricted to M. ulcerans.—Au-thor’s Abstract

Ergin, A., and Hascelik, G. Non-tuberculousmycobacteria (NTM) in patients withunderlying diseases: results obtained byusing polymerase chain reaction-restriction enzyme analysis between1997–2002. New Microbiol. 27(1) (2004)49–53.

In this study, we aimed to evaluate the fre-quency of non-tuberculous mycobacteria(NTM) isolated from clinical specimensusing Polymerase Chain Reaction-RestrictionEnzyme Analysis (PCR-REA) and to inves-tigate the patients who had clinically signif-

220 International Journal of Leprosy 2004

icant NTM infections in our hospitalthrough the five year period from May1997 to June 2002. A total of 364 myco-bacterial strains isolated from clinical spec-imens which gave positive growth index inthe BACTEC 460 radiometric system inHacettepe University Hospital Clinical Mi-crobiology Laboratory were evaluated byPCR-REA and clinical data were obtainedfrom the patient records. Three hundredand one of the strains (82.7%) were iden-tified as Mycobacterium tuberculosis and63 (17.3%) were identified as nontubercu-lous mycobacteria. Seven (11.1%) of 63NTM patients were regarded as havingclinical mycobacteriosis. Chronic obstruc-tive pulmonary disease and other pre-existing lung diseases were seen in 39(61.9%) of the patients, 11 (17.5%) of thepatients had chronic renal failure. Four(6.3%) and 9 (14.3%) of them had AIDSand carcinomas, respectively. PCR-REAwas found to be a reliable method for typ-ing of our mycobacterial isolates to thespecies level. These data may shed light onthe epidemiology of the mycobacterialspecies and help to select a proper treat-ment regimen.—Authors’ Abstract

Harmsen, D., Dostal, S., Roth, A., Nie-mann, S., Rothganger, J., Sammeth,M., Albert, J., Frosch, M., and Richter,E. RIDOM: Comprehensive and publicsequence database for identification ofMycobacterium species. BMC Infect.Dis. 3(1) (2003) 26.

BACKGROUND: Molecular identifica-tion of Mycobacterium species has two pri-mary advantages when compared to pheno-typic identification: rapid turn-around timeand improved accuracy. The informationcontent of the 5′ end of the 16S ribosomalRNA gene (16S rDNA) is sufficient foridentification of most bacterial species.However, reliable sequence-based identifi-cation is hampered by many faulty and somemissing sequence entries in publicly acces-sible databases. METHODS: In order to es-tablish an improved 16S rDNA sequencedatabase for the identification of clinical andenvironmental isolates, we sequenced bothstrands of the 5′ end of 16S rDNA (Esche-richia coli positions 54 to 510) from 199

mycobacterial culture collection isolates. Allvalidly described species (n = 89; up toMarch 21, 2000) and nearly all published se-quevar variants were included. If the 16SrDNA sequences were not discriminatory,the internal transcribed spacer (ITS) regionsequences (n = 84) were also determined.RESULTS: Using 5′-16S rDNA sequencinga total of 64 different mycobacterial species(71.9%) could be identified. With the addi-tional input of the ITS sequence, a further 16species or subspecies could be differenti-ated. Only Mycobacterium tuberculosiscomplex species, M. marinum/M. ulceransand the M. avium subspecies could not bedifferentiated using 5′-16S rDNA or ITS se-quencing. A total of 77 culture collectionstrain sequences, exhibiting an overlap of atleast 80% and identical by strain number tothe isolates used in this study, were found inthe GenBank. Comparing these with our se-quences revealed that an average of 4.31 nu-cleotide differences (S.D. ± 0.57) were pres-ent. CONCLUSIONS: The data from thisanalysis show that it is possible to differenti-ate most mycobacterial species by sequenceanalysis of partial 16S rDNA. The high-qual-ity sequences reported here, together withancillary information (e.g., taxonomic, med-ical), are available in a public database,which is currently being expanded in theRIDOM project http://www.ridom-rdna.de),for similarity searches.—Authors’Abstract

Hirano, K., Aono, A., Takahashi, M., andAbe, C. Mutations including IS6110 in-sertion in the gene encoding the MPB64protein of Capilia TB-negative Mycobac-terium tuberculosis isolates. J. Clin. Mi-crobiol. 42(1) (2004) 390–392.

A simple immunochromatographic assay,Capilia TB, using anti-MPB64 monoclonalantibodies, is a kit for discriminating be-tween the Mycobacterium tuberculosis com-plex and mycobacteria other than tuberclebacilli. The sensitivity of the kit was esti-mated to be 99.2% (381 of 384 samples).The sequencing analysis revealed that all ofthe Capilia TB-negative isolates had muta-tions within the mpb64 gene, leading to theproduction of an incomplete protein as a re-sult of a deletion of the C-terminal region ofthe protein.—Authors’Abstract

72, 2 Current Literature, Microbiology 221

Jenkin, G. A., Stinear, T. P., Johnson, P.D., and Davies, J. K. Subtractive hy-bridization reveals a type I polyketidesynthase locus specific to Mycobacteriumulcerans. J. Bacteriol. 185(23) (2003)6870–6882.

Mycobacterium ulcerans causes Buruliulcer, the third most prevalent mycobacte-rial infection of immunocompetent humansafter tuberculosis and leprosy. Recent workhas shown that the production by M. ulcer-ans of mycolactone, a novel polyketide,may partly explain the pathogenesis of Bu-ruli ulcer. To search for the genetic basis ofvirulence in M. ulcerans, we took advantageof the close genetic relationship between M.ulcerans and Mycobacterium marinum byperforming genomic suppressive subtractivehybridization of M. ulcerans with M. mar-inum. We identified several DNA fragmentsspecific to M. ulcerans, in particular, a typeI polyketide synthase locus with a highlyrepetitive modular arrangement. We postu-late that this locus is responsible for the syn-thesis of mycolactone in M. ulcerans.—Au-thors’Abstract

Marsollier, L., Stinear, T., Aubry, J.,Saint Andre, J. P., Robert, R., Legras,P., Manceau, A. L., Audrain, C., Bour-don, S., Kouakou, H., and Carbon-nelle, B. Aquatic plants stimulate thegrowth of and biofilm formation by My-cobacterium ulcerans in axenic cultureand harbor these bacteria in the environ-ment. Appl. Environ. Microbiol. 70(2)(2004) 1097–1103.

Mycobacterium ulcerans is the causativeagent of Buruli ulcer, one of the most com-mon mycobacterial diseases of humans.Recent studies have implicated aquaticinsects in the transmission of this pathogen,but the contributions of other elements ofthe environment remain largely unknown.We report here that crude extracts from twogreen algae added to the BACTEC 7H12Bculture medium halved the doubling timeof M. ulcerans and promoted biofilmformation. Using the 7H12B medium,modified by the addition of the algal ex-tract, and immunomagnetic separation, wealso demonstrate that M. ulcerans is as-

sociated with aquatic plants in an area ofthe Ivory Coast where Buruli ulcer is en-demic. Genotype analysis showed thatplant-associated M. ulcerans had the sameprofile as isolates recovered in the same re-gion from both aquatic insects and clinicalspecimens. These observations implicateaquatic plants as a reservoir of M. ulceransand add a new potential link in the chain oftransmission of M. ulcerans to humans.—Authors’ Abstract

Morita, Y. S., Patterson, J. H., Billman-Ja-cobe, H., and McConville, M. J. Biosyn-thesis of mycobacterial phosphatidylinos-itol mannosides. Biochem. J. 378(Pt 2)(2004) 589–597.

All mycobacterial species, includingpathogenic Mycobacterium tuberculosis,synthesize an abundant class of phos-phatidylinositol mannosides (PIMs) that areessential for normal growth and viability.These glycolipids are important cell-walland/or plasma-membrane components intheir own right and can also be hyperglyco-sylated to form other wall components, suchas lipomannan and lipoarabinomannan. Wehave investigated the steps involved in thebiosynthesis of the major PIM species in anew M. smegmatis cell-free system. A num-ber of apolar and polar PIM intermediateswere labelled when this system was contin-uously labelled or pulse-chase-labelled withGDP-[3H]Man, and the glycan head groupsand the acylation states of these specieswere determined by chemical and enzymictreatments and octyl-Sepharose chromatog-raphy respectively. These analyses showedthat (1) the major apolar PIM species, acyl-PIM2, can be synthesized by at least twopathways that differ in the timing of the firstacylation step, (2) early PIM intermediatescontaining a single mannose residue can bemodified with two fatty acid residues, (3)formation of polar PIM species from acyl-PIM2 is amphomycin-sensitive, indicatingthat polyprenol phosphate-Man, rather thanGDP-Man, is the donor for these reactions,(4) modification of acylated PIM4 withalpha1-2- or alpha1-6-linked mannoseresidues is probably the branch point in thebiosyntheses of polar PIM and lipoarabino-mannan respectively and (5) GDP strongly

222 International Journal of Leprosy 2004

inhibits the synthesis of early PIM interme-diates and increases the turnover of poly-prenol phosphate-Man. These findings areincorporated into a revised pathway for my-cobacterial PIM biosynthesis.—Authors’Abstract

Pina-Vaz, C., Costa-Oliveira, S., Rod-rigues, A. G., and Salvador, A. Novelmethod using a laser scanning cytometerfor detection of mycobacteria in clinicalsamples. J. Clin. Microbiol. 42(2) (2004)906–908.

In order to evaluate the capacity of laserscanning cytometry (LSC) to detect acid-fast bacilli directly on clinical samples, acomparison between Kinyoun-stainedsmears analyzed under light microscopy andpropidium iodide-auramine-stained smearsanalyzed by LSC was performed. The re-sults were compared with those for cultureon BACTEC MGIT 960. LSC is a new, re-liable methodology to detect MYCOBAC-TERIA.—Authors’Abstract

Portevin, D., De Sousa-D’Auria, C.,Houssin, C., Grimaldi, C., Chami, M.,Daffe, M., and Guilhot, C. A polyketidesynthase catalyzes the last condensationstep of mycolic acid biosynthesis in myco-bacteria and related organisms. Proc. Natl.Acad. Sci. U.S.A. 101(1) (2004) 314–319.

Mycolic acids are major and specific con-stituents of the cell envelope of Corynebac-terineae, a suborder of bacterial species in-cluding several important human pathogenssuch as Mycobacterium tuberculosis, Myco-bacterium leprae, or Corynebacterium diph-theriae. These long-chain fatty acids are in-volved in the unusual architecture andimpermeability of the cell envelope of thesebacteria. The condensase, the enzyme re-sponsible for the final condensation step inmycolic acid biosynthesis, has remained anenigma for decades. By in silico analysis ofvarious mycobacterial genomes, we identi-fied a candidate enzyme, Pks13, that con-tains the four catalytic domains required forthe condensation reaction. Orthologs of thisenzyme were found in other Corynebacter-ineae species. A Corynebacterium glutami-

cum strain with a deletion in the pks13 genewas shown to be deficient in mycolic acidproduction whereas it was able to producethe fatty acids precursors. This mutant straindisplayed an altered cell envelope structure.We showed that the pks13 gene was essen-tial for the survival of Mycobacterium smeg-matis. A conditional M. smegmatis mutantcarrying its only copy of pks13 on a ther-mosensitive plasmid exhibited mycolic acidbiosynthesis defect if grown at nonpermis-sive temperature. These results indicate thatPks13 is the condensase, a promising targetfor the development of new antimicrobialdrugs against Corynebacterineae.—Au-thors’Abstract

Rindi, L., Bonanni, D., Lari, N., andGarzelli, C. Most human isolates of My-cobacterium avium Mav-A and Mav-Bare strong producers of hemolysin, a pu-tative virulence factor. J. Clin. Microbiol.41(12) (2003) 5738–5740.

Hemolysin was quantified in 58 isolates ofMycobacterium avium from human, animal,and environmental sources. Human Mav-Aand Mav-B isolates were the strongest pro-ducers; in contrast, animal and environmen-tal Mav-A isolates and human, animal, andenvironmental Mav-C organisms were low-level producers. Hemolysin production wasnot restricted to isolates causing invasive in-fections.—Authors’Abstract

Song, T., Dove, S. L., Lee, K. H., and Hus-son, R. N. RshA, an anti-sigma factor thatregulates the activity of the mycobacterialstress response sigma factor SigH. Mol.Microbiol. 50(3) (2003) 949–959.

See Current Literature, Molecular andGenetic Studies, p. 261.

Tortoli, E. Mycobacterium kansasii, speciesor complex? Biomolecular and epidemio-logical insights. Kekkaku. 78(11) (2003)705–709.

Mycobacterium kansasii is one of the bestknown nontuberculous mycobacteria andlarge awareness exists about its involvement

72, 2 Current Literature, Microbiology 223

in diseases both of immunocompetent andimmunocompromised patients. Two pheno-typic variants within this species, which dif-fer for the virulence in guinea pig too, havebeen detected since 1962. It was however fol-lowing recent progress in genetic studies thata large variability emerged. Major contribu-tions to the disclosure of such findings camefrom the DNA probes hybridization, the nu-cleotide sequencing of 16 rDNA and internaltranscribed spacer (ITS), and from the analy-ses of repetitive DNA sequences polymor-phism. At present five subtypes of M. kansasiiare recognized, defined by the ITS sequenceand by the polymorphism revealed by differ-ent restriction enzyme technologies. Suchvariants differ from the epidemiological pointof view too, with type i being isolated fromhumans, type ii both from humans and envi-ronment, and types iii, iv and v, from the en-vironment only. A revision of the present tax-onomic status of M. kansasii and its splittinginto different species or subspecies seemsnowadays necessary. –Authors Abstract

Wade, M. M., and Zhang, Y. Mechanisms ofdrug resistance in Mycobacterium tuber-culosis. Front Biosci. 9 (2004) 975–994.

Tuberculosis is a worldwide health prob-lem posing increasing threat with the spreadof HIV infection and drug resistant Myco-bacterium tuberculosis strains. Conse-quently, control of this disease has becomea significant challenge despite the availabil-ity of chemotherapy and BCG vaccine. Drugresistance for all first-line anti-tuberculosisagents and some second-line agents has beenobserved. Moreover, the occurrence ofstrains of M. tuberculosis resistant to multi-ple anti-tuberculosis drugs is increasing.Mechanisms of action and resistance ofmajor anti-tuberculosis drugs are reviewed.In addition, the phenotypic drug resistance

such as dormant or persistent tubercle bacilliand its importance are also emphasized. Inorder to combat the threat of drug resistanttuberculosis and to more effectively controlthe disease, an understanding of the mech-anisms underlying drug resistance is neces-sary. This knowledge could be used for thedevelopment of molecular tests for rapid de-tection of drug resistant bacilli and futureanti-tuberculosis drugs.—Authors’Abstract

Xu, Z. Q., Barrow, W. W., Suling, W. J.,Westbrook, L., Barrow, E., Lin, Y. M.,and Flavin, M. T. Anti-HIV natural prod-uct (+)-calanolide A is active against bothdrug-susceptible and drug-resistant strainsof Mycobacterium tuberculosis. Bioorg.Med. Chem. 12(5) (2004) 1199–1207.

Naturally occurring anti-HIV-1 agent (+)-calanolide A was found to be activeagainst all of the strains of Mycobacteriumtuberculosis tested, including those resist-ant to the standard antitubercular drugs. Ef-ficacy evaluations in macrophages revealedthat (+)-calanolide A significantly inhibitedintracellular replication of M. tuberculosisH37Rv at concentrations below the MICobserved in vitro. Preliminary mechanisticstudies indicated that (+)-calanolide A rap-idly inhibits RNA and DNA synthesis fol-lowed by an inhibition of protein synthesis.Compared with known inhibitors, this sce-nario is more similar to effects observedwith rifampin, an inhibitor of RNA syn-thesis. Since (+)-calanolide A was activeagainst a rifampin-resistant strain, it is be-lieved that these two agents may involve dif-ferent targets. (+)-Calanolide A and its re-lated pyranocoumarins are the first class ofcompounds identified to possess antimy-cobacterial and antiretroviral activities, rep-resenting a new pharmacophore for anti-TBactivity.—Authors’Abstract

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Microbiology (Leprosy)

Matsuoka, M., Zhang, L., Budiawan, T.,Saeki, K., and Izumi, S. Genotyping ofMycobacterium leprae on the basis of thepolymorphism of TTC repeats for analy-sis of leprosy transmission. J. Clin. Mi-crobiol. 42(2) (2004) 741–745.

The polymorphism of TTC repeats in My-cobacterium leprae was examined using thebacilli obtained from residents in villages atNorth Maluku where M. leprae infectionsare highly endemic (as well as from patientsat North Sulawesi of Indonesia) to elucidate

the possible mode of leprosy transmission.TTC genotypes are stable for several gener-ations of passages in nude mice footpadsand, hence, are feasible for the genotypingof isolates and epidemiological analysis ofleprosy transmission. It was found thatbacilli with different TTC genotypes weredistributed among residents at the samedwelling in villages in which leprosy is en-demic and that some household contactsharbored bacilli with a different genotypefrom that harbored by the patient. Investi-gations of a father-and-son pair of patientsindicated that infections of bacilli with 10and 18 copies, respectively, had occurred.

Genotypes of TTC repeats were found todiffer between a son under treatment andtwo brothers. These results reveal the pos-sibility that in addition to exposure via thepresence of a leprosy patient with a multi-bacillary infection who was living withfamily members, there might have beensome infectious sources to which the resi-dents had been commonly exposed outsidethe dwellings. A limited discriminative ca-pacity of the TTC polymorphism in the epi-demiological analysis implies the need ofsearching other useful polymorphic loci fordetailed subdivision of clinical isolates.—Authors’Abstract

72, 2 Current Literature, Microbiology (Tuberculosis) 225

Microbiology (Tuberculosis)

Cheng, A. F., Yew, W. W., Chan, E. W.,Chin, M. L., Hui, M. M., and Chan, R.C. Multiplex PCR amplimer conformationanalysis for rapid detection of gyrA muta-tions in fluoroquinolone-resistant Myco-bacterium tuberculosis clinical isolates.Antimicrob. Agents Chemother. 48(2)(2004) 596–601.

See Current Literature, Molecular andGenetic Studies, p. 256.

Darwin, K. H., Ehrt, S., Gutierrez-Ramos,J. C., Weich, N., and Nathan, C. F. Theproteasome of Mycobacterium tubercu-losis is required for resistance to nitricoxide. Science. 302(5652) (2003) 1963–1996.

The production of nitric oxide and otherreactive nitrogen intermediates (RNI) bymacrophages helps to control infection byMycobacterium tuberculosis (Mtb). How-ever, the protection is imperfect and infec-tion persists. To identify genes that Mtb re-quires to resist RNI, we screened 10,100Mtb transposon mutants for hypersuscepti-bility to acidified nitrite. We found 12 mu-tants with insertions in seven genes repre-senting six pathways, including the repairof DNA (uvrB) and the synthesis of a flavincofactor (fbiC). Five mutants had insertionsin proteasome-associated genes. An Mtbmutant deficient in a presumptive protea-

somal adenosine triphosphatase was atten-uated in mice, and exposure to proteasomalprotease inhibitors markedly sensitizedwild-type Mtb to RNI. Thus, the mycobac-terial proteasome serves as a defenseagainst oxidative or nitrosative stress.—Au-thors’ Abstract

Ewann, F., Locht, C., and Supply, P. In-tracellular autoregulation of the Myco-bacterium tuberculosis PrrA responseregulator. Microbiology 150(Pt 1) (2004)241–246.

Two-component systems are major regu-latory systems for bacterial adaptation to en-vironmental changes. During the infectiouscycle of Mycobacterium tuberculosis, adap-tation to an intracellular environment iscritical for multiplication and survival of themicro-organism within the host. The M. tu-berculosis prrA gene, encoding the regula-tor of the two-component system PrrA-PrrB,has been shown to be induced upon macro-phage phagocytosis and to be transiently re-quired for the early stages of macrophage in-fection. In order to study the mechanisms ofregulation of the PrrA-PrrB two-componentsystem, PrrA and the cytoplasmic part of thePrrB histidine kinase were produced and pu-rified as hexahistidine-tagged recombinantproteins. Electrophoretic mobility shift as-says indicated that PrrA specifically binds tothe promoter of its own operon, with in-

creased affinity upon phosphorylation.Moreover, induction of fluorescence wasobserved after phagocytosis of a wild-typeM. tuberculosis strain containing the gfpreporter gene under the control of the prrA-prrB promoter, while this induction was notseen in a prrA/B mutant strain containingthe same construct. These results indicatethat the early intracellular induction ofprrA depends on the autoregulation of thistwo-component system.—Authors’ Ab-stract

Goulding, C. W., Apostol, M. I., Gleiter, S.,Parseghian, A., Bardwell, J., Gennaro,M., and Eisenberg, D. Gram-positiveDsbE proteins function differently fromGram-negative DsbE homologs. A struc-ture to function analysis of DsbE fromMycobacterium tuberculosis. J. Biol.Chem. 279(5) (2004) 3516–3524.

Mycobacterium tuberculosis, a Gram-positive bacterium, encodes a secreted Dsb-like protein annotated as Mtb DsbE(Rv2878c, also known as MPT53). BecauseDsb proteins in Escherichia coli and otherbacteria seem to catalyze proper folding dur-ing protein secretion and because folding ofsecreted proteins is thought to be coupled todisulfide oxidoreduction, the function ofMtb DsbE may be to ensure that secretedproteins are in their correctly folded states.We have determined the crystal structure ofMtb DsbE to 1.1 A resolution, which revealsa thioredoxin-like domain with a typicalCXXC active site. These cysteines are intheir reduced state. Biochemical characteri-zation of Mtb DsbE reveals that this disul-fide oxidoreductase is an oxidant, unlikeGram-negative bacteria DsbE proteins,which have been shown to be weak reduc-tants. In addition, the pK(a) value of theactive site, solvent-exposed cysteine isapproximately 2 pH units lower than that ofGram-negative DsbE homologs. Finally, thereduced form of Mtb DsbE is more stablethan the oxidized form, and Mtb DsbE isable to oxidatively fold hirudin. Structuraland biochemical analysis implies that MtbDsbE functions differently from Gram-negative DsbE homologs, and we discuss itspossible functional role in the bacterium.—Authors’Abstract

Huard, R. C., Chitale, S., Leung, M.,Lazzarini, L. C., Zhu, H., Shashkina,E., Laal, S., Conde, M. B., Kritski, A.L., Belisle, J. T., Kreiswirth, B. N.,Lapa e Silva, J. R., and Ho, J. L. TheMycobacterium tuberculosis complex-restricted gene cfp32 encodes an ex-pressed protein that is detectable in tu-berculosis patients and is positivelycorrelated with pulmonary interleukin-10. Infect. Immun. 71(12) (2003)6871–6883.

Human tuberculosis (TB) is caused by thebacillus Mycobacterium tuberculosis, a sub-species of the M. tuberculosis complex(MTC) of mycobacteria. Postgenomic dis-section of the M. tuberculosis proteome isongoing and critical to furthering our under-standing of factors mediating M. tuberculo-sis pathobiology. Towards this end, a 32-kDaputative glyoxalase in the culture filtrate(CF) of growing M. tuberculosis (originallyannotated as Rv0577 and hereafter desig-nated CFP32) was identified, cloned, andcharacterized. The cfp32 gene is MTC re-stricted, and the gene product is expressed exvivo as determined by the respective South-ern and Western blot testing of an assortmentof mycobacteria. Moreover, the cfp32 genesequence is conserved within the MTC, as nopolymorphisms were found in the testedcfp32 PCR products upon sequence analysis.Western blotting of M. tuberculosis subcel-lular fractions localized CFP32 predomi-nantly to the CF and cytosolic compart-ments. Data to support the in vivo expressionof CFP32 were provided by the serum recog-nition of recombinant CFP32 in 32% of TBpatients by enzyme-linked immunosorbentassay (ELISA) as well as the direct detectionof CFP32 by ELISA in the induced sputumsamples from 56% of pulmonary TB pa-tients. Of greatest interest was the observa-tion that, per sample, sputum CFP32 levels(a potential indicator of increasing bacterialburden) correlated with levels of expressionin sputum of interleukin-10 (an immunosup-pressive cytokine and a putative contributingfactor to disease progression) but not levelsof gamma interferon (a key cytokine in theprotective immune response in TB), as mea-sured by ELISA. Combined, these data sug-gest that CFP32 serves a necessary biologi-cal function(s) in tubercle bacilli and may

226 International Journal of Leprosy 2004

contribute to the M. tuberculosis pathogenicmechanism. Overall, CFP32 is an attractivetarget for drug and vaccine design as well asnew diagnostic strategies.—Authors’ Ab-stract

Jaeger, T., Budde, H., Flohe, L., Menge,U., Singh, M., Trujillo, M., and Radi,R. Multiple thioredoxin-mediated routesto detoxify hydroperoxides in Mycobac-terium tuberculosis. Arch. Biochem. Bio-phys. 423(1) (2004) 182–191.

Drug resistance and virulence of Myco-bacterium tuberculosis are in part related tothe pathogen’s antioxidant defense systems.KatG(–) strains are resistant to the first linetuberculostatic isoniazid but need to com-pensate their catalase deficiency by alterna-tive peroxidase systems to stay virulent. Sofar, only NADH-driven and AhpD-mediatedhydroperoxide reduction by AhpC has beenimplicated as such virulence-determiningmechanism. We here report on two novelpathways which underscore the importanceof the thioredoxin system for antioxidant de-fense in M. tuberculosis: (i) NADPH-drivenhydroperoxide reduction by AhpC that ismediated by thioredoxin reductase andthioredoxin C and (ii) hydroperoxide reduc-tion by the atypical peroxiredoxin TPx thatequally depends on thioredoxin reductasebut can use both, thioredoxin B and C. Ki-netic analyses with different hydroperoxidesincluding peroxynitrite qualify the redoxcascade comprising thioredoxin reductase,thioredoxin C, and TPx as the most efficientsystem to protect M. tuberculosis against ox-idative and nitrosative stress in situ.—Au-thors’Abstract

Kocincova, D., Sonden, B., de Mendonca-Lima, L., Gicquel, B., and Reyrat, J.M. The Erp protein is anchored at the sur-face by a carboxy-terminal hydrophobicdomain and is important for cell-wallstructure in Mycobacterium smegmatis.FEMS Microbiol. Lett. 231(2) (2004)191–196.

Erp (Exported Repetitive Protein), alsoknown as P36, Pirg and Rv3810, is a mem-ber of a mycobacteria-specific family of ex-

tracellular proteins. In pathogenic species,the erp gene has been described as a viru-lence factor. The Erp proteins comprisethree domains. The N- and C-terminal do-mains are similar in all mycobacterialspecies, while the central domain consists ofa repeated module that differs considerablybetween species. Here we show that the Erpprotein is loosely attached to the surface andthat the carboxy-terminal domain, whichdisplays hydrophobic features, anchors Erpat the surface of the bacillus. The hy-drophobic region is not necessary for thecomplementation of the altered colony mor-phology of a Mycobacterium smegmatiserp- mutant but proved to be necessary toachieve resistance to detergent at wild-typelevels.—Authors’Abstract

Kotlowski, R., Shamputa, I. C., El Aila, N.A., Sajduda, A., Rigouts, L., van Deun,A., and Portaels, F. PCR-based geno-typing of Mycobacterium tuberculosiswith new GC-rich repeated sequencesand IS6110 inverted repeats used asprimers. J. Clin. Microbiol. 42(1) (2004)372–377.

In the present study we attempted todevelop a PCR-based epidemiological toolfor the differentiation of Mycobacterium tu-berculosis isolates. Use of the designedprimers Mtb1 (5′-CCG-GCG-GGG-CCG-GCG-G) and Mtb2 (5′-CGG-CGG-CAA-CGG-CGG-C) targeting frequently repeated16-bp sequences in combination withprimers sited at the inverted repeats flankingIS6110 allowed differentiation of M. tuber-culosis isolates.—Authors’Abstract

Morlock, G. P., Metchock, B., Sikes, D.,Crawford, J. T., and Cooksey, R. C.ethA, inhA, and katG loci of ethionamide-resistant clinical Mycobacterium tuber-culosis isolates. Antimicrob. AgentsChemother. 47(12) (2003) 3799–3805.

Ethionamide (ETH) is a structural analogof the antituberculosis drug isoniazid (INH).Both of these drugs target InhA, an enzymeinvolved in mycolic acid biosynthesis. INHrequires catalase-peroxidase (KatG) activa-tion, and mutations in katG are a major INH

72, 2 Current Literature, Microbiology (Tuberculosis) 227

resistance mechanism. Recently an enzyme(EthA) capable of activating ETH has beenidentified. We sequenced the entire ethAstructural gene of 41 ETH-resistant Myco-bacterium tuberculosis isolates. We also se-quenced two regions of inhA and all or partof katG. The MICs of ETH and INH weredetermined in order to associate the muta-tions identified with a resistance phenotype.Fifteen isolates were found to possess ethAmutations, for all of which the ETH MICswere ≥50 microg/ml. The ethA mutationswere all different, previously unreported,and distributed throughout the gene. In eightof the isolates, a missense mutation in theinhA structural gene occurred. The ETHMICs for seven of the InhA mutants were≥100 microg/ml, and these isolates werealso resistant to ≥8 microg of INH per ml.Only a single point mutation in the inhApromoter was identified in 14 isolates. AkatG mutation occurred in 15 isolates, forwhich the INH MICs for all but 1 were ≥32microg/ml. As expected, we found no asso-ciation between katG mutation and the levelof ETH resistance. Mutations within theethA and inhA structural genes were asso-ciated with relatively high levels of ETH re-sistance. Approximately 76% of isolates re-sistant to ≥50 microg of ETH per ml hadsuch mutations.—Authors’Abstract

Movahedzadeh, F., Smith, D. A., Norman,R. A., Dinadayala, P., Murray-Rust, J.,Russell, D. G., Kendall, S. L., Rison, S.C., McAlister, M. S., Bancroft, G. J.,McDonald, N. Q., Daffe, M., Av-Gay,Y., and Stoker, N. G. The Mycobacte-rium tuberculosis ino1 gene is essentialfor growth and virulence. Mol. Micro-biol. 51(4) (2004) 1003–1014.

Inositol is utilized by Mycobacterium tu-berculosis in the production of its major thioland of essential cell wall lipoglycans. We haveconstructed a mutant lacking the gene encod-ing inositol-1-phosphate synthase (ino1),which catalyses the first committed step in in-ositol synthesis. This mutant is only viable inthe presence of extremely high levels of ino-sitol. Mutant bacteria cultured in inositol-freemedium for four weeks showed a reduction inlevels of mycothiol, but phosphatidylinositolmannoside, lipomannan and lipoarabinoman-

nan levels were not altered. The ino1 mutantwas attenuated in resting macrophages and inSCID mice. We used site-directed mutagene-sis to alter four putative active site residues;all four alterations resulted in a loss of activity,and we demonstrated that a D310N mutationcaused loss of the active site Zn2+ ion and aconformational change in the NAD+ cofac-tor.—Authors’Abstract

Parish, T. Starvation survival response ofMycobacterium tuberculosis. J. Bacteriol.185(22) (2003) 6702–6706.

The ability of Mycobacterium tuberculo-sis auxotrophs to survive long-term star-vation was measured. Tryptophan andhistidine auxotrophs did not survive single-amino-acid starvation, whereas a prolineauxotroph did. All three auxotrophs sur-vived complete starvation. THP-1 cells werealso able to restrict the growth of the trypto-phan and histidine auxotrophs.—Author’sAbstract

Pashley, C. A., and Parish, T. Efficientswitching of mycobacteriophage L5-based integrating plasmids in Mycobac-terium tuberculosis. FEMS Microbiol.Lett. 229(2) (2003) 211–215.

We previously used a mycobacteriophageL5-derived integrating vector to demon-strate that glnE and aroK are essential genesin Mycobacterium tuberculosis by showingthat we were unable to excise the integratedvector when it carried the only functionalcopy of these genes. We tested three systemsto replace the integrated copy with alterna-tive alleles. The most efficient method wasto transform the strain with a second copy ofthe integrating vector. Excision of the resi-dent vector and integration of the incomingvector occurred at an extremely high effi-ciency. This technique will allow us to studythe role and functionality of essential genesin this important human pathogen.—Au-thors’Abstract

Shimono, N., Morici, L., Casali, N.,Cantrell, S., Sidders, B., Ehrt, S., andRiley, L. W. Hypervirulent mutant of

228 International Journal of Leprosy 2004

Mycobacterium tuberculosis resultingfrom disruption of the mce1 operon.Proc. Natl. Acad. Sci. U.S.A. 100(26)(2003) 15918–15923.

An estimated one-third of the world’spopulation is latently infected with Myco-bacterium tuberculosis, the etiologic agent oftuberculosis. Here, we demonstrate that, un-like wild-type M. tuberculosis, a strain of M.tuberculosis disrupted in the mce1 operonwas unable to enter a stable persistent stateof infection in mouse lungs. Instead, the mu-tant continued to replicate and killed themice more rapidly than did the wild-typestrain. Histological examination of mouselungs infected with the mutant strain re-vealed diffusely organized granulomas withaberrant inflammatory cell migration.Murine macrophages infected ex vivo withthe mutant strain were reduced in theirability to produce tumor necrosis factoralpha, IL-6, monocyte chemoattractant pro-tein 1, and nitric oxide (NO), but not IL-4.The mce1 mutant strain complemented withthe mce1 genes stimulated tumor necrosisfactor alpha and NO production by murinemacrophages at levels stimulated by thewild-type strain. These observations indicatethat the mce1 operon mutant is unable tostimulate T helper 1-type immunity in mice.The hypervirulence of the mutant strain mayhave resulted from its inability to stimulate aproinflammatory response that would other-wise induce organized granuloma formationand control the infection without killing theorganism. The mce1 operon of M. tubercu-losis may be involved in modulating the hostinflammatory response in such a way that thebacterium can enter a persistent state withoutbeing eliminated or causing disease in thehost.—Authors’Abstract

Theus, S. A., Cave, M. D., and Eisenach,K. D. Activated THP-1 cells: an attractivemodel for the assessment of intracellulargrowth rates of Mycobacterium tubercu-losis isolates. Infect. Immun. 72(2)(2004) 1169–1173.

Capacity of certain Mycobacterium tu-berculosis isolates to grow more rapidly inhuman macrophages may be indicative ofincreased virulence. Significant differences

were observed in intracellular growth of twoisolates from sites of tuberculosis transmis-sion, with an outbreak-associated straingrowing faster than a strain causing diseasein only one person. Activated THP-1 cellsare a suitable alternative to peripheral bloodmonocyte models.—Authors’Abstract

Timm, J., Post, F. A., Bekker, L. G.,Walther, G. B., Wainwright, H. C.,Manganelli, R., Chan, W. T., Tsenova,L., Gold, B., Smith, I., Kaplan, G., andMcKinney, J. D. Differential expressionof iron-, carbon-, and oxygen-responsivemycobacterial genes in the lungs ofchronically infected mice and tuberculo-sis patients. Proc. Natl. Acad. Sci. U.S.A.100(24) (2003) 14321–14326.

Pathogenetic processes that facilitate theentry, replication, and persistence of Myco-bacterium tuberculosis (MTB) in the mam-malian host likely include the regulated ex-pression of specific sets of genes at differentstages of infection. Identification of genesthat are differentially expressed in vivo wouldprovide insights into host-pathogen interac-tions in tuberculosis (TB); this approachmight be particularly valuable for the studyof human TB, where experimental opportu-nities are limited. In this study, the levels ofselected MTB mRNAs were quantified invitro in axenic culture, in vivo in the lungs ofmice, and in lung specimens obtained fromTB patients with active disease. We report thedifferential expression of MTB mRNAs as-sociated with iron limitation, alternative car-bon metabolism, and cellular hypoxia, con-ditions that are thought to exist within thegranulomatous lesions of TB, in the lungs ofwild-type C57BL/6 mice as compared withbacteria grown in vitro. Analysis of the sameset of mRNAs in lung specimens obtainedfrom TB patients revealed differences inMTB gene expression in humans as com-pared with mice.—Authors’Abstract

Tufariello, J. M., Jacobs, W. R. Jr., andChan, J. Individual Mycobacterium tu-berculosis resuscitation-promoting factorhomologues are dispensable for growthin vitro and in vivo. Infect. Immun. 72(1)(2004) 515–526.

72, 2 Current Literature, Microbiology (Tuberculosis) 229

Mycobacterium tuberculosis possessesfive genes with significant homology to theresuscitation-promoting factor (Rpf) ofMicrococcus luteus. The M. luteus Rpf is asecreted approximately 16-kDa proteinwhich restores active growth to cultures ofM. luteus rendered dormant by prolongedincubation in stationary phase. More re-cently, the Rpf-like proteins of M. tubercu-losis have been shown to stimulate thegrowth of extended-stationary-phase cul-tures of Mycobacterium bovis BCG. Thesedata suggest that the Rpf proteins can influ-ence the growth of mycobacteria; however,the studies do not demonstrate specific func-tions for the various members of this proteinfamily, nor do they assess the function of M.tuberculosis Rpf homologues in vivo. To ad-dress these questions, we have disruptedeach of the five rpf-like genes in M. tuber-culosis Erdman, and analyzed the mutantsfor their growth in vitro and in vivo. In con-trast to M. luteus, for which rpf is an essen-tial gene, we find that all of the M. tubercu-losis rpf deletion mutant strains are viable;in addition, all show growth kinetics similarto Erdman wild type both in vitro and inmouse organs following aerosol infection.Analysis of rpf expression in M. tuberculo-sis cultures from early log phase throughlate stationary phase indicates that expres-sion of the rpf-like genes is growth phase-dependent, and that the expression patternsof the five M. tuberculosis rpf genes, whileoverlapping to various degrees, are not uni-form. We also provide evidence that myco-bacterial rpf genes are expressed in vivo inthe lungs of mice acutely infected with vir-ulent M. tuberculosis.—Authors’Abstract

Zahrt, T. C., Wozniak, C., Jones, D., andTrevett, A. Functional analysis of the My-

cobacterium tuberculosis MprAB two-component signal transduction system.Infect. Immun. 71(12) (2003) 6962–6970.

The mechanisms utilized by Mycobacte-rium tuberculosis to establish, maintain, orreactivate from latent infection in the hostare largely unknown but likely includegenes that mediate adaptation to conditionsencountered during persistence. Previously,a two-component signal transduction sys-tem, mprAB, was found to be required inM. tuberculosis for establishment andmaintenance of persistent infection in a tis-sue- and stage-specific fashion. To begin tocharacterize the role of this system in M.tuberculosis physiology and virulence, afunctional analysis of the mprA and mprBgene products was initiated. Here, evidenceis presented demonstrating that sensor ki-nase MprB and response regulator MprAfunction as an intact signal-transducingpair in vitro and in vivo. Sensor kinaseMprB can be autophosphorylated, can do-nate phosphate to MprA, and can act as aphospho-MprA phosphatase in vitro. Cor-respondingly, response regulator MprA canaccept phosphate from MprB or from smallphosphodonors including acetyl phosphate.Mutagenesis of residues His249 in MprBand Asp48 in MprA abolished the ability ofthese proteins to be phosphorylated invitro. Introduction of these alleles intoMycobacterium bovis BCG attenuated vir-ulence in macrophages in vivo. Together,these results support a role for the mprABtwo-component system in M. tuberculosisphysiology and pathogenesis. Characteriza-tion of two-component signal transductionsystems will enhance our understanding ofprocesses regulated by M. tuberculosis dur-ing acute and/or persistent infection in thehost.—Authors’ Abstract

230 International Journal of Leprosy 2004

Experimental Infections and Prevention

Ando, M., Yoshimatsu, T., Ko, C., Con-verse, P. J., and Bishai, W. R. Deletionof Mycobacterium tuberculosis sigmafactor E results in delayed time to deathwith bacterial persistence in the lungs ofaerosol-infected mice. Infect. Immun.71(12) (2003) 7170–7172.

The stress-induced extracytoplasmicsigma factor E (SigE) of Mycobacterium tu-berculosis shows increased expression afterheat shock, sodium dodecyl sulfate treat-ment, and oxidative stress, as well as afterphagocytosis in macrophages. We reportthat deletion of sigE results in delayed

lethality in mice without a significant re-duction of bacterial numbers in lungs.—Au-thors’Abstract

Arevalo, M. I., Escribano, E., Calpena,A., Domenech, J., and Queralt, J. Ther-mal hyperalgesia and light touch allody-nia after intradermal Mycobacteriumbutyricum administration in rat. Inflam-mation 27(5) (2003) 293–299.

We examined the time course (7 weeks)of thermal hyperalgesia and light touch al-lodynia in rats after intradermal administra-tion of Mycobacterium butyricum. Nocicep-tive thresholds to heat and light touch wereassessed. Paw edema and temperature,motor function, body weight, and propio-ception were also tested. Some rats devel-oped arthritis (named AA rats) but others didnot (named non-AA rats). Both groups werecompared with healthy animals. Persistenthyperalgesia was found in both groups; inAA rats it appeared before clinical evidenceof arthritis. Transient allodynia ocurred onlyafter edema development and fell whenedema decreased. Motor function was im-paired only in AA rats. The results of thisstudy demonstrate that hyperalgesia, but notallodynia, appeared after Mycobacteriumbutyricum in both groups, suggesting thatchanges in sensitivity were not merely theresult of local hypersensitivity of the in-flamed tissue, but may also be due to alter-ations in nociception in the central nervoussystem.—Authors’Abstract

Bastos, R. G., Dellagostin, O. A., Barletta,R. G., Doster, A. R., Nelson, E., Zuck-ermann, F., and Osorio, F. A. Immuneresponse of pigs inoculated with Myco-bacterium bovis BCG expressing a trun-cated form of GP5 and M protein ofporcine reproductive and respiratory syn-drome virus. Vaccine 22(3–4) (2004)467–474.

Pigs were immunised with recombinantBCG (rBCG) expressing a truncated form ofGP5 (lacking the first 30 NH(2)-terminal res-idues) (rBCGGP5) and M protein (rBCGM)of porcine reproductive and respiratorysyndrome virus (PRRSV). At 30 days post-

inoculation (dpi), pigs inoculated withrBCGGP5 and rBCGM developed a specifichumoral immune response against the viralproteins, as detected by commercial ELISAand Western blot tests, and at 60 dpi, threeout of five animals developed neutralizingantibodies with titers ranging from 1:4 to 1:8.At 67 dpi, an IFN-gamma response againstBCG antigens, but not against the viral pro-teins, was detected by ELISPOT in inocu-lated pigs. Following challenge with a path-ogenic strain of PRRSV, pigs inoculated withrBCG showed lower (p <0.05) temperature,viremia and virus load in bronchial lymphnodes than control animals, suggesting theestablishment of partial protection againstPRRSV infection.—Authors’Abstract

Chen, L., Wang, J., Zganiacz, A., andXing, Z. Single intranasal mucosal My-cobacterium bovis BCG vaccination con-fers improved protection compared tosubcutaneous vaccination against pul-monary tuberculosis. Infect. Immun.72(1) (2004) 238–246.

Whether the intranasal (i.n.) route of My-cobacterium bovis BCG vaccination pro-vides better protection against pulmonarytuberculosis than subcutaneous (s.c.) vacci-nation remains an incompletely solvedissue. In the present study, we comparedboth immune responses and protectionelicited by single BCG vaccinations via thei.n. or s.c. route in BALB/c mice. While bothi.n. and s.c. vaccination triggered compara-ble levels of primary immune activation inthe spleen and draining lymph nodes, i.n.vaccination led to a greater antigen-specificgamma interferon recall response in spleno-cytes than s.c. vaccination upon secondaryrespiratory mycobacterial challenge, ac-companied by an increased frequency ofantigen-specific lymphocytes. There wasalso a quicker cellular response in the lungsof i.n. vaccinated mice upon mycobacterialchallenge. Mice vaccinated i.n. were foundto be much better protected, particularly inthe lung, than s.c. vaccinated counterpartsagainst pulmonary tuberculosis at both 3and 6 months postvaccination. These resultssuggest that the i.n. route of vaccination im-proves the protective effect of the currentBCG vaccine.—Authors’Abstract

72, 2 Current Literature, Experimental Infections 231

Chung, S. W., Choi, S. H., and Kim, T. S.Induction of persistent in vivo resistanceto Mycobacterium avium infection inBALB/c mice injected with interleukin-18-secreting fibroblasts. Vaccine 22(3–4)(2004) 398–406

Interferon-gamma (IFN-gamma) is closelyassociated with the generation of cell-mediated immunity and resistance to intracel-lular parasites. Interleukin-18 (IL-18) isknown to strongly induce IFN-gamma pro-duction by T cells and natural killer (NK)cells. To determine whether the paracrinesecretion of IL-18 can efficiently stimulate theresistance to Mycobacterium avium complex(MAC) infection, 3T3 fibroblasts were stablytransfected to secrete bioactive IL-18 and theireffects on MAC infection were investigated ingenetically susceptible BALB/c mice, com-pared with that of free recombinant IL-18. Im-munization with IL-18-secreting fibroblasts(3T3/IL-18) during intranasal infection withMAC resulted in a significant decrease in bac-terial load of lung during the entire 8-weekobservation period, while rIL-18 reduced thebacterial load at initial 1 week but not by 8weeks postinfection. Immunization with the3T3/IL-18 cells induced and maintained sig-nificantly higher levels of cytotoxic activityand nitric oxide production by lung cells thanthose of rIL-18 immunization. Furthermore,lung cells in mice injected with the 3T3/IL-18cells showed persistent production of IFN-gamma throughout the 8-week period, sug-gesting that the 3T3/IL-18 cells induced theresistance to MAC infection via IFN-gammaproduction. This work suggests that IL-18-secreting fibroblasts may serve as a vehicle forparacrine secretion of IL-18 in immunother-apy of MAC infection.—Authors’Abstract

Collins, D. M., Kawakami, R. P., Buddle, B.M., Wards, B. J., and de Lisle, G. W. Dif-ferent susceptibility of two animal speciesinfected with isogenic mutants of Myco-bacterium bovis identifies phoT as havingroles in tuberculosis virulence and phos-phate transport. Microbiology 149(Pt 11)(2003) 3203–3212.

The Mycobacterium tuberculosis complexincludes Mycobacterium bovis, whichcauses tuberculosis in most mammals, in-

cluding humans. In previous work, it wasshown that M. bovis ATCC 35721 has a mu-tation in its principal sigma factor gene,sigA, causing a single amino acid change af-fecting binding of SigA with the accessorytranscription factor WhiB3. ATCC 35721 isavirulent when inoculated subcutaneouslyinto guinea pigs but can be restored to viru-lence by integration of wild-type sigA toproduce M. bovis WAg320. Subsequently, itwas surprising to discover that WAg320 wasnot virulent when inoculated intratracheallyinto the Australian brushtail possum (Tri-chosurus vulpecula), a marsupial that isnormally very susceptible to infection withM. bovis. In this study, an in vivo comple-mentation approach was used with ATCC35721 to produce M. bovis WAg322, whichwas virulent in possums, and to identify thevirulence-restoring gene, phoT. There aretwo point deletions in the phoT gene ofATCC 35721 causing frameshift inactiva-tion, one of which is also in the phoT ofBCG. Knockout of phoT from ATCC 35723,a virulent strain of M. bovis, produced M.bovis WAg758, which was avirulent in bothguinea pigs and possums, confirming thatphoT is a virulence gene. The effect on vir-ulence of mode of infection versus animalspecies susceptibility was investigated byinoculating all the above strains by aerosolinto guinea pigs and mice and comparingthese to the earlier results. Characterizationof PhoT indicated that it plays a role inphosphate uptake at low phosphate concen-trations. At least in vitro, this role requiresthe presence of a wild-type sigA gene andappears separate from the ability of phoT torestore virulence to ATCC 35721. This studyshows the advantages of using different an-imal models as tools for the molecular bio-logical investigation of tuberculosis viru-lence.—Authors’Abstract

Collins, D. M., Kawakami, R. P., Wards,B. J., Campbell, S., and de Lisle, G. W.Vaccine and skin testing properties of twoavirulent Mycobacterium bovis mutantswith and without an additional esat-6 mu-tation. Tuberculosis (Edinb). 83(6) (2003)361–366.

SETTING: Molecular techniques are nowavailable to develop new live tuberculosis

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vaccines by producing avirulent strains ofthe Mycobacterium tuberculosis complexwith known genes deleted. OBJECTIVES:Determine if removal of esat-6 from newlive tuberculosis vaccines with known at-tenuating mutations affects their vaccine ef-ficacy and if it could enable the developmentof discriminating diagnostic tests. DESIGN:Remove the esat-6 gene by allelic exchangefrom two illegitimate mutants of Mycobac-terium bovis that had previously been shownto have similar vaccine efficacy to BCG in aguinea pig vaccination model. Determinethe effect this removal has on virulence, vac-cine efficacy and skin test reactivity inguinea pigs. RESULTS: Two double knock-out strains of M. bovis were produced andtheir virulence and vaccine efficacy werecompared to their parent strains. Removal ofthe esat-6 gene had no significant effect onvaccine efficacy. In skin tests, animals inoc-ulated with the double knockout strains re-acted to PPD but not ESAT-6, whereas thoseinoculated with the parent strains had simi-lar skin test reactivity to both PPD and esat-6.CONCLUSION: Removal of esat-6 fromnew live tuberculosis vaccine candidates hasno significant effect on vaccine propertiesbut does enable the use of skin tests to dis-tinguish between vaccination and infec-tion.—Authors’Abstract

Derrick, S. C., Repique, C., Snoy, P.,Yang, A. L., and Morris, S. Immuniza-tion with a DNA vaccine cocktail protectsmice lacking CD4 cells against an aero-genic infection with Mycobacterium tu-berculosis. Infect. Immun. 72(3) (2004)1685–1692.

Tuberculosis (TB) is the most commonopportunistic disease and a potentially fatalcomplication among immunocompromisedindividuals infected with human immunode-ficiency virus (HIV). Effective vaccinationagainst TB in persons with HIV has beenconsidered unlikely because of the centralrole that CD4 cells play in controlling tu-berculous infections. Here we show that thevaccination of CD8(–/–) mice with a TBDNA vaccine cocktail did not significantlyenhance protective responses to a Mycobac-terium tuberculosis infection. In contrast,immunization with a DNA vaccine cocktail

or with the current TB vaccine, Mycobac-terium bovis BCG, induced considerableantituberculosis protective immunity inimmune-deficient mice lacking CD4 cells.In vaccinated CD4(–/–) animals, substan-tially reduced bacterial burdens in organsand much improved lung pathology wereseen 1 month after an aerogenic M. tuber-culosis challenge. Importantly, the postchal-lenge mean times to death of vaccinatedCD4(–/–) mice were significantly extended(mean with DNA cocktail, 172 ± 7 days;mean with BCG, 156 ± 22 days) comparedto that of naive CD4(–/–) mice (33 ± 6days). Furthermore, the treatment of DNA-vaccinated CD4(–/–) mice with an anti-CD8or anti-gamma interferon (IFN-gamma) an-tibody significantly reduced the effect of im-munization, and neither IFN-gamma(–/–)nor tumor necrosis factor receptor-deficientmice were protected by DNA immunization;therefore, the primary vaccine-inducedprotective mechanism in these immune-deficient mice likely involves the secretionof cytokines from activated CD8 cells. Thesubstantial CD8-mediated protective immu-nity that was generated in the absence ofCD4 cells suggests that it may be possible todevelop effective TB vaccines for use inHIV-infected populations.—Authors’ Ab-stract

Gorodezky, C., Alaez, C., Munguia, A.,Cruz, R., Vazquez, A., Camacho, A.,Flores, O., Rodriguez, M., and Ro-driguez, O. Molecular mechanisms ofMHC linked susceptibility in leprosy: to-wards the development of synthetic vac-cines. Tuberculosis (Edinb). 84(1–2)(2004) 82–92.

Tuberculoid (TT) and lepromatous lep-rosy (LL) develop in the human host de-pending on his ability to trigger a specificcellular immune response(CIR). Differentgenes have been demonstrated in suscepti-bility/protection and may explain the formsof leprosy. The major histocompatibilitycomplex (MHC) play an important role. Theaim of the study was to explore the contri-bution of human leukocyte antigen (HLA)DRB1, DQA1, DQB1 and DQ promotergenes in LL Mexican patients. Six families(26 LL, three TT patients and 27 controls)

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were analyzed; 114 unrelated patients werecompared with 204 controls. Class I typingwas done by the standard microlymphocy-totoxicity and class II typing using PCR-SSOP. Haplotype segregation correlatedwith specific CIR in vivo and in vitro usinglepromin. Haplotype sharing was significantlydeviated in the affected sibs (p = 0.01). Sixhealthy sibs were non-responders to leprominand four of them were DQ1 homozgotes.DQ1 was significantly associated with LL andwith non-responders. We set up macrophageactivation experiments after infecting thesecells with 5 × 10(6) bacilli to demonstrate ifelimination occurred in the context or DQ1.When DQ1 was present on macrophages andon T cells, bacteria were poorly eliminatedfrom the cell (32%) while when absent, 76%of the individuals were able to eliminate thebacilli (p = 0.03). DRB1*1501 DQA1*0102-DQB1*0602 (DQ1 subtype) was significantlyincreased in the patients, indicating its partic-ipation in susceptibility. QBP 5.11/5.12 pro-moter present in the mentioned haplotype, andQAP 1.4, linked to DRB1*1301/02 haplo-types were also associated. Two mecha-nisms are suggested: the promoter polymor-phisms may influence allele expression andthus the amount of peptides presented to theT-cell receptor, leading to a deficient CIR:HLA restriction is important for vaccinedesign; the way peptides anchor theDRB1*1501 groove may be relevant to theactivation of TH1 cells, which contribute toan efficient presentation of peptides induc-ing a protective T-cell response.—Authors’Abstract

Holten-Andersen, L., Doherty, T. M.,Korsholm, K. S., and Andersen, P.Combination of the cationic surfactant di-methyl dioctadecyl ammonium bromideand synthetic mycobacterial cord factoras an efficient adjuvant for tuberculosissubunit vaccines. Infect. Immun. 72(3)(2004) 1608–1617.

Recombinant, immunodominant antigensderived from Mycobacterium tuberculosiscan be used to effectively vaccinate againstsubsequent infection. However, the efficacyof these recombinant proteins is dependenton the adjuvant used for their delivery. Thisproblem affects many potential vaccines,

not just those for tuberculosis, so the dis-covery of adjuvants that can promote thedevelopment of cell-mediated immunity isof great interest. We have previously shownthat the combination of the cationic surfac-tant dimethyl dioctadecyl ammonium bro-mide and the immunomodulator modifiedlipid A synergistically potentiates Th1 T-cellresponses. Here we report a screening pro-gram for other adjuvants with reported Th1-promoting activity and identify a secondnovel adjuvant formulation that drives thedevelopment of Th1 responses with an ex-tremely high efficacy. The combination ofdimethyl dioctadecyl ammonium bromideand the synthetic cord factor trehalosedibehenate promotes strong protective im-mune responses, without overt toxicity,against M. tuberculosis infection in a vacci-nation model and thus appears to be a verypromising candidate for the development ofhuman vaccines.—Authors’Abstract

Hsieh, M. J., Junqueira-Kipnis, A. P.,Hoeffer, A., Turner, O. C., and Orme,I. M. Incorporation of CpG oligodeoxy-nucleotide fails to enhance the protectiveefficacy of a subunit vaccine against My-cobacterium tuberculosis.Vaccine 22(5–6)(2004) 655–659.

Vaccines which offer better protectionthan BCG are now badly needed for con-trolling tuberculosis infection throughoutthe world. Immunological adjuvants capableof inducing a TH1 type of protective re-sponse are necessary to augment the im-mune response, particularly in the case ofsubunit vaccines. It is now well establishedthat oligodeoxynucleotides (ODN) contain-ing cytidine phosphate guanosine (CpG)motifs enhance cell-mediated responses invivo by increasing the production of the TH1cytokines IL-12 and interferon gamma(IFNgamma). To determine if this wouldimprove subunit vaccination of mice CpGODN were added to a subunit vaccine con-sisting of the culture filtrate proteins (CFP)of Mycobacterium tuberculosis H37Rv. Itwas observed that although adding CpGODN to the vaccines promoted substantiallyincreased IFNgamma production by lymphnode cells draining sites of inoculation, thisfailed to translate after aerosol challenge

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into any degree of enhancement of bacterialclearance in the lungs, influx of IFN-positive T cells, or changes in histopathol-ogy. These data suggest that the vaccine en-hancing effects of CpG ODN are relativelytransient.—Authors’Abstract

Johansen, P., Raynaud, C., Yang, M., Col-ston, M. J., Tascon, R. E., and LowrieD. B. Anti-mycobacterial immunity in-duced by a single injection of M. lepraeHsp65-encoding plasmid DNA inbiodegradable microparticles. Immunol.Lett. 90(2–3) (2003) 81–85.

A single sub-cutaneous injection of a plas-mid DNA encoding a mycobacterial heatshock protein 65 (Hsp65) entrapped inbiodegradable poly(lactic-co-glycolic acid)microspheres produced high titers of anti-bodies, measured 5 months after the injec-tion in BALB/c mice. Splenocytes secretedIFN-gamma and exerted an anti-bacterial ef-fect on macrophages infected in vitro withMycobacterium tuberculosis. The results areencouraging with regard to obtaining goodcompliance and vaccination coverage withcandidate plasmid DNA vaccines, especiallyin developing countries.—Authors’Abstract

Junqueira-Kipnis, A. P., Turner, J.,Gonzalez-Juarrero, M., Turner, O. C.,and Orme, I. M. Stable T-cell populationexpressing an effector cell surface pheno-type in the lungs of mice chronically in-fected with Mycobacterium tuberculosis.Infect. Immun. 72(1) (2004) 570–575.

Analysis of T-cell subsets accumulating inthe lungs of C57BL/6 mice chronically in-fected with Mycobacterium tuberculosis re-vealed that both CD4 and CD8 T-cell popu-lations expressed a cell surface phenotypeconsistent with that of effector T cells andthat a significant proportion of these cellswere in the process of secreting gamma in-terferon.—Authors’Abstract

Kanaujia, G. V., Motzel, S., Garcia, M. A.,Andersen, P., and Gennaro, M. L.Recognition of ESAT-6 sequences by an-tibodies in sera of tuberculous nonhuman

primates. Clin. Diagn. Lab. Immunol.11(1) (2004) 222–226.

Previous work in our laboratory showedthat the ESAT-6 protein of Mycobacteriumtuberculosis and Mycobacterium bovis in-duces strong antibody responses in a largeproportion ( approximately 90%) of experi-mentally or naturally infected nonhumanprimates. Here, the antibody response toESAT-6 in tuberculous monkeys was char-acterized at the epitope level by measuringantibodies to overlapping, synthetic peptidesspanning the ESAT-6 sequence. The anti-body response against the COOH-terminalportion of the protein was the strongest inboth experimentally and naturally infectedanimals. Moreover, these antibodies becamedetectable the earliest during experimentalinfection, suggesting an ordered expansionof ESAT-6-specific B-cell clones in thecourse of infection. The data support use ofsynthetic peptides in lieu of the full-lengthESAT-6 protein in diagnostic antibody de-tection assays.—Authors’Abstract

Kanaujia, G. V., Garcia, M. A., Bouley, D.M., Peters, R., and Gennaro, M. L.Detection of early secretory antigenictarget-6 antibody for diagnosis of tuber-culosis in non-human primates. Comp.Med. 53(6) (2003) 602–606.

Tuberculosis is one of the most economi-cally devastating, zoonotic infections ofcaptive non-human primates. The limita-tions of the tuberculin skin test, which iscurrently used to diagnose tuberculosis inliving non-human primates, make it neces-sary to find new, simple, and economical di-agnostic methods. We describe use of anenzyme-linked immunoassay to detect IgGantibodies against early secretory antigenictarget (ESAT)-6, a small protein secreted byvirulent tubercle bacilli, in paired (pre- andpost-outbreak) sera from 57 non-human pri-mates involved in an outbreak of Mycobac-terium bovis infection in a research colony.Of 25 animals with tuberculosis lesions atnecropsy, 22 (88%) had high serum levels ofthe ESAT-6 antibody. The ESAT-6 antibodywas found in 16% (5/32) of post-outbreaksera from animals in which tuberculosiscould not be confirmed at necropsy. The

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strong association between the ESAT-6 an-tibody and tuberculosis in non-human pri-mates documented in this study, togetherwith the robustness of the serologic assay,make the ESAT-6 ELISA a valuable tool fordiagnosis of tuberculosis in captive non-human primates.—Authors’Abstract

Kumar, H., Malhotra, D., Goswami, S.,and Bamezai, R. N. How far have wereached in tuberculosis vaccine develop-ment? Crit. Rev. Microbiol. 29(4) (2003)297–312.

Tuberculosis, a bacterial disease prevalentsince ancient times, continues to cause themost deaths globally compared with allother diseases. The causative agent Myco-bacterium tuberculosis is responsible fordifferent types of tuberculosis in humans;however, pulmonary tuberculosis is themost common and causes the most deaths.Mycobacterium tuberculosis is an intracel-lular pathogenic bacterium, which has de-veloped sophisticated mechanisms to sur-vive inside host mononuclear phagocytesand thus evade the host immune system.This is attributed primarily to an inadequateimmune response toward infecting bacteria,which results in temporary growth inhibi-tion rather than death and subsequently al-lows the bacteria to multiply immensely,leading to full-blown disease in an indi-vidual. This disease has become a challengedue to poor diagnosis, a low-efficiency tu-berculosis vaccine (Mycobacterium bovisBacillus Calmette-Guerin [BCG]), a long-term antibacterial chemotherapy regimen(approximately 6 months), and an emer-gence of multiple drug resistant strains ofMycobacterium tuberculosis especially inpeople with human immune deficiency virus(HIV) infection, for whom researchersworldwide must develop effective short-term chemotherapy and an effective vac-cine. In this review different aspects of vac-cines in tuberculosis are discussed, andthese include the traditional BCG vaccine,the modern auxotrophic vaccine, the subunitor acellular vaccine; and a DNA vaccine. Wediscuss also the potential of mycobacteriallipids as a vaccine or as an adjuvant in thefuture. Since complete genome informationof Mycobacterium tuberculosis H37Rv and

bioinformatics tools are available, it is pos-sible to develop new strategies for a betterand effective tuberculosis vaccine, whichcan replace the traditional BCG vaccine.—Authors’Abstract

Lasco, T. M., Yamamoto, T., Yoshimura, T.,Allen, S. S., Cassone, L., and McMurray,D. N. Effect of Mycobacterium bovis BCGvaccination on Mycobacterium-specificcellular proliferation and tumor necrosisfactor alpha production from distinctguinea pig leukocyte populations. Infect.Immun. 71(12) (2003) 7035–7042.

In this study, we focused on threeleukocyte-rich guinea pig cell populations,bronchoalveolar lavage (BAL) cells, resi-dent peritoneal cells (PC), and splenocytes(SPC). BAL cells, SPC, and PC were stim-ulated either with live attenuated Mycobac-terium tuberculosis H37Ra or with live orheat-killed virulent M. tuberculosis H37Rv(multiplicity of infection of 1:100). Eachcell population was determined to prolifer-ate in response to heat-killed virulentH37Rv, whereas no measurable proliferativeresponse could be detected upon stimulationwith live mycobacteria. Additionally, thisproliferative capacity (in SPC and PC popu-lations) was significantly enhanced uponprior vaccination with Mycobacterium bovisBCG. Accordingly, in a parallel set of ex-periments we found a strong positive corre-lation between production of antigen-specificbioactive tumor necrosis factor alpha (TNF-alpha) and prior vaccination with BCG. Anonspecific stimulus, lipopolysaccharide,failed to induce this effect on BAL cells,SPC, and PC. These results showed that pro-duction of bioactive TNF-alpha from myco-bacterium-stimulated guinea pig cell culturespositively correlates with the vaccination sta-tus of the host and with the virulence of themycobacterial strain.—Authors’Abstract

Lasco, T. M., Turner, O. C., Cassone, L.,Sugawara, I., Yamada, H., McMurray,D. N., and Orme, I. M. Rapid accumu-lation of eosinophils in lung lesions inguinea pigs infected with Mycobacteriumtuberculosis. Infect. Immun. 72(2) (2004)1147–1149.

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Guinea pig eosinophils were positivelyidentified in bronchoalveolar lavage popu-lations and in the lung granulomas of My-cobacterium tuberculosis-infected guineapigs. It is possible that the rapid influx ofthese cells, and their subsequent degranula-tion during acute pulmonary tuberculosis,may play a key role in the susceptibility ofthis animal model.—Authors’Abstract

Lee, J., Choi, K., Olin, M. R., Cho, S. N.,and Molitor, T. W. Gammadelta T cellsin immunity induced by Mycobacteriumbovis bacillus Calmette-Guerin vaccina-tion. Infect. Immun. 72(3) (2004) 1504–1511.

Mycobacterium bovis bacillus Calmette-Guerin (BCG) vaccination is efficacious fornewborns or adults with no previous expo-sure to environmental mycobacteria. To de-termine the relative contribution and the na-ture of gammadelta T-cell receptor-positiveT cells in newborns, compared to CD4(+) Tcells, in immunity induced by M. bovis BCGvaccination, 4-week-old specific-pathogen-free pigs were vaccinated with M. bovisBCG and monitored by following the gamma-delta T-cell immune responses. A flow cy-tometry-based proliferation assay and intra-cellular staining for gamma interferon(IFN-gamma) were used to examine gam-madelta T-cell responses. Pigs were found tomount Th1-like responses to M. bovis BCGvaccination as determined by immunopro-liferation and IFN-gamma production. Thegammadelta T-cell lymphoproliferation andIFN-gamma production to stimulation withmycobacterial antigens were significantlyenhanced by M. bovis BCG vaccination.The relative number of proliferating gam-madelta T cells after stimulating peripheralblood mononuclear cells with Mycobacte-rium tuberculosis H37Rv culture filtrateprotein was higher than that of CD4(+) Tcells at an early time point after M. bovisBCG vaccination, but CD4(+) T cells werefound to be more abundant at a later timepoint. Although the gammadelta T-cell re-sponses were dependent on the presence ofCD4(+) T cells for the cytokine interleukin-2,the enhanced gammadelta T cells were dueto the intrinsic changes of gammadelta Tcells caused by M. bovis BCG vaccination

rather than being due solely to help fromCD4(+) T cells. Our study shows that gam-madelta T cells from pigs at early ages arefunctionally enhanced by M. bovis BCGvaccination and suggests an important rolefor this T-cell subset in acquired immunityconferred by M. bovis BCG vaccination.—Authors’Abstract

Lima, K. M., dos Santos, S. A., Santos, R.R., Brandao, I. T., Rodrigues, J. M. Jr.,and Silva, C. L. Efficacy of DNA-hsp65vaccination for tuberculosis varies withmethod of DNA introduction in vivo.Vaccine 22(1) (2003) 49–56.

A DNA vaccine codifying the mycobac-terial hsp65 can prevent infection with My-cobacterium tuberculosis in a prophylacticsetting and also therapeutically reduce thenumber of bacteria in infected mice. Theprotective mechanism is thought to be re-lated to Th1-mediated events that result inbacterial killing. To determine the bestmethod of hsp65 introduction for vaccina-tion efficacy against tuberculosis (TB), weevaluated the immunogenicity and protec-tion of DNA-hsp65 administered by genegun bombardment or intramuscular (i.m.)injection of naked DNA. Immunization bygene gun induced immune response withplasmid doses 100-fold lower than those re-quired for intramuscular immunization.However, in contrast to intramuscular im-munization, which was protective in thesestudies, gene gun immunization did not pro-tect BALB/c mice against challenge infec-tion.—Authors’Abstract

Nuermberger, E. L., Yoshimatsu, T.,Tyagi, S., Bishai, W. R., and Grosset, J.H. Paucibacillary tuberculosis in miceafter prior aerosol immunization withMycobacterium bovis BCG. Infect.Immun. 72(2) (2004) 1065–1071.

To develop a murine model of pau-cibacillary tuberculosis for experimentalchemotherapy of latent tuberculosis infec-tion, mice were immunized with viable My-cobacterium bovis BCG by the aerosol orintravenous route and then challenged sixweeks later with virulent Mycobacterium tu-

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berculosis. The day after immunization, thecounts were 3.71 ± 0.10 log(10) CFU in thelungs of aerosol-immunized mice and 3.65± 0.11 and 4.93 ± 0.07 log(10) CFU in thelungs and spleens of intravenously immu-nized mice, respectively. Six weeks later, thelungs of all BCG-immunized mice had manygross lung lesions and splenomegaly; thecounts were 5.97 ± 0.14 and 3.54 ± 0.07log(10) CFU in the lungs and spleens ofaerosol-immunized mice, respectively, and4.36 ± 0.28 and 5.12 ± 0.23 log(10) CFU inthe lungs and spleens of intravenously im-munized mice, respectively. Mice were thenaerosol challenged with M. tuberculosis byimplanting 2.37 ± 0.13 log(10) CFU in thelungs. Six weeks after challenge, M. tuber-culosis had multiplied so that the countswere 6.41 ± 0.27 and 4.44 ± 0.14 log(10)CFU in the lungs and spleens of controlmice, respectively. Multiplication of M. tu-berculosis was greatly limited in BCG-immunized mice. Six weeks after challenge,the counts were 4.76 ± 0.24 and 3.73 ± 0.34log(10) CFU in the lungs of intravenouslyimmunized and aerosol-immunized mice,respectively. In contrast to intravenouslyimmunized mice, there was no detectabledissemination to the spleen in aerosol-immunized mice. Therefore, immunizationof mice with BCG by the aerosol route priorto challenge with a low dose of M. tubercu-losis resulted in improved containment ofinfection and a stable paucibacillary infec-tion. This model may prove to be useful forevaluation of new treatments for latent tu-berculosis infection in humans.

Pinto, R., Saunders, B. M., Camacho, L.R., Britton, W. J., Gicquel, B., andTriccas, J. A. Mycobacterium tuberculo-sis defective in phthiocerol dimyco-cerosate translocation provides greaterprotective immunity against tuberculosisthan the existing bacille Calmette-Guerinvaccine. J. Infect. Dis. 189(1) (2004)105–112.

We demonstrate that Mycobacterium tu-berculosis that is unable to export the com-plex lipid phthiocerol dimycocerosate has adecreased capacity to replicate in mice andaffords sustained protective immunityagainst M. tuberculosis infection Protection

was significantly better than that provided bythe existing vaccine, Mycobacterium bovisbacille Calmette-Guerin (BCG), and this im-proved protective efficacy was maintainedfor at least 24 weeks after vaccination. Pro-tection afforded by this attenuated strain co-incided with a number of factors that werenot associated with BCG vaccination: long-term persistence of the strain within thehost, sustained and potent induction of an-timycobacterial interferon-gamma-secretingcells equal to that induced by virulent M. tu-berculosis, and elicitation of T cells recog-nizing dominant M. tuberculosis antigensabsent from BCG. These results suggest thatthe BCG vaccine may be too attenuated toafford effective protective immunity againsttuberculosis, and vaccine strains that canprovide sustained delivery of mycobacterialantigens are promising antituberculosis vac-cine candidates.—Authors’Abstract

Portaels, F., Aguiar, J., Debacker, M.,Guedenon, A., Steunou, C., Zinsou, C.,and Meyers, W. M. Mycobacteriumbovis BCG vaccination as prophylaxisagainst Mycobacterium ulcerans os-teomyelitis in Buruli ulcer disease. Infect.Immun. 72(1) (2004) 62–65.

Mycobacterium ulcerans disease, or Bu-ruli ulcer (BU), causes significant morbidityin West Africa. Clinically, the disease pres-ents in the skin as either nonulcerative or ul-cerative forms and often invades bones eithersubjacent to the skin lesion (contiguous os-teomyelitis) or remote from the skin lesion(metastatic osteomyelitis). Osteomyelitisrepresents a severe form of the disease thatoften requires numerous surgical interven-tions, even amputations. Surgery is acceptedas the present definitive treatment for BU. Inthe absence of an effective drug treatment,the need for the development of preventiveand control strategies becomes paramount.No specific vaccine, however, is presentlyavailable for BU. Of 372 consecutive pa-tients in Benin presenting with BU (con-firmed by microbiological and histopatho-logical analyses) whose Mycobacteriumbovis BCG scar statuses were known, 196children (<15 years old) and 108 adults hadneonatal BCG vaccination scars. Of 196children with BCG scars, 17 (8.7%) had os-

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teomyelitis, while 7 of 28 children withoutBCG scars (25.0%) had osteomyelitis. Of108 adults with BCG scars, 17 (15.7%) hadosteomyelitis, while 14 of 40 adults withoutBCG scars (35.0%) had osteomyelitis. Ourresults show that effective BCG vaccinationat birth provides significant protectionagainst the development of M. ulcerans os-teomyelitis in children and adults. Therefore,health authorities should give attention to theenhancement of neonatal BCG vaccinationcoverage in all countries of Africa where BUis endemic. Protection against severe formsof BU and childhood tuberculosis wouldlikewise be improved by this intervention.—Authors’Abstract

Prem Raj, P., Srivastava, S., Jain, S. K.,Srivastava, B. S., and Srivastava, R.Protection by live Mycobacterium ha-bana vaccine against Mycobacterium tu-berculosis H37Rv challenge in mice. In-dian J. Med. Res. 117 (2003) 139–145

BACKGROUND & OBJECTIVES: Inrecent years the efficacy of BCG vaccineagainst tuberculosis has been questionedand there is no alternative vaccine available.Several strategies are being applied to get asatisfactory vaccine. Two approaches aregenerally considered: the subunit vaccinesand the whole cell vaccines. The objectiveof this investigation was to evaluate an avir-ulent mycobacteria, Mycobacterium ha-bana, as a whole cell vaccine to protect micefrom infection of M. tuberculosis H37Rv.METHODS: AKR and immunocompro-mised SJL/J mice were immunized with liveM. habana vaccine. These mice were chal-lenged with M. tuberculosis H37Rv eightweeks later along with unimmunized controlmice. Protection by M. habana vaccine wasmeasured through several parameters, whichincluded survival of challenged mice, dis-semination of challenge strain and histo-pathology of lung tissues. RESULTS: M.habana vaccinated animals were healthierthan the unvaccinated mice after challengewith M. tuberculosis and survived with sig-nificant increase in mean survival time. Theviable count of challenge strain was at least100-fold less in vaccinated mice than thecontrol mice. The lung tissues in unvacci-nated mice showed marked bronchopneu-

monia with clusters of acid fast bacilli,whereas vaccinated mice showed smallareas of damage and evidence of protectionsubsequently. INTERPRETATION & CON-CLUSION: It may be concluded from theevidence presented here that mice vacci-nated with M. habana were protected fromchallenge with M. tuberculosis in both nor-mal and immunocompromised states.—Au-thors’Abstract

Price, N. M., Gilman, R. H., Uddin, J.,Recavarren, S., and Friedland, J. S.Unopposed matrix metalloproteinase-9expression in human tuberculous gran-uloma and the role of TNF-alpha-dependent monocyte networks. J. Im-munol. 171(10) (2003) 5579–5586.

Tuberculosis is characterized by granu-loma formation and caseous necrosis, butthe factors causing tissue destruction arepoorly understood. Matrix metalloproteinase(MMP)-9 (92-kDa gelatinase) secretion frommonocytes is stimulated by Mycobacteriumtuberculosis (M. tb) and associated withlocal tissue injury in tuberculosis patients.We demonstrate strong immunohistochem-ical MMP-9 staining in monocytic cells atthe center of granuloma and adjacent tocaseous necrosis in M. tb-infected patientlymph nodes. Minimal tissue inhibitor ofMMPs-1 staining indicated that MMP-9 ac-tivity is unopposed. Because granulomascharacteristically contain few mycobacteria,we investigated whether monocyte-monocytecytokine networks amplify MMP-9 se-cretion. Conditioned medium from M. tb-infected primary human monocytes orTHP-1 cells (CoMTB) stimulated MMP-9gene expression and a >10-fold increase inMMP-9 secretion by monocytes at 3–4 days(p <0.009, vs controls). Although CoMTBstimulated dose-dependent MMP-9 secretion,MMP-1 (52-kDa collagenase) was not in-duced. Anti-TNF-alpha Ab but not IL-1Rantagonist pretreatment decreased CoMTB-induced MMP-9 secretion by 50% (p =0.0001). Anti-TNF-alpha Ab also inhibitedMMP-9 secretion from monocytic cells by50%, 24 hr after direct M. tb infection (p =0.0002). Conversely, TNF-alpha directlystimulated dose-dependent MMP-9 secre-tion. Pertussis toxin inhibited CoMTB-

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induced MMP-9 secretion and enhanced theinhibitory effect of anti-TNF-alpha Ab (p =0.05). Although chemokines bind to G pro-tein-linked receptors, CXCL8, CXCL10,CCL2, and CCL5 did not stimulate mono-cyte MMP-9 secretion. However, the re-sponse to cholera toxin confirmed that Gprotein signaling pathways were intact. Insummary, MMP-9 within tuberculous gran-uloma is associated with tissue destruction,and TNF-alpha, critical for antimycobacte-rial granuloma formation, is a key autocrineand paracrine regulator of MMP-9 secre-tion.—Authors’Abstract

Rickman, L., Saldanha, J. W., Hunt, D. M.,Hoar, D. N., Colston, M. J., Millar, J. B.,and Buxton, R. S. A two-componentsignal transduction system with a PASdomain-containing sensor is required forvirulence of Mycobacterium tuberculosisin mice. Biochem. Biophys. Res. Com-mun. 314(1) (2004) 259–267.

Mycobacterium tuberculosis, the causativeorganism of tuberculosis, encounters oxida-tive stress during phagocytosis by themacrophage and following macrophage ac-tivation during an acquired immune re-sponse, and also from internally generatedsources of radical oxygen intermediatesthrough intermediary metabolism. We haveidentified the SenX3 protein, a sensor in 1 ofthe 11 complete pairs of two-component sig-nal transduction systems in M. tuberculosis,as a possible orthologue of the Mak2p pro-tein from the fission yeast Schizosaccha-romyces pombe that is known to sense per-oxide stress. Moreover, the SenX3-RegX3two-component system was the top scoringhit in a homology search with the Escheri-chia coli ArcB-ArcA global control systemof aerobic genes. Using structural modellingtechniques we have determined that SenX3contains a PAS-like domain found in a va-riety of prokaryotic and eukaryotic sensorsof oxygen and redox. Mutants with knock-outs of senX3 or of the accompanying tran-scriptional regulator regX3 were constructedand found to have reduced virulence in amouse model of tuberculosis infection, themutant bacteria persisting for up to 4 monthspost-infection; complemented mutants hadregained virulence confirming that it was

mutations of this two-component systemthat were responsible for the avirulent phe-notype. This work identifies the PAS domainas a possible drug target for tuberculosis andmutations in the senX3-regX signal trans-duction system as potentially useful compo-nents of live vaccine strains.—Authors’Ab-stract

Shim, T. S., Turner, O. C., and Orme, I. M.Toll-like receptor 4 plays no role in sus-ceptibility of mice to Mycobacterium tu-berculosis infection. Tuberculosis (Edinb).83(6) (2003) 367–371

Although various members of the patternrecognition Toll-like receptor (TLR) familyhave been implicated in host resistance toMycobacterium tuberculosis infection, it re-mains unclear if the TLR4 receptor plays animportant role. We demonstrate here that in-fection of TRL4-competent and TLR4-deficient mice on the C3H inbred mousestrain background had similar outcomes,measured in terms of the course of the dis-ease, cell accumulation patterns in the lungs,and lung histopathology. These data argueagainst a significant role for TLR4 in im-munity to tuberculosis in the mousemodel.—Authors’Abstract

Shirtcliffe, P. M., Goldkorn, A., Weather-all, M., Tan, P. L., and Beasley, R. Pilotstudy of the safety and effect of intranasaldelipidated acid-treated Mycobacteriumvaccae in adult asthma. Respirology 8(4)(2003) 497–503.

OBJECTIVE: There is epidemiologicaland experimental evidence that exposure tomycobacteria has the potential to suppress thedevelopment of atopy and/or asthma. Delip-idated, deglycolipidated and arabinogalactan-depleted autoclaved Mycobacterium vaccae(delipidated acid-treated M. vaccae) hasbeen shown to suppress allergen-inducedairway eosinophilia in mice. METHODOL-OGY: Thirty-seven adults with stable mod-erately severe asthma who were skin pricktest-positive to house dust mite were ran-domized to receive two doses 2 weeks apartof delipidated acid-treated M. vaccae (firstdose 0.4 mg and second dose 0.8 mg) or

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phosphate buffered saline, given as drops in-tranasally. Safety, tolerability and markersof asthma severity (including peak flow,FEV1, major and minor exacerbations,symptom scores and beta-agonist use), andnasal symptom scores, blood eosinophil andIgE levels were monitored for 8 weeks. RE-SULTS: Delipidated acid-treated M. vaccaewas safe and well tolerated although therewas an occasional mild local reaction. Therewere no statistically significant differencesbetween the treatment group and placebo forany of the outcome variables. CONCLU-SIONS: There is a requirement to elucidatethe reasons why mycobacterial-based vac-cines have not shown equivalent efficacy inhuman trials compared with animal models.The role of factors such as duration of dis-ease, route of administration and the activecomponent of mycobacteria need to be ad-dressed.—Authors’Abstract

Singh, R., Rao, V., Shakila, H., Gupta, R.,Khera, A., Dhar, N., Singh, A., Koul, A.,Singh, Y., Naseema, M., Narayanan, P.R., Paramasivan, C. N., Ramanathan,V. D., and Tyagi, A. K. Disruption ofmptpB impairs the ability of Mycobacte-rium tuberculosis to survive in guineapigs. Mol. Microbiol. 50(3) (2003) 751–762.

Protein tyrosine kinases and tyrosinephosphatases from several bacterial patho-gens have been shown to act as virulencefactors by modulating the phosphorylationand dephosphorylation of host proteins. Theidentification and characterization of two ty-rosine phosphatases namely MptpA andMptpB from Mycobacterium tuberculosishas been reported earlier. MptpB is secretedby M. tuberculosis into extracellular mileuand exhibits a pH optimum of 5.6, similar tothe pH of the lysosomal compartment of thecell. To determine the role of MptpB in thepathogenesis of M. tuberculosis, we con-structed a mptpB mutant strain by homolo-gous recombination and compared theability of parent and the mutant strain to sur-vive intracellularly. We show that disruptionof the mptpB gene impairs the ability of themutant strain to survive in activated macro-phages and guinea pigs but not in restingmacrophages suggesting the importance of

its role in the host-pathogen interaction. In-fection of guinea pigs with the mutant strainresulted in a 70-fold reduction in the bacil-lary load of spleens in infected animals ascompared with the bacillary load in animalsinfected with the parental strain. Upon rein-troduction of the mptpB gene into the mu-tant strain, the complemented strain wasable to establish infection and survive inguinea pigs at rates comparable to theparental strain. These observations demon-strate a role of MptpB in the pathogenesis ofM. tuberculosis.—Authors’Abstract

Stanford, J., Stanford, C., and Grange, J.Immunotherapy with Mycobacteriumvaccae in the treatment of tuberculosis.Front Biosci. 9 (2004) 1701–1719.

All the trials of immunotherapy of tuber-culosis with killed Mycobacterium vaccae,published or not, that are known to the au-thors are reviewed here. Following an intro-duction giving a brief account of some ear-lier immunotherapies for tuberculosis, theorigins of the concept of immunotherapywith M. vaccae are considered. Progress istraced from the early work with irradiation-killed organisms in leprosy to the study inLondon of modulation of tuberculin skin-test responses, and the first comparative tri-als in The Gambia and Kuwait. In the last ofthese studies, dosages and different prepa-rations were compared. As a result of thissubsequent studies have used 109 heat-killed organisms, equivalent to 1mg wet-weight of bacilli, as a standard dose. A seriesof small trials in Argentina, India, Nigeria,Romania, South Africa and Vietnam havepioneered the way forward, disclosing geo-graphic variability, with South Africa as theonly country where almost no effects wererecorded. Together the studies have shownthat a single dose may not be sufficient.These studies have confirmed the mode ofaction of M. vaccae to be regulation of cell-mediated immunity with enhancement ofTh1 and down-regulation of Th2, and theyhave shown benefits in faster bacteriologi-cal conversion, reduction in ESR, recoveryof body weight and resolution of radiologi-cal opacities, leading to better recovery fromthe disease even when given to patients re-ceiving directly observed therapy, short-

72, 2 Current Literature, Experimental Infections 241

course (DOTS). Three major randomised,placebo-controlled and partly blinded trialshave been carried out in Africa. The first, inSouth Africa showed no M. vaccae-relatedeffects. The second trial, in Uganda, con-firmed the observations made in the earlierstudies of faster sputum conversion and bet-ter radiological clearance. The third trial, inZambia and Malawi, showed a trend to-wards benefits in the treatment of HIVseronegative patients but failed to show ben-eficial effects in HIV seropositive patients.Studies in patients with multi-drug-resistanttuberculosis have shown that multiple dosesof immunotherapy are required in mostcases, and that these markedly improvecure-rates for these patients. This is espe-cially so when they are also treated withchemotherapy tailored to the resistance pat-tern of their infecting organisms. A smallstudy has just commenced in which repeateddoses of M. vaccae are being administeredto a group of patients who have failed treat-ment with DOTS-Plus (directly observedtherapy with drugs selected on the basis ofdrug susceptibility profiles). Late in the in-vestigation came publications from Chinasupporting and confirming the data in bothdrug-sensitive and drug-resistant disease, bythe use of multiple injections of their owndifferent preparation of M. vaccae. The trialthat is now beginning in Vietnam of 3 dosesof M. vaccae in the treatment of newly di-agnosed pulmonary tuberculosis, is accom-panied by a chemotherapeutic regimen witha shortened continuation phase. If this im-portant study is successful, immunotherapywith killed M. vaccae should be introducedinto the treatment regimens for tuberculosisworldwide.

Sugawara, I., Yamada, H., Mizuno, S.,Takeda, K., and Akira, S. Mycobacterialinfection in MyD88-deficient mice. Mi-crobiol. Immunol. 47(11) (2003) 841–847.

MyD88 is an adaptor protein that plays amajor role in TLR/IL-1 receptor family sig-naling. To understand the role of MyD88 inthe development of murine tuberculosis invivo, MyD88 knockout (KO) mice aeriallywere infected with Mycobacterium tubercu-losis. Infected MyD88 mice were not highlysusceptible to M. tuberculosis infection, but

they developed granulomatous pulmonarylesions with neutrophil infiltration whichwere larger than those in wild-type (WT)mice (p <0.01). The pulmonary tissue levelsof mRNA for iNOS and IL-18 were slightlylower, but levels of mRNA for IL-1 beta,IL-2, IL-4, IL-6, IL-10, IFN-gamma, andTGF-beta were higher in MyD88 KO mice.IFN-gamma, TNF-alpha, IL-1 beta, and IL-12 also were high in the sera of MyD88 KOmice. There were no statistically significantdifferences in the expression of TNF-alpha,IL-12, and ICAM-1 mRNA between MyD88KO and WT mice. Thus, MyD88 deficiencydid not influence the development of murinetuberculosis. NF-kappa B activity wassimilar in the alveolar macrophages fromthe lung tissues of MyD88 KO and WTmice. Also, there may be a TLR2-specific,MyD88-independent IL-1 receptor/TLR-mediated pathway to activate NF-kappa B inthe host defense against mycobacterial in-fection.—Author’s Abstract

Tomioka, H., Shimizu, T., Sato, K., Sano,C., Kamei, T., Emori, M., and Saito, H.Comparative roles of macrophages andNK cells in the host innate resistance ofmice to Mycobacterium fortuitum infec-tion. J. Infect. 48(1) (2004) 74–80.

OBJECTIVES: Profiles of host innate re-sistance to Mycobacterium fortuitum (MFT)infection in mice and the roles of macro-phages (Mphis) and NK cells in host resist-ance to MFT infection were studied.METHODS: MFT-infected mice with orwithout the treatments to reduce Mphis andNK cells were examined for survival and thebacterial loads in the kidneys during thecourse of infection. RESULTS: A uniqueprofile of strain difference was found in theinnate resistance of mice to MFT. A/J,C3H/He and DBA/2 mice were susceptible,while BALB/c, B10A and C57BL/6 micewere resistant, in terms of survival afterMFT infection. Such profiles of host resis-tance to MFT were essentially correlatedwith the ability of individual strain mice toprevent the bacterial growth in the early pe-riods after infection. These profiles were dif-ferent from the strain difference controlledby Bcg gene. Studies using carrageenan,anti-asialo GM1 antibody, and NK cell-

242 International Journal of Leprosy 2004

deficient beige mice indicated the importantroles of Mphis and NK cells in the host in-nate defense against MFT. CONCLU-SIONS: These findings suggest that Bcggene does not control the host resistance toMFT and that both Mphis and NK cells playcrucial roles in the host innate resistance toMFT infection.—Authors’Abstract

Walker, C., Sawicka, E., and Rook, G. A.Immunotherapy with mycobacteria. Curr.Opin. Allergy Clin. Immunol. 3(6) (2003)481–486.

SUMMARY: PURPOSE OF REVIEW:To summarize and evaluate critically recentprogress with mycobacteria as a potentialnovel disease modifying treatment strategyin asthma. RECENT FINDINGS: The linkbetween exposure to pathogenic or sapro-phytic mycobacteria and protection fromallergic diseases is still controversial, andrecent epidemiological studies, which ad-dressed only exposure to Mycobacteriumtuberculosis or bacillus Calmette-Guerin,did not help to clarify this issue. Moreover,the clear efficacy of mycobacterial treatmentseen in animal models has not been repro-duced in human asthma, and a recent smallstudy testing the hypothesis that heat-killedMycobacterium vaccae attenuates asthmaticreactions after allergen challenge did notprovide convincing results. However, it hasbeen shown that treatment of mice with M.vaccae induces the generation of allergen-specific T regulatory cells capable of sup-pressing allergen-mediated eosinophiliclung inflammation, suggesting that a generaldeficiency of T regulatory cell activity mightbe responsible for the increased prevalenceof asthma. This hypothesis is supported byfindings that a lack of T regulatory cells, asfound in genetic disorders of man andmouse attributable to a mutation of Foxp3,a transcription factor specifically expressedby T regulatory cells, is associated withmanifestations of severe atopy and autoim-munity, precisely the spectrum of diseaseslinked to the hygiene hypothesis. SUM-MARY: Further studies on the relationshipbetween mycobacteria and atopic disordersare needed, but there is reason to believe

that the novel findings and molecularmechanisms associated with mycobacterialinfections will further strengthen the cur-rently unproved therapeutic value of im-munotherapy with mycobacteria.—Au-thors’ Abstract

Waters, W. R., Palmer, M. V., Nonnecke,B. J., Whipple, D. L., and Horst, R. L.Mycobacterium bovis infection of vita-min D-deficient NOS2–/– mice. Microb.Pathog. 36(1) (2004) 11–17.

Vitamin D deficiency is associated withan increased risk for tuberculosis infec-tion. Studies using in vitro systems indi-cate that 1,25-dihydroxyvitamin D(3) [i.e.,1,25(OH)(2)D(3)], the most active form ofthe vitamin, enhances mycobacterial killingby increasing nitric oxide (NO) production.To evaluate concurrently the role of1,25(OH)(2)D(3) and NO on the host re-sponse to tuberculosis infection, mice defi-cient in NO synthase 2 (NOS2(–/–)) and/orvitamin D were aerosol-challenged withMycobacterium bovis and subsequentlyevaluated for mycobacterial colonizationand lesion formation. Infected NOS2(–/–)mice developed severe necrotizing pyo-granulomatous inflammation of the lungswith heavy M. bovis colonization and sys-temic dissemination of the bacillus. Colo-nization and lung lesion area of NOS2(–/–)mice exceeded that of NOS2(+/+) mice. Ad-ditionally, disease progression was morerapid in NOS2(–/–) mice than in NOS2(+/+)mice. Lung colonization and lesion area ofvitamin D deficient mice exceeded that ofvitamin D replete mice, regardless of NOS2phenotype. However, effects of vitamin Don colonization, but not lesion area, weremore pronounced in NOS2(+/+) mice thanin NOS2(–/–) mice. These findings are con-sistent with the current hypothesis that1,25(OH)(2)D(3) enhances mycobacterialkilling through a NO-dependent mecha-nism. As responses of NOS2(–/–) mice wereaffected by 1,25(OH)(2)D(3) deficiency, al-beit to a lesser extent than were those ofNOS2(+/+) mice, NO-independent actionsof 1,25(OH)(2)D(3) also likely exist.—Au-thors’Abstract

72, 2 Current Literature, Experimental Infections 243

Bierrenbach, A. L., Cunha, S. S., Barreto,M. L., Pereira, S. M., Dourado, I., Ichi-hara, M. Y., Brito, S. C., and Rodrigues,L. C. Tuberculin reactivity in a populationof school children with high BCG vacci-nation coverage. Pan American J. Pub.Health 13(5) (2003) 285–293.

A study was conducted to investigate theinfluence of BCG vaccination or revaccina-tion on tuberculin skin test reactivity, inorder to guide the correct interpretation ofthis test in a setting of high neonatal BCGvaccination coverage and an increasingBCG revaccination coverage at school age.In 1997, we conducted tuberculin skin test-ing and BCG scar reading in 1148 childrenaged 7–14 years old in the city of Salvador,Bahia, Brazil. We measured the positive ef-fect of the presence of one or two BCG scarson the proportion of tuberculin skin test re-sults above different cut-off levels (indura-tion sizes of ≥5 mm, ≥10 mm, and ≥15 mm)and also using several ranges of indurationsize (0, 1–4, 5–9, 10–14, and ≥15 mm). Wealso measured the effects that age, gender,and the school where the child was enrolledhad on these proportions. The proportion oftuberculin results ≥10 mm was 14.2% (95%confidence interval (CI) = 8.0%–20.3%) forchildren with no BCG scar, 21.3% (95% CI= 18.5%–24.1%) for children with one BCGscar, and 45.0% (95% CI = 32.0%–58.0%)for children with two BCG scars. There wasevidence for an increasing positive effect ofthe presence of one and two BCG scars onthe proportion of results ≥5 mm and ≥10mm. Similarly, there was evidence for an in-creasing positive effect of the presence ofone and two scars on the proportion of tu-berculin skin test results in the ranges of 5–9mm and of 10–14 mm. The BCG scar effecton the proportion of results ≥5 mm and ≥10mm did not vary with age. There was no ev-idence for BCG effect on the results ≥15mm. In Brazilian school children, BCG-induced tuberculin reactivity is indistin-guishable, for results under 15 mm, fromreactivity induced by Mycobacterium tuber-culosis infection. BCG revaccination atschool age increases the degree of BCG-induced tuberculin reactivity found among

school children. This information should betaken into account in tuberculin skin testsurveys intended to estimate M. tuberculo-sis prevalence or to assess transmission pat-terns as well as in tuberculin skin testing ofindividuals used as an auxiliary tool indiagnosing tuberculosis. Taking this infor-mation into consideration is especially im-portant when there is increasing BCG re-vaccination coverage.

Dony, J. F., Ahmad, J., and Khen Tiong,Y. Epidemiology of tuberculosis and lep-rosy, Sabah, Malaysia. Tuberculosis(Edinb). 84(1–2) (2004) 8–18.

The objectives in this epidemiology re-view are to measure and report the extent ofmorbidity and mortality due to tuberculosis(TB), the proportion of new sputum smearpositive cases in districts and the status ofcohort analysis as of 1999. As for leprosy,the main objective is to determine morbid-ity and the treatment outcomes of MultipleDrug Therapy (MDT). Based on the resultsobtained, a comprehensive action plan forprevention, control and monitoring of tu-berculosis and leprosy cases and patients isbeing produced and implemented through-out the state. The analysis concentrated onpatients diagnosed at all out-patient unitsand admitted in all of the state’s hospitals.The patient particulars were recorded usinga standardized format based on TB and Lep-rosy Health Management Information Sys-tem (TB HMIS). TB was the second highestby notification of communicable diseases inMalaysia in 2001. 29% or about one-third ofthe national TB cases are from Sabah. How-ever, it has been noted that there was an av-erage decline of 2.6% in annual notificationsince 10 years ago to date. There was also areduction of 11.4% in 2001 as compared toannual notification in 2000. Immigrants con-tribute more than 24% in detection of newcases since 1990. Treatment success rate interm of completion of treatment to date is82%. Mortality rate has steadily declinedfrom 14 deaths to 7 deaths per 100,000population. Leprosy in Sabah also con-tributes to 30% of the yearly total caseload

244 International Journal of Leprosy 2004

Epidemiology and Prevention

of Malaysia and has the highest notifica-tion rate of 2 per every 100,000 populationas compared to other states. The averageregistered leprosy cases over the past 5years are 239 cases and the prevalence rateis 0.7/10,000 population. The state hassuccessfully achieved its goal to decreaseleprosy as per the World Health Organiza-tion (WHO) goal of yearly overall preva-lence rate of less than 1 case for every10,000 population. However, the districtsof Kudat, Tawau, Lahad Datu, Kota Kina-balu and Semporna are still within theprevalence rate of more than one per10,000 population. This review highlightssome interesting findings which can be in-

corporated into the State and Districts ac-tion plans and strategies. It is also notedthat in order to translate National Plansand Strategies into effective action at thecommunity level, health workers need rel-evant up-to-date knowledge of the patternof health and disease, and of their deter-minants, in each district. The Sabah HealthDepartment continues to organize and sup-port programs related to management andcontrol of tuberculosis and leprosy to pro-gressively reduce the incidence of thesediseases in the community by breaking thechain of transmission of Mycobacteriumtuberculosis and M. leprae, respectively.—Authors’ Abstract

72, 2 Current Literature, Other Mycobacterial Diseases 245

Other Mycobacterial Diseases

Arkwright, P. D., and David, T. J. Effectof Mycobacterium vaccae on atopic der-matitis in children of different ages. Br. J.Dermatol. 149(5) (2003) 1029–1034.

BACKGROUND: Exposure to environ-mental microorganisms is associated withvariations in the prevalence and severity ofatopic diseases. We have previously shownthat administration of a Mycobacterium vac-cae suspension significantly reduced theseverity of atopic dermatitis (AD) in chil-dren aged 5–18 years. OBJECTIVES: Thisstudy aimed to extend these observations toyounger children. METHODS: Fifty-sixchildren aged 2–6 years with moderate tosevere AD were enrolled in a randomized,double-blind, placebo-controlled trial andgiven one intradermal injection of eitherkilled M. vaccae suspension or buffer solu-tion (placebo). Skin surface area affectedand dermatitis severity score were assessedbefore and 1, 3 and 6 months after treatment.RESULTS: Although a 38–54% reduction insurface area affected by dermatitis wasnoted at all time points after M. vaccae ad-ministration (p = 0.005), this improvementwas not significantly different from that ob-served in the placebo group. Meta-analysisof this and our previous cohort (97 childrenaged 2–18 years) showed that M. vaccaewas associated with a significant improve-ment in clinical severity at all ages, whereaswithin the placebo group, younger but notolder children showed a similar improve-

ment. CONCLUSIONS: Despite a reductionin clinical severity associated with M. vac-cae at all ages, no benefit could be foundafter administering M. vaccae to childrenwith AD aged 2–6 years when comparedwith placebo. M. vaccae may offer greaterbenefit in children over 5 years old, whoseAD appears less likely to regress sponta-neously.—Authors’Abstract

Coussens, P. M., Jeffers, A., and Colvin,C. Rapid and transient activation of geneexpression in peripheral blood mononu-clear cells from Johne’s disease positivecows exposed to Mycobacterium paratu-berculosis in vitro. Microb. Pathog. 36(2)(2004) 93–108.

Mycobacterium avium subspecies paratu-berculosis (M. paratuberculosis) is thecausative agent of Johne’s disease in rumi-nants. M. paratuberculosis is a slow-growing intracellular bacterium and infec-tions with M. paratuberculosis can persist ina subclinical state for several years. An earlyand appropriate T cell-mediated cytotoxicresponse (Th1-like) to M. paratuberculosisinfection is often replaced with an antibodyor Th 2-like response as infected animalsmove toward a progressively more clinicalstate. The reasons for this shift in immuneresponse are unknown. Recent studies sug-gest that in vitro exposure of peripheralblood mononuclear cells (PBMCs) from

Johne’s disease positive cows to M. paratu-berculosis for 18–24 hr results in suppressedexpression of numerous immune cell genes.This effect appears at odds with the notionthat immune cells from infected cows wouldrespond to M. paratuberculosis-specificantigens in a vigorous and positive manner.In this report, we detail experiments de-signed to test the hypothesis that many pos-itive changes in PBMC gene expression in-duced by M. paratuberculosis in vitro aretransient, being rapidly suppressed by as yetunknown mechanisms. Our results demon-strate that, indeed, in vitro stimulation withM. paratuberculosis induces rapid changesin infected cow PBMC gene expression(within 2–4 hr of exposure) and that manyof these changes are no longer evident by8–16 hr of exposure to M. paratuberculosis.Although precise mechanisms responsiblefor rapid M. paratuberculosis-mediatedactivation of PBMC gene expression and theloss thereof remain to be determined, ournovel results suggest that PBMCs fromJohne’s disease positive cows are indeedcapable of vigorously responding to M.paratuberculosis and that, for many genes,this response is tempered within 8 hr of ex-posure.—Authors’Abstract

Darie, H. Infection par Mycobacterium ul-cerans: aspects epidemiologiques, clin-iques et thérapeutiques. Bull. Soc. Pathol.Exot. 96(5) (2003) 368–371.

Mycobacteriun ulcerans causing Buruliulcer is an environmental mycobacteria re-sponsible for an infectious necrotizing pan-niculitis. The epidemiology of this disablingdisease is strongly linked to the aquaticecosystem. Occurring mainly in children, it isan emergent public health threat in manyhumid rural tropical areas. Human contami-nation probably follows a direct pecutaneousroute from humid environment, but some in-sects may play a role in transmission. Theclinical features develop in three phases: pre-ulcer, ulcer with unstick margins, healingleading to functional sequelae. Treatment re-lies on antibiotics in order to sterilize the in-fectious focus, together with the surgical re-pair of lost skin and joint deformities, as wellas early physiotherapy. Despite uncertaintiesof in vivo efficacy of antibiotics, it seems

logical to administer chemotherapy with bothRifampicin and Aminoglycosid or Fluro-quinolon and Aminoglycosid. Surgical treat-ment depends on the size of the ulcer, as wellas available techniques and skills on the field.Wide excision and graft are often recom-mended, however limited excision followedby small islet grafts may be successful.—Bulletin de la Société de Pathologie Exotique

den Broeder, A. A., Vervoort, G., van Assen,S., Verduyn Lunel, F., de Lange, W. C.,and de Sevaux, R. G. Disseminated My-cobacterium gordonae infection in a renaltransplant recipient. Transpl. Infect. Dis.5(3) (2003) 151–155.

The use of more intensive immunosup-pressive regimens and the increasing num-ber of patients that are exposed to immuno-suppressive strategies in transplantationmedicine have changed the spectrum of in-fections that is encountered by the clinician.We describe a 62-year-old female renaltransplant recipient receiving immunosup-pressive therapy who developed complaintsof weight loss, diarrhoea, cough, and fever.Increased C-reactive protein and pancy-topenia were found. The presence of Myco-bacterium gordonae, a non-tuberculous my-cobacterium, was eventually demonstratedin bronchoalveolar lavage fluid, bone mar-row, spleen, and liver. Determination of thepathogen was accelerated using a LineProbe Assay, a reverse hybridisation tech-nique using an RNA fragment specific fordifferent mycobacterium species. Treatmentwas initiated using a combination of clarith-romycin, ethambutol, and rifampicin. Theinitial response to treatment was good, butsplenectomy and change of immunosup-pressive and antimycobacterial therapy werenecessary for long-term control of the in-fection. Problems in the diagnosis and treat-ment of this uncommon pathogen are dis-cussed.—Authors’Abstract

Ferrara, E., Lemire, J., Grimm, P. C.,Reznik, V. M., Mendoza, S. A., Leake,J. A., and Benador, N. M. Mycobacte-rial peritonitis in pediatric peritoneal dial-ysis patients. Pediatr. Nephrol. 19(1)(2004) 114–117.

246 International Journal of Leprosy 2004

Peritonitis is the most common complica-tion and the leading cause of death in pedi-atric peritoneal dialysis (PD) patients. Ac-cording to the most recent data availablefrom the North American Pediatric RenalTransplant Cooperative Study (NAPRTCS),approximately 25% of pediatric PD patientswho die succumb to infection. There are noreported cases of Mycobacterium tubercu-losis (MTB) or Mycobacterium avium-intracellulare peritonitis in the NAPRTCSregistry. With an increasing incidence ofMTB worldwide and the impairment of cel-lular immunity in chronic renal failure pa-tients, it is not surprising that mycobacte-rium peritonitis can occur in PD patients.We report two pediatric PD patients withmycobacterial peritoneal infection diag-nosed over an 11-year period at our institu-tion. One patient presented with a malfunc-tioning Tenckhoff catheter and again 3 yearslater with hyponatremia and ascites. Theother presented with recurrent culture-negative peritonitis. These cases illustratethe importance of more extensive evaluationof PD complications, to include evaluationfor mycobacterium with special media orperitoneal biopsy, in the above clinical set-tings if the routine work-up is unreveal-ing.—Authors’Abstract

Fogla, R., Rao, S. K., and Padmanabhan,P. Interface keratitis due to Mycobacte-rium fortuitum following laser in situkeratomileusis. Indian J. Ophthalmol.51(3) (2003) 263–265

A case of unilateral interface keratitis dueto Mycobacterium fortuitum following si-multaneous bilateral LASIK procedure forlow myopia is reported. Excimer photother-apeutic keratectomy was performed to thestromal bed to reduce the infective load. In-tensive topical therapy with topicalamikacin and ciprofloxacin resulted in reso-lution of the keratitis.—Authors’Abstract

Foroumadi, A., and Soltani, F. Antituber-culosis agents. IX. In vitro antimycobac-terial activity of N-(2-phenyl-2-oxoethyl)and N-[2-(4-fluorophenyl)-2- oxoethyl]-ciprofloxacin derivatives against somedrug-resistant strains of Mycobacterium

tuberculosis and Mycobacterium aviumisolates. Boll. Chim. Farm. 142(6) (2003)248–250.

The in vitro antimycobacterial activityof two ciprofloxacin analogues (2a and 2b)containing 2-phenyl-2-axoethyl and 2-(4-fluorophenyl)-2-axoethyl groups attachedto N-4 position of piperazin ring, was eval-uated against M. tuberculosis strains re-sistant to Isoniazid (MIC 2a and 2b = 3.13micrograms/ml), Ethambutol (MIC 2a and2b = 1.56 micrograms/ml), Rifampin (MIC2a and 2b = 1.56 micrograms/ml), Kana-mycin (MIC 2a and 2b = 1.56 micro-grams/ml) and ciprofloxacin (MIC 2a and2b>25 micrograms/ml). Furthermore, theminimum bactericidal concentration (MBC)of 2a and 2b was determined against M. tu-berculosis H37Rv (MBC2a = 6.25 and 2b= 25 micrograms/ml) and strains resistantto isoniazid (MBCs >25 micrograms/ml)and rifampin (MBC2a = 3.13 and 2b =6.25 micrograms/ml). Also, in this studythe activity of 2a and 2b was determinedagainst a strain of M. avium (%Inhibition 2a= 89 and 2b = 95%). Expanded primaryscreening was conducted for 2b (havingMIC <6.25 micrograms/ml) against fiveclinical isolates of M. avium using theMABA and BACTEC 460 systems (MIC =2–4 micrograms/ml). The significant anti-Mycobacterium avium activity of 2b sug-gested further M. avium assays and so, 2bwas then retested at lower concentrationsagainst 30 strains of M. avium includingfive strains resistant to clarithromycin (MIC= 2–16 micrograms/ml).—Authors’Ab-stract

Fox, L. P., Geyer, A. S., Husain, S., Della-Latta, P., and Grossman, M. E. Myco-bacterium abscessus cellulitis and multi-focal abscesses of the breasts in atranssexual from illicit intramammary in-jections of silicone. J. Am. Acad. Derma-tol. 50(3) (2004) 450–454.

We report the case of a 29-year-old trans-sexual who developed Mycobacterium ab-scessus infection after receiving intramam-mary liquid silicone injections in thenonphysician office setting. Our patient rep-resents 1 of 14 confirmed and 11 suspected

72, 2 Current Literature, Other Mycobacterial Diseases 247

cases in New York City of M. abscessus in-fection after illicit cosmetic procedures. Asinjectable cosmetic procedures are becom-ing increasingly popular, dermatologistsshould be aware of both the common andunusual complications. Furthermore, allphysicians should be alerted to the currentcluster of M. abscessus infections after in-jections for cosmetic purposes by nonmed-ical practitioners in New York City.—Au-thors’Abstracts

Hong, H., Gates, P. J., Staunton, J., Stin-ear, T., Cole, S. T., Leadlay, P. F., andSpencer, J. B. Identification using LC-MSn of co-metabolites in the biosynthe-sis of the polyketide toxin mycolactoneby a clinical isolate of Mycobacteriumulcerans. Chem. Commun. (Camb). 22(2003) 2822–2823.

LC-MSn analysis of mycolactone toxinfrom extracts of Mycobacterium ulceranshas shown that minor co-metabolites, in-cluding two previously unreported, differstructurally from mycolactone only in asmall portion of the polyketide side-chain.—Authors’Abstract

Hong, T., Butler, W. R., Hollis, F., Floyd,M. M., Toney, S. R., Tang, Y. W., Steele,C., and Leggiadro, R. J. Characteriza-tion of a novel rapidly growing Myco-bacterium species associated with sepsis.J. Clin. Microbiol. 41(12) (2003) 5650–5653.

A rapidly growing mycobacterium wasisolated five times from blood cultures froma 6-year-old female patient with relapsedpre-B-cell acute lymphocytic leukemia. Allfive isolates had identical nucleotide se-quences for the first 500 bp of the 16S rRNAgene, indicative of a single species. High-performance liquid chromatography analy-sis of mycolic acids indicated that thespecies was similar to Mycobacteriumsmegmatis. Sequence analysis of the 16SrRNA gene (1,455 bp) for one isolatedemonstrated that the species was closelyrelated to Mycobacterium diernhoferi.Based on the phenotypic features and phy-logenetic analysis, it was concluded that the

isolates represented a novel rapidly growingMycobacterium species. The name “Myco-bacterium hackensackense” is proposed forthis unique strain, 147-0552(T), which wasdeposited in the American Type CultureCollection as ATCC BAA-823(T).—Au-thors’Abstract

Jenkin, G. A., Stinear, T. P., Johnson, P.D., and Davies, J. K. Subtractive hy-bridization reveals a type I polyketidesynthase locus specific to Mycobacteriumulcerans. J. Bacteriol. 185(23) (2003)6870–6882.

See Current Literature, Microbiology, p.222.

Koranyi, K. I., and Ranalli, M. A. Myco-bacterium aurum bacteremia in an im-munocompromised child. Pediatr. Infect.Dis. J. 22(12) (2003) 1108–1109.

Mycobacterium aurum was cultured fromthe Broviac catheter of a 5-year-old childwith metastatic Wilms tumor. Removal ofthe catheter resulted in prompt resolution ofthe fever and sterilization of the blood cul-ture. This rapidly growing mycobacterium,previously believed to be a commensal, cancause disease in the immunocompromisedhost.—Authors’Abstract

Marie, I., Heron, F., Lecomte, F., Jarlier,V., Truffot-Pernot, C., Laquerriere, A.,Huerre, M., Levesque, H., and Cour-tois, H. Multiple cerebral abscesses as acomplication of Mycobacterium fortui-tum infection. Eur. J. Intern. Med. 14(6)(2003) 386–389.

Mycobacterium fortuitum is a rapidlygrowing, nontuberculous mycobacteria thathas rarely been associated with central nerv-ous system impairment. We describe thecase of a patient who developed multiplecerebral abscesses revealing Mycobacteriumfortuitum infection. Brain biopsy specimensshowed suppurative, noncaseating, granulo-matous inflammation consisting of epithe-lioid histiocytes and multinucleated giantcells. All clinical signs and CT scan cerebral

248 International Journal of Leprosy 2004

lesions disappeared after institution of ap-propriate antimycobacterial therapy.—Au-thors’Abstract

Marsollier, L., Prevot, G., Honore, N.,Legras, P., Manceau, A. L., Payan, C.,Kouakou, H., and Carbonnelle, B. Sus-ceptibility of Mycobacterium ulcerans toa combination of amikacin/rifampicin. IntJ. Antimicrob. Agents. 22(6) (2003) 562–566.

The effectiveness of rifampicin (RIF),amikacin (AMK) and their combinationwere estimated in the treatment of mice ex-perimentally infected by Mycobacterium ul-cerans and the risk of relapse after the treat-ment was evaluated. After 7 weeks oftreatment with RIF or with the combinationof AMK/RIF and 8 weeks with AMK alone,no viable bacilli were found in the infectedtissues and these remained uninfected dur-ing the following 6 months. Among the micetreated with AMK alone, three mice re-lapsed, but the minimal inhibitory concen-tration of AMK for these isolates remainedunchanged. With RIF alone, two mice re-lapsed and the minimal inhibitory concen-tration of these isolated strains was higher.However, with all the mice treated with bothRIF and AMK, no relapse was observed.—Authors’Abstract

Martinelli, C., Farese, A., Carocci, A.,Giorgini, S., Tortoli, E., and Leoncini, F.First case of Mycobacterium haemophiluminfection in an AIDS patient in Italy. J. Eur.Acad. Dermatol. Venereol. 18(1) (2004)83–85.

Mycobacterium haemophilum, a stronglyacid- and alcohol-fast bacillus belonging tothe group of non-tuberculous mycobacteriawas first described in 1978 as the cause ofcutaneous ulcerating lesions in a womanwith Hodgkin’s disease. Infection due to M.haemophilum is rare but increasing in preva-lence in immnunosuppressed subjects, par-ticularly in patients with acquired immun-odeficiency syndrome (AIDS) patients. Theskin is the most common site of infectionwith erythematous or violaceous papulesand/or nodules that are usually painless at

first, but some elements develop into ab-scesses or ulcers that can become verypainful. The incidence of M. haemophilumis unknown, but cases of infection have beenreported in Australia, Canada, the UnitedStates, France, Israel, the United Kingdomand Taiwan; to date no cases have been re-ported in Italy, thus the case reported here isapparently the first one observed in ourcountry.—Authors’Abstract

Menard, A., Couppié, P., Sainte-Marie,D., Pradinaud, R. Diagnostic par PCRde l’infection due à Mycobacerium ul-cerans: à propos de trois cas observés enGuyane Française. Bull. Soc. Pathol.Exot. 96(5) 403–405.

Mycobacterium ulcerans infection is thethird most important mycobacterial infec-tion in the world. It has been described inmany different countries including FrenchGuiana. The diagnosis of M. ulcerans infec-tion by culture is often difficult because cul-ture is hard to perform in endemic areas andtheir sensitivity is not reliable. As a resultthe diagnosis of this infection is often de-layed. However, molecular methods are nowavailable to diagnose rapidly infections byM. ulcerans and distinguish it from othermycobacteria. We report three cases of skininfection due to M. ulcerans observed inFrench Guiana. Diagnosis was initiallymade by polymerase chain reaction and wasconfirmed later by culture (in two patients)and inoculation to mice (in one patient). Afaster diagnosis of M. ulcerans infectionshould lead to a better prognosis of this in-fection.—Bulletin de la Société de Patholo-gie Exotique

Nolt, D., Michaels, M. G., and Wald, E. R.Intrathoracic disease from nontubercu-lous mycobacteria in children: two casesand a review of the literature. Pediatrics112(5) (2003) e434.

Pulmonary disease caused by nontuber-culous mycobacteria (NTM) is a relativelyrare occurrence in immunocompetent chil-dren. Two cases of endobronchial NTM in-fection in immunocompetent children aredescribed. In addition, 41 other children

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with NTM pulmonary disease reported inthe English literature between 1930 and2003 are reviewed. Clinical manifestationsare either purely respiratory or respiratorywith more widespread systemic symptoms.Compared with children with only respira-tory complaints, children with constitutionalsymptoms from NTM pulmonary disease 1)had symptoms for a shorter period beforepresentation (10 vs 28 days), 2) had more ra-diographic evidence of pulmonary disease,and 3) were treated longer with antimy-cobacterial agents (11.5 months vs 6months). The most common causative or-ganism was Mycobacterium avium com-plex. Pediatricians should be increasinglyaware of NTM in the differential diagnosisof persistent pulmonary disease in previ-ously healthy children.—Authors’Abstract

Olivier, K. N.; NTM in CF Study Group.The natural history of nontuberculousmycobacteria in patients with cystic.Pediatr. Respir. Rev. 5 Suppl (2004)A:S213–A:S216.

With the increasing lifespan of personswith cystic fibrosis (CF), the emergence ofa variety of previously seldom seenpathogens, including the nontuberculousmycobacteria (NTM), has been seen. Deter-mining the impact of these indolent organ-isms on the natural history of cystic fibrosislung disease has been difficult. We initiateda two-stage study in 1993 to assess theprevalence and clinical impact of these or-ganisms among persons with CF in US CFCenters. These organisms were frequentlyrecovered from older patients with relativelymild disease. While over the short, 15-month course of follow-up no significantdifferences in the rate of decline of lungfunction attributable to NTM were seen,concerning changes and progression ofhigh-resolution computed tomography find-ings were seen in patients from whom theseorganisms were repeatedly recovered.—Au-thors’Abstract

Portaels, F., Aguiar, J., Debacker, M.,Guedenon, A., Steunou, C., Zinsou, C.,and Meyers, W. M. Mycobacteriumbovis BCG vaccination as prophylaxis

against Mycobacterium ulcerans os-teomyelitis in Buruli ulcer disease. Infect.Immun. 72(1) (2004) 62–65.

See Current Literature, Experimental In-fections, p. 238.

Pumberger, W., Hallwirth, U., Pawlowsky,J., and Pomberger, G. Cervicofaciallymphadenitis due to atypical mycobacte-ria: a surgical disease. Pediatr. Dermatol.21(1) (2004) 24–29.

Despite the increasing prevalence of cervi-cofacial lymphadenitis due to atypical myco-bacteria (AMB) in children, the true nature ofAMB infection in clinical practice is poorlyunderstood. The purpose of our study was todefine the most common signs and symp-toms, and to establish a workable scheme ofdiagnosis and treatment. Patients fulfilling thecriteria of AMB infection (i.e., clinical signs,positive cultures or polymerase chain reac-tion, histologic features) were included in thestudy. All children underwent a standard sur-gical procedure, depending on pretreatmentand the course of the disease. Sixteen infantspresented with characteristic unilateral lym-phadenopathy predominantly involving thesubmandibular area (13/16). Eight childrenhad been initially treated at various institu-tions by fine-needle puncture or incision, and7 of the 16 patients had received antitubercu-lous multidrug treatment for a varying lengthof time. Complete excision of the affectedlymph nodes was the definitive treatment inall patients. Three children had transient mar-ginal mandibular nerve paralysis that re-solved within a few months in all cases.Recognition of the characteristic features ofAMB adenitis may permit early diagnosisand appropriate surgical treatment.—Au-thors’Abstract

Redaelli de Zinis, L. O., Tironi, A., Nassif,N., and Ghizzardi, D. Temporal bone in-fection caused by atypical mycobacterium:case report and review of the literature.Otol. Neurotol. 24(6) (2003) 843–849.

OBJECTIVE: To discuss the clinical as-pects and management of nontuberculousmycobacteriosis of the temporal bone.

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STUDY DESIGN: Case report and reviewof the literature. SETTING: University hos-pital, tertiary referral center. PATIENT,INTERVENTION, AND RESULTS: Theauthors describe an uncommon case of non-tuberculous mycobacteriosis of the tempo-ral bone in an immunosuppressed 62-year-old woman with facial nerve paralysiscaused by disease complication. The casewas cured with radical tympanomastoidec-tomy and prolonged multiple antibiotic ther-apy. CONCLUSIONS: Nontuberculousmycobacteriosis should be suspected in im-munosuppressed patients with intractablemiddle ear granulations. Cultural and histo-logic examinations are the mainstay for di-agnosis. Long-standing multiantibiotic ther-apy together with aggressive surgery shouldbe considered as appropriate manage-ment.—Authors’Abstract

Roupe, G. [Buruli ulcer—Africa’s latestmycobacterial scourge]. Lakartidningen.100(45) (2003) 3596–3597. [Article inSwedish]

Buruliulcer is an extensive ulceration usu-ally on the extremities. The ulcer can spreadto subcutaneous fat, muscle and even bonecausing osteomyelitis and death. It is the thethird most common mycobacterial diseasein humans after tuberculosis and leprosy.The bacterium grows in still standing waterand infects children through small ulcera-tions in their skin. Mycobacterium ulceransmay also be transmitted by the bite ofaquatic bugs (Naucordiae), which harbor thebacterium in their salivary glands. The dis-ease affects poor people in rural, tropicalareas where deforestation has led to flood-ing rivers, stagnant bodies of water andmarsh. Benin, Cote d’Ivoire and Ghana inWest Africa are seriously hit. Skin trans-plantation is the treatment of choice. Treat-ment with antibiotics has been disappoint-ing.—Author’s Abstract

Saeki, S., Matsuse, H., Shimoda, T., Soe-jima, Y., Ohno, H., and Kohno, S. [Acase of pulmonary Mycobacterium gor-donae infection with pleural effusion].Nihon Kokyuki Gakkai Zasshi. 42(1)(2004) 103–107. [Article in Japanese].

A 65-year-old woman, treated with pred-nisolone (5 mg daily) for rheumatoid arthritis,visited our hospital because of right chestpain. Chest CT showed small nodular shad-ows in the right lung accompanied with rightpleural effusion. A pulmonary Mycobacte-rium gordonae infection was diagnosed, sinceM. gordonae was identified twice from hersputum. She was treated with rifampicin,ethambutol and streptomycin for two months,and then streptomycin was replaced with clar-ithromycin. Three months after the initialtreatment, M. gordonae was eradicated fromher sputum. Pleural puncture revealed bloody,exudative, lymphocytotic pleural effusion, butno malignant cells were identified. Althoughpathological diagnosis by thoracoscopic pleu-ral biopsy could not be performed, it is likelythat the pleural effusion was associated withthe pulmonary M. gordonae infection in thepresent case.—Authors’Abstract

Saiman, L. The mycobacteriology of non-tuberculous mycobacteria. Paediatr. Respir.Rev. 5 Suppl. (2004) A:S221– A:S223.

The genus Mycobacterium consists of>50 species that have been associated withhuman disease. Mycobacterium are cate-gorised into M. tuberculosis and NTM thatare also subdivided into rapid growers andnon-rapid growers. Five major clinical syn-dromes have been described that are attrib-utable to mycobacterium. These include:pulmonary disease; lymphadenitis; skin, softtissue, and skeletal infections; catheter-related blood-stream infections in immuno-compromised hosts; and disseminated dis-ease in persons with AIDS. There is verylimited documentation of person-to-persontransmission of NTM. Nosocomial infec-tions and outbreaks caused by inadequatedisinfection/sterilization of medical devicesor environmental contamination of medica-tions or medical devices are well described.Staining for AFB, culture, histopathologic,or genetic amplification technologies areused to detect and identify mycobacterium.Pulsed-field gel electrophoresis is themethod of choice to determine strain relat-edness. At present, susceptibility testing fornon-tuberculous mycobacteria is not fullystandardized and has not been correlatedwith clinical outcomes.—Authors’Abstract

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Sechi, L. A., Mura, M., Tanda, E., Lissia,A., Fadda, G., and Zanetti, S. Mycobac-terium avium sub. paratuberculosis in tis-sue samples of Crohn’s disease patients.New Microbiol. 27(1) (2004) 75–77.

Crohn’s disease is a non-specific chronictransmural inflammatory disease. The dis-ease was associated with a frameshift mu-tation in the NOD2 gene. Nevertheless,other researchers associated the presence ofM. paratuberculosis within the intestinal tis-sues of patients with the disease. An adapted“in situ hybridization” technique was usedto detect IS900 M. paratuberculosis DNA inparaffin embedded tissue from Crohns tissuedisease samples. We were able to identifyM. paratuberculosis DNA in around 69% ofthe paraffine embedded intestinal samples ofCrohn’s disease patients analysed. The pres-ence of M. paratuberculosis DNA in the in-testinal samples analysed does not neces-sarily mean that M. paratuberculosis isresponsible for Crohn’s disease. Our resultssupport the hypothesis that infection may becaused by cell wall defective M. paratuber-culosis since no bacteria were detected byZiehl Neelsen stain.—Authors’Abstract

Sermet-Gaudelus, I., Le Bourgeouis, M.,Pierre-Audigier, C., Offredo, C., Guille-mot, D., Halley, S., Akoua-Koffi, C.,Vincent, V., Sivadon-Tardy, V., Fer-roni, A., Berche, P., Scheinmann, P.,Lenoir, G., and Gaillard, J.-L. Myco-bacterium abcessus with Cystic Fibrosis.Emerging Infect. Dis. 9(12) (2003) 1587–1591.

We prospectively studied 298 patientswith cystic fibrosis (mean age 11.3 years;range 2 months to 32 years; sex ratio, 0.47)for nontuberculous mycobacteria in respira-tory samples from January 1, 1996, to De-cember 31, 1999. Mycobacterium abscessuswas by far the most prevalent nontubercu-lous mycobacterium: 15 patients (6 male, 9female; mean age 11.9 years; range 2.5–22years) had at least one positive sample forthis microorganism (versus 6 patients posi-tive for M. avium complex), including 10with >3 positive samples (versus 3 patientsfor M. avium complex). The M. abscessusisolates from 14 patients were typed by

pulsed-field gel electrophoresis: each of the14 patients harbored a unique strain, rulingout a common environmental reservoir orperson-to-person transmission. Water sam-ples collected in the cystic fibrosis centerwere negative for M. abscessus. This majormycobacterial pathogen in children andteenagers with cystic fibrosis does not ap-pear to be acquired nosocomially.—Emerg-ing Infectious Diseases

Shiratsuch, H., and Basson, M. D. Cas-pase activation may be associated withMycobacterium avium pathogenicity.Am. J. Surg. 186(5) (2003) 547–551.

BACKGROUND: Mycobacterium aviumcauses disseminated infection in immuno-compromised patients and triggers aprocess resembling Crohn’s disease ingoats. Colony morphotypes predict patho-genicity. Smooth-transparent (SmT) mor-photypes are more virulent and induce lessinterleukin (IL)-1beta and IL-18 productionthan avirulent smooth-domed (SmD) mor-photypes. Caspases are essential for IL-1beta and IL-18 production. METHODS:Caspase activation was examined in humanmonocytes after M. avium infection. RE-SULTS: Fresh monocytes constitutively ex-pressed caspase-1 mRNA and pro-caspase-1. The M. avium infection increasedmonocyte caspase-1 mRNA expression.Furthermore, SmD-infected monocytes ex-pressed 2.3-fold higher levels (p <0.05, N =3) of activated caspases than SmT-infectedmonocytes. Caspase-1 inhibition signifi-cantly reduced IL-1beta production bySmT- and SmD-infected monocytes (p<0.05, N = 4). Caspase-3 inhibition inhib-ited IL-1beta production 43.5% ± 8.0% (p<0.02, N = 4) by SmD-infected but notSmT-infected monocytes. CONCLU-SIONS: Decreased mature IL-1beta releaseby SmT-infected monocytes may reflect se-lective induction of caspase-1 activity butnot caspase-3. Differential caspase expres-sion in monocytes after infection may con-tribute to M. avium pathogenicity in hu-mans.—Authors’ Abstract

Sugihara, E., Hirota, N., Niizeki, T.,Tanaka, R., Nagafuchi, M., Koyanagi,

252 International Journal of Leprosy 2004

T., Ono, N., Rikimaru, T., and Aizawa,H. Usefulness of bronchial lavage for thediagnosis of pulmonary disease causedby Mycobacterium avium-intracellularecomplex (MAC) infection. J. Infect.Chemother. 9(4) (2003) 328–332.

To evaluate the usefulness of bronchiallavage for the diagnosis of pulmonary diseasedue to Mycobacterium avium-intracellularecomplex (MAC) infection, we examined theclinical records and bacteriologic findings ofpatients admitted to our hospital between1999 and 2002 who fulfilled the 1997American Thoracic Society (ATS) criteriafor MAC pulmonary infection. Broncho-scopic examinations were performed inthose patients with MAC pulmonary diseasewho showed negative sputum smears formycobacteria on 3 consecutive days (N =14) or who could not expectorate sputum (n= 2). The bronchial lavage sample wassmear-positive for acid-fast bacilli in 8 ofthe 16 patients (50.0%), polymerase chainreaction (PCR)-positive for MAC in 10 of15 (66.7%), and culture-positive for MACin 15 of 16 (93.7%). The brushing samplewas positive for MAC in 5 of 14 patients(35.7%), and transbronchial lung biopsy(TBLB)-positive for MAC in 2 of 5(40.0%). MAC was isolated by culture ofbronchial lavage samples in a higher per-centage of patients than that in whom MACwas isolated by sputum culture, and wecould make an early diagnosis of MAC pul-monary disease based on the smear and PCRresults for bronchial lavage samples.Bronchial lavage is useful to screen sputumsmear-negative patients suspected of havingMAC pulmonary disease.—Authors’ Ab-stract

Teelken, M. A., Stienstra, Y., Ellen, D. E.,Quarshie, E., Klutse, E., van derGraaf, W. T. A., van der Werf, T. S. Bu-ruuli ulcer: differences in treatment out-come between two centers in Ghana.Acta Tropica 88(1) (2003) 51–56.

Objectives: Assess treatment effects byfollowing up patients treated for Buruli ulcerin two hospitals with different treatment as-pects, including widely differing surgicalpractices. Patients/methods: Treated patients

were retrospectively identified from hospitalrecords. Between 1994 and July 2000, 136patients had been admitted for Buruli ulcerin both hospitals, and lived in areas coveredin the research period. 78 (57%) Patientswere included in the study. Treatment andstatus of the patient were analyzed. Results:27 (35%) Patients were not healed. Of the 33patients treated in hospital A, six (18%)were not healed at follow-up, whereas of the45 patients treated in hospital B, 21 (47%)were not healed. The length of stay in hos-pital A was significantly longer (p = 0.002),and more operations on average were doneper patient (p = 0.002). In a univariate analy-sis, treatment in hospital A; the use of ri-fampicin (p = 0.013); and BCG vaccinationstatus (p = 0.04) were all significantly asso-ciated with ulcer healing. Using a logistic re-gression model for multivariate analysis,only treatment as given in hospital A, withstandard prctice of wide surgical excision,appeared to predict ulcer healing independ-ently (p = 0.02). Conclusions: This studyshows large differences in treatment out-come between the two hospitals; the resultssupport the hypothesis that extent of surgicaltreatment influences the chance of healing ofBuruli ulcer.—Tropical Disease Bulletin

Ticca, F., Comparcola, D., Graziani, M.C., Lancella, L., Marsella, P., Nicolosi,L., Pierro, V., Rivosecchi, M. R., Ticca,C., and Tieri, L. [Otomastoiditis causedby Mycobacterium avium: a case report].Infez. Med. 5(2) (1997) 114–117. [Arti-cle in Italian]

Non tuberculous Mycobacterial (NTM)infections mainly affect immunocompro-mised patients, appearing as disseminated orpulmonary disease. In immunocompetentchildren the most common form of infectionwith NTM is cervical adenitis. Ear infectionseems to be a rare disease. We present a caseof otomastoiditis caused by Mycobacteriumavium in a 15 months old child, immunolog-ically normal. Patient was referred for per-sistent right otitis unresponsive to routinemedical therapy. TC scan of the ear and tem-poral bones revealed: soft tissue in externalauditory canal, Eustachian canal, and middleear overlying ossicles with erosion of tegmentympani. Tuberculin skin test was positive

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with 5 units PPD and culture yielded M.avium. The patient undergo timpanomas-toidectomy and medical therapy with antitu-berculous drugs and steroids, subsequentlyhe was given Clarithromycin and Rifabutin.M. avium is an ubiquitous low gradepathogen found in soil, water, dust and food.There is no evidence of direct transmission.Only a few cases of otomastoiditis due to M.avium have previously been reported. Thecase presented underlines the importance ofmicrobiological investigations. When a NTMinfection is suspected surgeons and infec-tious diseases specialists should cooperate tofind an optimal treatment regimen of this un-usual disease.—Authors’Abstract

Toy, B. R. Foreign-body reaction with My-cobacterium abscessus superinfection.Dermatol. Online J. 9(4) (2003) 29.

Mycobacterium abscessus is a rare causeof skin and soft tissue infections that oftenresults from inoculation with contaminatedforeign material. A 41-year-old woman isdescribed regarding an outbreak of M. ab-scessus following soft tissue augmentation.Clinical features and treatment options arereviewed.—Author’s Abstract

Winthrop, K. L., Albridge, K., South, D.,Albrecht, P., Abrams, M., Samuel, M.

C., Leonard, W., Wagner, J., andVugia, D. J. The clinical managementand outcome of nail salon-acquired My-cobacterium fortuitum skin infection.Clin. Infect. Dis. 38(1) (2004) 38–44.

Nontuberculous mycobacterial infectionsare becoming more common. Recently, My-cobacterium fortuitum and other rapidlygrowing mycobacteria have been found tocause severe skin and soft-tissue infectionsin association with nail salon whirlpool foot-baths. We recently investigated a large out-break of M. fortuitum furunculosis amongwomen who received pedicures at a singlenail salon. To better define the clinicalcourse of such infections, we collected clin-ical details from physicians who were treat-ing outbreak patients. We constructed mul-tivariable linear models to evaluate theeffect of antibiotic treatment on disease du-ration. Sixty-one patients were included inthe investigation. The mean disease durationwas 170 days (range, 41–336 days). Forty-eight persons received antibiotic therapy fora median period of 4 months (range, 1–6months), and 13 persons were untreated.Isolates were most susceptible to cipro-floxacin and minocycline. Early administra-tion of therapy was associated with shorterduration of disease only in persons withmultiple boils (p <.01). One untreated,healthy patient had lymphatic disease dis-semination.—Authors’Abstract

254 International Journal of Leprosy 2004

Molecular and Genetic Studies

Adekambi, T., Colson, P., and Drancourt,M. rpoB-based identification of nonpig-mented and late-pigmenting rapidlygrowing mycobacteria. J. Clin. Micro-biol. 41(2) 2003 5699–5708.

See Current Literature, Microbiology, p. 219.

Al-Attiyah, R., and Mustafa, A. S.Computer-assisted prediction of HLA-DR binding and experimental analysis forhuman promiscuous Th1-cell peptides inthe 24 kDa secreted lipoprotein (LppX)

of Mycobacterium tuberculosis. Scand. J.Immunol. 59(1) 2004 16–24.

The secreted 24 kDa lipoprotein (LppX)is an antigen that is specific for Mycobacte-rium tuberculosis complex and M. leprae.The present study was carried out to identifythe promiscuous T helper 1 (Th1)-cell epi-topes of the M. tuberculosis LppX (MT24,Rv2945c) antigen by using 15 overlappingsynthetic peptides (25 mers overlapping by10 residues) covering the sequence of thecomplete protein. The analysis of Rv2945csequence for binding to 51 alleles of nineserologically defined HLA-DR molecules,

by using a virtual matrix-based predictionprogram (propred), showed that eight of the15 peptides of Rv2945c were predicted tobind promiscuously to ≥10 alleles frommore than or equal to three serologically de-fined HLA-DR molecules. The Th1-cell re-activity of all the peptides was assessed inantigen-induced proliferation and interferon-gamma (IFN-gamma)-secretion assays withperipheral blood mononuclear cells (PBMCs)from 37 bacille Calmette-Guerin (BCG)-vaccinated healthy subjects. The resultsshowed that 17 of the 37 donors, which rep-resented an HLA-DR-heterogeneous group,responded to one or more peptides ofRv2945c in the Th1-cell assays. Althougheach peptide stimulated PBMCs from one ormore donors in the above assays, the bestpositive responses (12/17 (71%) responders)were observed with the peptide p14 (aa196–220). This suggested a highly promis-cuous presentation of p14 to Th1 cells. Inaddition, the sequence of p14 is completelyidentical among the LppX of M. tuberculo-sis, M. bovis and M. leprae, which furthersupports the usefulness of Rv2945c and p14in the subunit vaccine design against bothtuberculosis and leprosy.—Authors’ Ab-stract

Beyene, D., Aseffa, A., Harboe, M., Ki-dane, D., Macdonald, M., Klatser, P.R., Bjune, G. A., and Smith, W. C.Nasal carriage of Mycobacterium lepraeDNA in healthy individuals in Lega Robivillage, Ethiopia. Epidemiol. Infect.131(2) (2003) 841–848.

See Current Literature, Clinical Sciences,p. 192.

Bhanu, N. V., van Soolingen, D., vanEmbden, J. D., and Seth, P. Two Myco-bacterium fortuitum strains isolated from pulmonary tuberculosis patients inDelhi harbor IS6110 homologue. Diagn.Microbiol. Infect. Dis. 48(2) (2004)107–110.

We report 2 isolates of Mycobacteriumfortuitum from patients with pulmonary tu-berculosis lesions hybridizing to IS6110probe in restriction fragment length poly-

morphism (RFLP) typing. Results of poly-merase chain reaction-hybridization formatsusing the non-specific region of IS6110 forthe molecular detection of mycobacteria inclinical material should be interpreted withcaution.—Authors’Abstract

Bisen, P. S., Garg, S. K., Tiwari, R. P.,Tagore, P. R., Chandra, R., Karnik, R.,Thaker, N., Desai, N., Ghosh, P. K.,Fraziano, M., and Colizzi, V. Analysisof the shotgun expression library of theMycobacterium tuberculosis genome forimmunodominant polypeptides: potentialuse in serodiagnosis. Clin. Diagn. Lab.Immunol. 10(6) (2003) 1051–1058.

A recombinant DNA strategy was appliedto analyze and screen the shotgun expres-sion library from a clinically confirmed localvirulent isolate of Mycobacterium tubercu-losis with sera from tuberculosis patients,which led to expression and purification ofhighly immunoreactive and specific myco-bacterial antigens expressed during thecourse of active disease which could be ofdiagnostic significance. An enzyme-linkedimmunoassay for diagnosis of tuberculosiswas devised by using a shotgun immunoex-pression library in the lambdagt11 vector.DNA from a virulent M. tuberculosis patientisolate (TBW-33) confirmed with theBACTEC 460 system was sheared and ex-pressed to generate shotgun polypeptides.beta-Galactosidase fusion proteins capableof demarcating active tuberculosis infec-tions from Mycobacterium bovis BCG-vaccinated healthy subjects or people har-boring environmental mycobacteria wereselected by comparative immunoreactivitystudies. Promising mycobacterial DNA cas-settes were subcloned and expressed into theglutathione S-transferase (GST) fusion vec-tor pGEX-5X-1 with a strong tac promoterand were expressed in Escherichia coliBL21. These fusion proteins were severed ata built-in factor Xa recognition site to sepa-rate the GST tags and were utilized in an in-direct enzyme-linked immunoassay forserodiagnosis of patients with active tuber-culosis. The system offered a clear demar-cation between BCG-vaccinated healthysubjects and patients with active tuberculo-sis and proved to be effective in detecting

72, 2 Current Literature, Molecular and Genetic Studies 255

pulmonary as well as extrapulmonary tu-berculosis, with an overall sensitivity of84.33% and an overall specificity of93.62%.—Authors’Abstract

Chattopadhyay, C., Sau, S., and Mandal,N. C. Cloning and characterization of thepromoters of temperate mycobacterio-phage L1. J. Biochem. Mol. Biol. 36(6)(2003) 586–592.

Four putative promoters of the temperatemycobacteriophage L1 were cloned by de-tecting the beta-galactosidase reporter ex-pression in E. coli transformants that carriedL1 specific operon-fusion library. All of thefour L1 promoters were also found to ex-press differentially in the homologous envi-ronment of mycobacteria. Of the four pro-moters, two were suggested to be theputative early promoters of L1 since theyexpress within 0 to 10 min of the initiationof the lytic growth of L1. One of the puta-tive early promoters showed a relatively bet-ter and almost identical activity in both E.coli and M. smegmatis. By a sequenceanalysis, we suggest that the L1 insert thatcontained the stronger early promoter pos-sibly carries two convergent E. colisigma70-like L1 promoters, which are sep-arated from each other by about 300 nu-cleotides. One of them is the early promoterof L1 as it showed a 100% similarity withthe early Pleft promoter of the homoimmunephage L5. The second promoter, designatedP4, was suggested for its appreciable levelof reporter activity in the absence of the -10element of the Pleft equivalent of L1. By an-alyzing most of the best characterized my-cobacteriophages-specific promoters, in-cluding the L1 promoter P4, we suggest thatboth the -10 and -35 hexamers of the my-cobacteriophage promoters are highly con-served and almost similar to the consensus -10 and -35 hexamers of the E. coli sigma70promoters.—Authors’Abstract

Cheng, A. F., Yew, W. W., Chan, E. W.,Chin, M. L., Hui, M. M., and Chan, R.C. Multiplex PCR amplimer conforma-tion analysis for rapid detection of gyrAmutations in fluoroquinolone-resistantMycobacterium tuberculosis clinical iso-

lates. Antimicrob. Agents Chemother.48(2) (2004) 596–601.

A new strategy known as multiplex PCRamplimer conformation was developed fordetection of mutation in the gyrA gene of138 clinical isolates of Mycobacterium tu-berculosis. The method generated a single-stranded and heteroduplex DNA bandingpattern of multiplex PCR amplimers of theregion of interest that was extremely sensi-tive to specific mutations, thus enablingmuch more sensitive and reliable mutationanalysis compared to the standard single-stranded conformation polymorphism tech-nique. The genetic profiles of the gyrA geneof the 138 isolates as detected by MPACwere confirmed by nucleotide sequencingand were found to correlate strongly withthe in vitro susceptibilities of the mutantstrains to six fluoroquinolones (ofloxacin,levofloxacin, sparfloxacin, moxifloxacin,gatifloxacin, and sitafloxacin). All 32 iso-lates that contained gyrA mutations exhib-ited cross-resistance to the six fluoro-quinolones (ofloxacin MIC for 90% ofstrains >16 mg/liter), although moxi-floxacin, gatifloxacin, and sitafloxacin (MICfor 90% of strains ≤4 mg/liter) were appar-ently more active than ofloxacin, lev-ofloxacin, and sparfloxacin (MIC for 90% ofstrains >/==″ BORDER=″0″ >16 mg/liter).All gyrA mutations were clustered in codons90, 91, and 94, and aspartic acid 94 wasmost frequently mutated. Twenty-three iso-lates without gyrA mutations were alsofound to exhibit reduced susceptibility toofloxacin (MIC for 90% of strains = 4mg/liter), but largely remained susceptibleto other drugs (MIC for 90% of strains ≤1mg/liter). Another 83 isolates without muta-tions were fully susceptible to all six fluoro-quinolones (ofloxacin MIC for 90% ofstrains = 1 mg/liter). In conclusion, high-level phenotypic resistance to fluoro-quinolones among M. tuberculosis clinicalisolates, which appears to be predominantlydue to gyrA mutations, may be readily de-tected by genotyping techniques such asmultiplex PCR amplimer conformation.—Authors’Abstract

Jamieson, S. E., Miller, E. N., Black, G. F.,Peacock, C. S., Cordell, H. J., Howson,

256 International Journal of Leprosy 2004

J. M., Shaw, M. A., Burgner, D., Xu,W., Lins-Lainson, Z., Shaw, J. J.,Ramos, F., Silveira, F., and Blackwell,J. M. Evidence for a cluster of genes onchromosome 17q11-q21 controlling sus-ceptibility to tuberculosis and leprosy inBrazilians. Genes Immun. 5(1) (2004)46–57.

The region of conserved synteny on mousechromosome 11/human 17q11-q21 is knownto carry a susceptibility gene(s) for intra-macrophage pathogens. The region is rich incandidates including NOS2A, CCL2/MCP-1,CCL3/MIP-1alpha, CCL4/MIP-1beta,CCL5/RANTES, CCR7, STAT3 and STAT5A/5B.To examine the region in man, we studied92 multicase tuberculosis (627 individuals)and 72 multicase leprosy (372 individuals)families from Brazil. Multipoint nonpara-metric analysis (ALLEGRO) using 16 mi-crosatellites shows two peaks of linkage forleprosy at D17S250 (Z(lr) score 2.34; p =0.01) and D17S1795 (Z(lr) 2.67; p = 0.004)and a single peak for tuberculosis atD17S250 (Z(lr) 2.04; p = 0.02). Combinedanalysis shows significant linkage (peakZ(lr) 3.38) at D17S250, equivalent to an al-lele sharing LOD score 2.48 (p = 0.0004).To determine whether one or multiple genescontribute, 49 informative single nucleotidepolymorphisms were typed in candidategenes. Family-based allelic association test-ing that was robust to family clusteringdemonstrated significant associations withtuberculosis susceptibility at four loci sepa-rated by intervals (NOS2A-8.4 Mb-CCL18-32.3 kb-CCL4-6.04 Mb-STAT5B) up toseveral Mb. Stepwise conditional logisticregression analysis using a case/pseudo-control data set showed that the four genescontributed separate main effects, consistentwith a cluster of susceptibility genes across17q11.2.—Authors’Abstract

Kalate, R. N., Tambe, S. S., and Kulkarni,B. D. Artificial neural networks for pre-diction of mycobacterial promoter se-quences. Comput. Biol. Chem. 27(6)(2003) 555–564.

A multilayered feed-forward ANN ar-chitecture trained using the error-back-propagation (EBP) algorithm has been de-

veloped for predicting whether a given nu-cleotide sequence is a mycobacterial pro-moter sequence. Owing to the high predic-tion capability (congruent with 97%) of thedeveloped network model, it has been fur-ther used in conjunction with the caliper ran-domization (CR) approach for determiningthe structurally/functionally important re-gions in the promoter sequences. The resultsobtained thereby indicate that: (i) upstreamregion of -35 box, (ii) -35 region, (iii) spacerregion and, (iv) -10 box, are important formycobacterial promoters. The CR approachalso suggests that the -38 to -29 region playsa significant role in determining whether agiven sequence is a mycobacterial promoter.In essence, the present study establishesANNs as a tool for predicting mycobacterialpromoter sequences and determining struc-turally/functionally important sub-regionstherein.—Authors’Abstract

Lee, C. K., Gi, H. M., Cho, Y., Kim, Y. K.,Lee, K. N., Song, K. J., Song, J. W.,Park, K. S., Park, E. M., Lee, H., andBai, G. H. The genomic heterogeneityamong Mycobacterium terrae complexdisplayed by sequencing of 16S rRNAand hsp 65 genes. Microbiol. Immunol.48(2) (2004) 83–90.

The species identification within Myco-bacterium terrae complex has been knownto be very difficult. In this study, the ge-nomic diversity of M. terrae complex witheighteen clinical isolates, which were ini-tially identified as M. terrae complex byphenotypic method, was investigated, in-cluding that of three type strains (M. terrae,M. nonchromogenicum, and M. triviale).16S rRNA and 65-kDa heat shock protein(hsp 65) gene sequences of mycobacteriawere determined and aligned with elevenother references for the comparison usingsimilarity search against the GenBank andRibosomal Database Project II (RDP) data-bases. 16S rRNA and hsp 65 genes of M.terrae complex showed genomic hetero-geneity. Amongst the eighteen clinical iso-lates, nine were identified as M. nonchro-mogenicum, eight as M. terrae, one as M.mucogenicum with the molecular character-istic of rapid growth. M. nonchromogenicumcould be subdivided into three subgroups,

72, 2 Current Literature, Molecular and Genetic Studies 257

while M. terrae could be subdivided intotwo subgroups using a 5 bp criterion (>1%difference). Seven isolates in two subgroupsof M. nonchromogenicum were Mycobacte-rium sp. strain MCRO 6, which was closelyrelated to M. nonchromogenicum. The hsp65 gene could not differentiate one M.nonchromogenicum from M. avium or oneM. terrae from M. intracellulare. The nu-cleotide sequence analysis of 16S rRNA andhsp 65 genes was shown to be useful inidentifying the M. terrae complex, but hsp65 was less discriminating than 16SrRNA.—Authors’Abstract

Liu, W., Zhang, C. Y., Tian, L., Li, C. Z.,Wu, X. M., Zhao, Q. M., Zhang, P. H.,Yang, S. M., Yang, H., and Cao, W. C. [Acase-control study on natural-resistance-associated macrophage protein 1 genepolymorphisms and susceptibility to pul-monary tuberculosis]. Zhonghua Yu FangYi Xue Za Zhi. 37(6) (2003) 408–411. [Ar-ticle in Chinese]

OBJECTIVE: To investigate associationbetween the natural-resistance-associatedmacrophage protein 1 (NRAMP1) genepolymorphisms and susceptibility to pul-monary tuberculosis (TB) in Chinese Hanpopulation. METHODS: Hospital-basedcase-control study design was adopted.Polymerase chain reaction (PCR) and re-striction fragment length polymorphism(RFLP) technique were used to type threeNRAMP1 polymorphisms (INT4, D543Nand 3′UTR). Information on related factorsof tuberculosis was collected using a pre-tested standard questionnaire. Univariateand multivariate unconditional logisticanalyses were conducted using SPSS forwindow software package. Totally, 110cases of TB were selected during April 2001to June 2002, with an average age of (27.7 ±12.7) years. Also, 180 cases of healthy con-trol were selected, aged (27.3 ± 9.2) years inaverage. Locus of NRAMP1 polymorphismwas analysed with univariate method. RE-SULTS: Univariate analysis demonstratedthat the D543N G/A and 3′UTR TGTG+/delgenotype occurred more frequently in thecases than in the controls, with crude oddsratios (OR) (95% CI) of 2.22 (1.03–4.78)and 1.93 (1.14–3.26), respectively. No sig-

nificant association was observed betweenTB and INT4 polymorphisms. In multivari-ate analysis, associations of TB and D543NG/A and 3′UTR TGTG+/del genotypes re-mained, adjusted for exposure history andbacille Camette-Guerin immunization. Ad-justed OR (95% CI) was 3.04 (1.12–8.27)and 2.36 (1.20–4.64), respectively. Still, nosignificant association between INT4 poly-morphisms and TB was found. CONCLU-SION: Polymorphisms of D543N and3′UTR locus in NRAMP1 gene might affecttheir susceptibility to TB in Chinese Hanpopulation.—Authors’Abstract

Majeed, A. A., Ahmed, N., Rao, K. R.,Ghousunnissa, S., Kauser, F., Bose, B.,Nagarajaram, H. A., Katoch, V. M.,Cousins, D. V., Sechi, L. A., Gilman, R.H., and Hasnain, S. E. AmpliBASEMTTM: a Mycobacterium tuberculosisdiversity knowledgebase. Bioinformatics20(6) (2004) 989–992.

SUMMARY: AmpliBASE MT trade markis an online databank of high-resolution DNAfingerprints representing fluorescent amplifiedfragment length polymorphism (FAFLP) pro-files or amplitypes developed for the Myco-bacterium tuberculosis complex strains from48 different countries. AmpliBASE MT trademark is based on a relational database man-agement system that is hyperlinked to visual-ize genotyping results in the form of DNAfingerprint images for individual strains. Aflexible search system based on systematiccomparisons of fragment sizes in base pairs al-lows inter-laboratory comparison of FAFLPprofiles. Besides this, the database also dis-plays previously published data on IS6110profiles, spoligotypes, MIRU-VNTRs andlarge sequence polymorphisms along with theFAFLP records that will give the overall com-parisons. Being the first of its kind, Am-pliBASE MT trade mark is expected to be avery helpful tool in strengthening the conceptof ‘geographic genomics’ and will be veryhelpful to molecular epidemiologists andthose interested in diagnostic development fortuberculosis.—Authors’Abstract

Marmiesse, M., Brodin, P., Buchrieser, C,Gutierrez, C., Simoes, N., Vincent, V.,

258 International Journal of Leprosy 2004

Glaser, P., Cole, S. T., and Brosch, R.Macro-array and bioinformatic analysesreveal mycobacterial ‘core’ genes, varia-tion in the ESAT-6 gene family and newphylogenetic markers for the Mycobacte-rium tuberculosis complex. Microbiol-ogy. 150(Pt 2) (2004) 483–496.

To better understand the biology and thevirulence determinants of the two major my-cobacterial human pathogens Mycobacte-rium tuberculosis and Mycobacterium lep-rae, their genome sequences have beendetermined recently. In silico comparisonsrevealed that among the 1439 genes com-mon to both M. tuberculosis and M. leprae,219 genes code for proteins that show nosimilarity with proteins from other organ-isms. Therefore, the latter ‘core’ genes couldbe specific for mycobacteria or even for theintracellular mycobacterial pathogens. Toobtain more information as to whether thesegenes really were mycobacteria-specific,they were included in a focused macro-array, which also contained genes from pre-viously defined regions of difference (RD)known to be absent from Mycobacteriumbovis BCG relative to M. tuberculosis. Hy-bridization of DNA from 40 strains of the M.tuberculosis complex and in silico compar-ison of these genes with the near-completegenome sequences from Mycobacteriumavium, Mycobacterium marinum and Myco-bacterium smegmatis were undertaken toanswer this question. The results showedthat among the 219 conserved genes, veryfew were not present in all the strains tested.Some of these missing genes code for pro-teins of the ESAT-6 family, a group ofhighly immunogenic small proteins whosepresence and number is variable among thegenomically highly conserved members ofthe M. tuberculosis complex. Indeed, the re-sults suggest that, with few exceptions, the‘core’ genes conserved among M. tubercu-losis H37Rv and M. leprae are also highlyconserved among other mycobacterialstrains, which makes them interesting po-tential targets for developing new specificanti-mycobacterial drugs. In contrast, thegenes from RD regions showed great vari-ability among certain members of the M. tu-berculosis complex, and some new specificdeletions in Mycobacterium canettii, Myco-bacterium microti and seal isolates were

identified and further characterized duringthis study. Together with the distribution ofa particular 6 or 7 bp micro-deletion in thegene encoding the polyketide synthasepks15/1, these results confirm and furtherextend the revised phylogenetic model forthe M. tuberculosis complex recently pre-sented.—Authors’Abstract

Matsuoka, M., Zhang, L., Budiawan, T.,Saeki, K., and Izumi, S. Genotyping ofMycobacterium leprae on the basis of thepolymorphism of TTC repeats for analy-sis of leprosy transmission. J. Clin. Mi-crobiol. 42(2) (2004) 741–745.

See Current Literature, Microbiology,Leprosy, p. 224.

Miller, E. N., Jamieson, S. E., Joberty, C.,Fakiola, M., Hudson, D., Peacock, C.S., Cordell, H. J., Shaw, M. A., Lins-Lainson, Z., Shaw, J. J., Ramos, F., Sil-veira, F., and Blackwell, J. M. Genome-wide scans for leprosy and tuberculosissusceptibility genes in Brazilians. GenesImmun. 5(1) (2004) 63–67.

Genome-wide scans were conducted fortuberculosis and leprosy per se in Brazil. Atstage 1, 405 markers (10 cM map) weretyped in 16 (178 individuals) tuberculosisand 21 (173 individuals) leprosy families.Nonparametric multipoint analysis detected8 and 9 chromosomal regions respectivelywith provisional evidence (p <0.05) for link-age. At stage 2, 58 markers from positiveregions were typed in a second set of 22(176 individuals) tuberculosis families, with22 additional markers typed in all families;42 positive markers in 50 (192 individuals)new leprosy families, and 30 additionalmarkers in all families. Three regions(10q26.13, 11q12.3, 20p12.1) retained sug-gestive evidence (peak LOD scores 1.31,1.85, 1.78; p = 0.007, 0.0018, 0.0021) forlinkage to tuberculosis, 3 regions (6p21.32,17q22, 20p13) to leprosy (HLA-DQA, 3.23,p = 5.8 × 10(–5); D17S1868, 2.38, p =0.0005; D20S889, 1.51, p = 0.004). Thepeak at D20S889 for leprosy is 3.5 Mb dis-tal to that reported at D20S115 for leprosyin India. (151 words).—Authors’Abstract

72, 2 Current Literature, Molecular and Genetic Studies 259

Mostowy, S., Cousins, D., and Behr, M. A.Genomic interrogation of the dassiebacillus reveals it as a unique RD1 mu-tant within the Mycobacterium tubercu-losis complex. J. Bacteriol. 186(1) (2004)104–149.

Despite their remarkable genetic homol-ogy, members of the Mycobacterium tuber-culosis complex express very different phe-notypes, most notably in their spectra ofclinical presentation. For example, M. tuber-culosis is regarded as pathogenic to humans,whereas members having deleted RD1, suchas Mycobacterium microti and Mycobacte-rium bovis BCG, are not. The dassie bacillus,an infrequent variant of the M. tuberculosiscomplex characterized as being most similarto M. microti, is the causative agent of tuber-culosis (TB) in the dassie (Procavia capen-sis). Intriguingly, the dassie bacillus is notpathogenic to rabbits or guinea pigs and hasnever been documented to infect humans. Al-though it was identified more than a half-century ago, the reasons behind its attenua-tion are unknown. Because large sequencepolymorphisms have presented themselves asthe most obvious genomic distinction amongmembers of the M. tuberculosis complex, theDNA content of the dassie bacillus wasinterrogated by Affymetrix GeneChip toidentify regions that are absent from it butpresent in M. tuberculosis H37Rv. Compari-son has led to the identification of nine re-gions of difference (RD), five of which areshared with M. microti (RDs 3, 7, 8, 9, and10). Although the dassie bacillus does notshare the other documented deletions in M.microti (RD1(mic), RD5(mic), MID1, MID2,and MID3), it has endured unique deletionsin the regions of RD1, RD5, N-RD25, andRv3081-Rv3082c (virS). RD1(das), affectingonly Rv3874-Rv3877, is the smallest naturaldeletion of the RD1 region uncovered andpoints to genes within this region that arelikely implicated in virulence. Newfounddeletions from the dassie bacillus are dis-cussed in relation to their evolutionary andbiological significance.—Authors’Abstract

Remus, N., Alcais, A., and Abel, L. Humangenetics of common mycobacterial in-fections. Immunol. Res. 28(2) (2003)109–129.

There is increasing interest in and un-derstanding of the role of human geneticfactors controlling susceptibility/resistanceto infectious diseases. This is of particularimportance for the two most common my-cobacterial infections, tuberculosis and lep-rosy, because this will allow a genetic dis-section of antimycobacterial immunity andshould open new fields of preventive andtherapeutic measures. In this review wewill initially discuss various methods of ge-netic epidemiology that have been and arebeing developed to identify human genescontrolling infectious diseases, and then il-lustrate the findings obtained in the numer-ous studies performed in tuberculosis andleprosy. Although the most convincing re-sults were observed for HLA-DR2 andNRAMP1 (or a closely linked gene) in pul-monary tuberculosis and leprosy subtypesand for a 10p13 locus in paucibacillary lep-rosy, the molecular basis of their effectsremains to be established.—Authors’Abstract

Sampson, S. L., Richardson, M., VanHelden, P. D., and Warren, R. M.IS6110-mediated deletion polymorphismin isogenic strains of Mycobacterium tu-berculosis. J. Clin. Microbiol. 42(2)(2004) 895–898.

Previous studies have described IS6110-mediated polymorphism as an importantdriving force in Mycobacterium tuberculo-sis genome evolution and have provided in-direct evidence for IS6110-driven deletionevents. This study provides the first descrip-tion of an IS6110-mediated deletion event intruly isogenic strains. We also provide fur-ther support for the hypothesis that the re-gion from Rv1754 to Rv1765 is a hot spotfor IS6110 insertion and deletion events.—Authors’Abstract

Sen Gupta, R., Hillemann, D., Kubica, T.,Zissel, G., Muller-Quernheim, J.,Galle, J., Vollmer, E., and Goldmann,T. HOPE-fixation enables improvedPCR-based detection and differentiationof Mycobacterium tuberculosis complexin paraffin-embedded tissues. Pathol.Res. Pract. 199(9) (2003) 619–623.

260 International Journal of Leprosy 2004

See Current Literature, Immunopathol-ogy, p. 203.

Song, T., Dove, S. L., Lee, K. H., and Hus-son, R. N. RshA, an anti-sigma factor thatregulates the activity of the mycobacterialstress response sigma factor SigH. Mol.Microbiol. 50(3) (2003) 949–959.

SigH, an alternative sigma factor of My-cobacterium tuberculosis, is a central regu-lator of the response to oxidative and heatstress. Exposure to these stresses results inincreased expression of sigH itself, and ofgenes encoding additional regulators and ef-fectors of the bacterial response to thesestresses. In this work we show that RshA, aprotein encoded by a gene in the sigHoperon, is an anti-sigma factor of SigH. Wedemonstrate that RshA binds to SigH in vitroand in vivo. This protein-protein interaction,as well as the ability of RshA to inhibit SigH-dependent transcription, is redox-dependent,with RshA functioning as a negative regula-tor of SigH activity only under reducing con-ditions. The interaction of SigH and RshA isalso disrupted in vitro by elevated tempera-ture. RshA, a protein of 101 amino acids,contains five conserved cysteine residues ofwhich two appear to be essential for RshA toinhibit SigH activity, suggesting that thesecysteines may be important for the redoxstate dependence of RshA function. Our re-sults indicate that RshA is a sensor that re-sponds to oxidative stress, and also to heatstress, resulting in activation of SigH and ex-pression of the SigH-dependent genes thatallow M. tuberculosis to adapt to thesestresses.—Authors’Abstract

Stratmann, J., Strommenger, B., Goethe,R., Dohmann, K., Gerlach, G. F.,Stevenson, K., Li, L. L., Zhang, Q.,Kapur, V., and Bull, T. J. A 38-kilobasepathogenicity island specific for Myco-bacterium avium subsp. paratuberculosisencodes cell surface proteins expressed inthe host. Infect. Immun. 72(3) (2004)1265–1274.

We have used representational differenceanalysis to identify a novel Mycobacteriumavium subsp. paratuberculosis-specific ABC

transporter operon (mpt), which comprisessix open reading frames designated mptAto -F and is immediately preceded by twoputative Fur boxes. Functional genomics re-vealed that the mpt operon is flanked on oneend by a fep cluster encoding proteins in-volved in the uptake of Fe(3+) and on theother end by a sid cluster encoding non-ribosome-dependent heterocyclic siderophoresynthases. Together these genes form a 38-kbM. avium subsp. paratuberculosis-specificlocus flanked by an insertion sequence sim-ilar to IS1110. Expression studies usingWestern blot analyses showed that MptC ispresent in the envelope fraction of M. aviumsubsp. paratuberculosis. The MptD proteinwas shown to be surface exposed, using aspecific phage (fMptD) isolated from aphage-peptide library, by differential screen-ing of Mycobacterium smegmatis transfor-mants. The phage fMptD-derived peptidecould be used in a peptide-mediated capturePCR with milk from infected dairy herds,thereby showing surface-exposed expres-sion of the MptD protein in the host. To-gether, these data suggest that the 38-kblocus constitutes an M. avium subsp. paratu-berculosis pathogenicity island.—Authors’Abstract

van den Braak, N., Simons, G., Gorkink,R., Reijans, M., Eadie, K., Kremers,K., van Soolingen, D., Savelkoul, P.,Verbrugh, H., and van Belkum, A. Anew high-throughput AFLP approach foridentification of new genetic polymor-phism in the genome of the clonal mi-croorganism Mycobacterium tuberculo-sis. J. Microbiol. Methods 56(1) (2004)49–62.

We have here applied high-throughputamplified fragment length polymorphism(htAFLP) analysis to strains belonging tothe five classical species of the Mycobacte-rium tuberculosis complex. Using 20 strains,three enzyme combinations and eight selec-tive amplification primer pairs, 24 AFLP re-actions were performed per strain. Overall,this resulted in 480 DNA fingerprints andmore than 1200 htAFLP-amplified PCRfragments were visualised per strain. Thecumulative dendrogram correctly clusteredstrains from the various species, albeit

72, 2 Current Literature, Molecular and Genetic Studies 261

within a distance of 6.5% for most of them.The single isolate of Mycobacterium canet-tii presented separately at 19% distance. Allover, 169 fragments (14%) appeared to bepolymorphic. Sixty-eight were specific forM. canetti and forty-five for Mycobacteriumbovis. For the 10 different M. tuberculosisstrains included in the present analysis, 56polymorphic markers were identified. Uponsequencing 20 of these marker regions andcomparisons with the H37Rv genome se-quence, 25% appeared to share homology tomembers of the antigenically variablePE/PPE surface protein encoding genefamily confirming previous findings on thegenetic heterogeneity within these genes. Inaddition, homologues for phage genes andinsertion element-encoded genes were de-tected. Forty-five percent of the sequencesderived from ORFs with a currently un-known function, which was corroborated bygenome sequence comparison for the clini-cal M. tuberculosis CD 1551 isolate. Se-quence variation in M. tuberculosis was as-sessed in more detail for a subset of theseloci by newly designed PCR restriction frag-ment length polymorphism (RFLP) tests anddirect sequencing. Fourteen novel PCRRFLP tests were developed and twelvenovel single nucleotide polymorphisms(SNPs) were identified, all suited for epi-demiological analysis of M. tuberculosis.The tests allowed for identification of the

major Mycobacterium species and M. tu-berculosis variants and clones.—Authors’Abstract

Wang, J. P., Rought, S. E., Corbeil, J.,and Guiney, D. G. Gene expression pro-filing detects patterns of human macro-phage responses following Mycobacteriumtuberculosis infection. FEMS Immunol.Med. Microbiol. 39(2) (2003) 163–172.

High-density oligonucleotide microarraysallow simultaneous monitoring of the ex-pression of a large number of cellular genes.Microarrays were used to screen the globalhuman monocyte-derived macrophage tran-scriptional response to infection with theintracellular pathogen Mycobacterium tu-berculosis. The microarray detected repro-ducible patterns of regulated gene expres-sion. Analysis of the expression data showedinduction of cytokines and chemokines,ribosomal proteins, and the interferon-response gene Stat1. Several changes werevalidated by quantitative reverse transcriptionpolymerase chain reaction and immunoblotassays. Augmentation of the respiratoryburst and preservation of the response to in-terferon-gamma were also demonstrated.These data supplement existing knowledgeon macrophage responses to tuberculosis in-fection.—Authors’Abstract

262 International Journal of Leprosy 2004

*Aide aux Lepreux Emmaus-Suisse, 9Spitalgasse, CH-3011 Berne, Switzer-land.

*American Leprosy Missions, One ALMWay, Greenville, South Carolina 29601,U.S.A.

*Amici dei Lebbrosi, Fondazione ItalianaRaoul Follereau, Via Borselli 4, 40135Bologna, Italy.

Damien-Dutton Society, 616 Bedford Av-enue, Bellmore, New York 11710, U.S.A.

*Damien Foundation (DF/APD), 16 RueStevin, B-1040 Bruxelles, Belgium.

*Deutsches Aussatzigen-Hilfswerk e. V.,Postfach 9062, D-97090 Würzburg 11,Germany.

*Le Secours aux Lépreux (Canada), 1275Rue Hodge, Bureau 12, Montreal H4N3H4, Canada.

*Netherlands Leprosy Relief, Wibaut-straat 137K, 1097 DN Amsterdam, TheNetherlands.

*Pacific Leprosy Foundation, 115 Sher-borne Street, Bag 4730, Christchurch,New Zealand.

*Sasakawa Memorial Health Founda-tion, Senpaku Shinko Bldg., 1-15-16Toranomon, Minato-ku, Tokyo 105,Japan.

INTERNATIONAL JOURNAL OF LEPROSY Volume 72, Number 2Printed in the U.S.A.

(ISSN 0148-916x)

ACKNOWLEDGMENT

The Board of Directors of the INTERNATIONAL JOURNAL OF LEPROSY gratefully ac-knowledges the financial assistance from special grantors and sustaining memberswhich, with the special donations of certain members, has made possible the con-tinuation of publication of the JOURNAL directly by the International Leprosy Asso-ciation. Without this assistance the official organ of the ILA, so essential to leprosyworkers everywhere, could not be published.

SPECIAL GRANTORS

* ILEP member/association.

263

SUSTAINING MEMBERS

*Foundation Follereau (France), 33 Ruede Dantzig, 75015 Paris, France.

Japanese Leprosy Association, c/o NationalInstitute for Leprosy Research, 2-1, 4-Chome, Aobacho, Higashimuryamashi,Tokyo 189, Japan.