JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 2004, p. 1680–1685 Vol. 42, No. 40095-1137/04/$08.00�0 DOI: 10.1128/JCM.42.4.1680–1685.2004Copyright © 2004, American Society for Microbiology. All Rights Reserved.

New Approach for Serological Testing for Leptospirosis by UsingDetection of Leptospira Agglutination by Flow

Cytometry Light Scatter AnalysisS. Yitzhaki,1* A. Barnea,2 A. Keysary,1 and E. Zahavy3*

Departments of Infectious Diseases1, Biotechnology,2 and Physical Chemistry,3

Israel Institute for Biological Research, Ness-Ziona 74100, Israel

Received 22 July 2003/Returned for modification 20 September 2003/Accepted 16 December 2003

Leptospirosis is considered an important reemerging infectious disease worldwide. The standard and mostwidespread method for the diagnosis of leptospirosis is the microscopic agglutination test (MAT). This test islaborious and time-consuming, and the interpretation of the results is subjective. In the present work wedescribe an application of flow cytometry (FCM) as a tool for the serological diagnosis of leptospirosis. Theanalysis is based on the sensitivity of FCM to the size and shape of the bacteria analyzed by measurement oflight scatter parameters: forward scatter (FSC) and side scatter (SSC). The addition of positive serum to aninfecting leptospiral serovar results in a shift of the light scatter parameter to a different location with higherFSC and SSC values, indicating the formation of leptospiral aggregates. By using immunofluorescent staining,we have shown that the large particles formed are the agglutinated leptospires. Quantification of the agglu-tination process has been achieved by calculating an agglutination factor (Af), based on changes in the lightscatter parameters measured by FCM. Af enables us to determine the specificity of the serological reaction ofthe patient serum with each leptospiral serovar. In this work, 27 serum samples from 18 leptospirosis patientswere tested by both the MAT and the FCM techniques, in which each serum sample was tested against 13serovars. Twenty-six human serum samples derived from patients with a variety of other defined illnesses wereused as negative controls and enabled us to define the Af threshold value as <9.3 for negative patients, whileany value higher than that would be a positive result for leptospirosis. Compared to MAT, the FCM techniquewas found to be more specific and sensitive, especially in identifying the serogroup in the acute phase of thedisease. The whole process was found to be rapid and took less than 1.5 h. Moreover, FCM analysis is objectiveand can be automated for the handling of large numbers of samples.

Leptospirosis is considered one of the most widespread zoo-noses worldwide (18, 34). The disease is caused by spirochetesof the genus Leptospira. The genus Leptospira is classified se-rologically into two species, the pathogenic species Leptospirainterrogans and the saprophytic species Leptospira biflexa.There are more than 200 serovars of L. interrogans and morethan 60 serovars of L. biflexa (16).

Leptospirosis usually results from contact with the urine ofinfected animals (13). The diagnosis of leptospirosis is mainlybased on serological tests, with the microscopic agglutinationtest (MAT) considered the standard methodology (8, 12). Theserological test for Leptospira is based on the formation ofbacterial aggregates resulting from the addition of serum sam-ples to the Leptospira suspension. The agglutination leads to asignificant change in the analyzed particles, as observed bydark-field microscopy by the MAT procedure.

A variety of serological tests other than MAT have beendeveloped for the diagnosis of leptospirosis. Among them arethe complement fixation test (33), several enzyme-linked im-munosorbent assay formats (1, 29), the macroscopic slide ag-

glutination test (14), the microcapsule agglutination test (9),the indirect hemagglutination assay (20), the dipstick assay(27), and other methods (3, 15, 22, 30). Each assay has its ownadvantages, drawbacks, and limitations (4, 18). Despite itswidespread use, MAT has several limitations. The test is dif-ficult to perform and control, the results are difficult to inter-pret, and it is time-consuming and labor-intensive (31). Theinterpretation of MAT results is subjective and may causequality assurance difficulties. One of the disadvantages of se-rologic testing by MAT compared to that by other techniquesis its low sensitivity, particularly with early acute-phase speci-mens (3, 5, 10). In this work, we describe the use of the flowcytometry (FCM) technique for the serological diagnosis ofleptospirosis. It is shown that the diagnosis of leptospirosis andthe definition of the serogroup involved are feasible, based onthe changes in the light scatter parameters forward scatter(FSC) and side scatter (SSC). By the FCM technique, the sizesand the shapes of the cells can be determined by measurementof FSC and SSC (17, 26, 32). Whereas FSC is related to the cellsize and the optical refraction index of the outer membrane ofthe cell, SSC is related to the cell’s granularity. Analysis ispossible due to the highly developed new generation of flowcytometric analyzers with the capability of observing particleswith diameters of 0.5 �m, which is as small as a variety ofbacterial species (2, 7, 11, 23–25, 28, 35).

FCM analysis was found to be objective, sensitive, and rapid.The duration of the whole process, i.e., the times for incuba-

* Corresponding authors. Mailing address for Shmuel Yitzhaki: De-partment of Infectious Diseases, Israel Institute for Biological Re-search, P.O. Box 19, Ness-Ziona 74100, Israel. Phone: 972-8-9381414.Fax: 972-8-9381639. E-mail: yitzhaki@iibr.gov.il. Mailing address forEran Zahavy: Department of Physical Chemistry, Israel Institute forBiological Research, P.O. Box 19, Ness-Ziona 74100, Israel. Phone:972-8-9381457. Fax: 972-8-9381743. E-mail: eran@iibr.gov.il.

1680

on July 22, 2015 by guesthttp://jcm

.asm.org/

Dow

nloaded from

tion of the sera, analysis, and interpretation of the results, wasless than 1.5 h.

MATERIALS AND METHODS

Human sera and MAT. Human sera were sent from medical centers through-out Israel to the Israel Institute for Biological Research, the central referencelaboratory for leptospirosis in Israel. Each serum sample was tested against 21different serovars by MAT by the standard procedure (21). Agglutination wasexamined by dark-field microscopy at a magnification of �100. The reported titerwas calculated as the reciprocal of the highest dilution of serum that agglutinatedat least 50% of the cells for each serovar used. A MAT-confirmed case wasdefined as a fourfold increase in antibody titer or a single titer �1:200, accordingto the case definition of the Centers for Disease Control and Prevention (6).

In the present study patients positive for leptospirosis were considered thosesuspected of having leptospirosis by the clinicians because they exhibited clinicalsymptoms typical of the disease. Their sera were sent to the Israel Institute forBiological Research and were found to be positive by MAT. Also, nine of thesepatients were known to work in areas of leptospirosis outbreaks from whichL. interrogans serovar hardjo was isolated.

Sera from 26 patients whose paired sera were found to be negative by MATserved as negative controls: 4 serum samples from patients with murine typhus,4 serum samples from patients with Mediterranean spotted fever, 4 serum sam-ples from patients with Q fever, 5 serum samples from patients with syphilis, and9 serum samples from patients with other clinical symptoms resembling lepto-spirosis.

Bacterial culture. Twenty-one reference serovars of living leptospiral spiro-chetes were used; among these were 19 pathogenic serovars (L. interrogans) and2 nonpathogenic (L. biflexa) serovars. Details about the serovars are listed inTable 1.

FCM. FCM analysis was performed with a FACSCalibur analyzer (BectonDickinson Immuno Cytometry Systems, San Jose, Calif.) equipped with a 15-mWargon laser as the excitation light source. The detectors used in this work are aphotodiode for FSC (� � 488/10 nm), a photomultiplier for SSC (� � 488/10nm), and a photomultiplier for green fluorescence (FL1; emission � � 530/30nm). The instrument settings included logarithmic amplifiers on all detectors. Allexperiments were performed for a fixed time of analysis (30 s). Acquisition andanalysis were performed with CellQuest software.

Preparation of samples for FCM analysis. Human sera were diluted (1:100) insaline-formaldehyde (0.14%) and filtered through a low-protein-binding 0.45-�m-pore-size syringe filter. Leptospiral organisms were grown for a week inEMJH medium (nos. 279410 and 279510; Becton Dickinson), counted with aPetroff-Hausser counting chamber under a dark-field microscope to confirm thepresence of 1 � 108 to 2 � 108 bacteria/ml, and then harvested. Each serovar wasdiluted 1/10 in saline-formaldehyde (0.14%), incubated in an equal volume ofserum at room temperature for 60 min, and analyzed by FCM.

Quantification of agglutination. Quantification of the agglutination processbased on the light scatter parameters was achieved by comparison of the agglu-tinated to the nonagglutinated Leptospira dot plots. Nonagglutinated leptospirescould be observed as a subpopulation under region R2 on a dot plot of the lightscatter parameters (FSC and SSC) (Fig. 1). Agglutinated leptospires could beobserved as a subpopulation under region R3 on the same dot plot (Fig. 1).

An arithmetic equation (equation 1) was developed in order to quantify theagglutination factor (Af):

Af �R3 � meanFSC(R3)

R2 (1)

where meanFSC(R3) is the mean FSC value of the events included in region R3,and R3 and R2 are the number of events in each region. This equation is basedon three major parameters affecting the light scatter parameters of the aggluti-nated and nonagglutinated species on the FCM dot plots: (i) the decrease in thepopulation in region R2, (ii) the increase in the population in region R3, and (iii)the increase in the mean FSC for region R3. The number of particles in regionR3, after agglutination, is also dependent on the size of the bacterial clusterformed. In cases in which large particles are formed, fewer particles will becounted in region R3; however, the mean FSC will increase and will compensatefor the low counts in region R3. The parameters R3, meanFSCR3, and R2 usedin equation 1 are normalized to the values of nonagglutinated leptospires bymeasuring the values for the samples with different serovars before and afterserum addition.

RESULTS

Determination of leptospiral agglutination by FCM. Thechanges in light scatter parameters (FSC and SSC) were ini-tially studied by monitoring the incubation of L. interrogansserovar icterohaemorrhagiae with hyperimmune rabbit anti-serovar icterohaemorrhagiae serum and fluorescent anti-rabbitimmunoglobulin G–fluorescein isothiocyanate (FITC) conju-gates.

Figure 1A shows the light scatter parameters of nonaggluti-nated Leptospira. Even though the leptospiral size is in themicron range, and hence their signals were at the limit ofsensitivity of FCM, the Leptospira population could be ob-served by using logarithmic amplification of the FSC and SSCparameters. The Leptospira population was defined as regionR2, in which a concentration dependency of the bacteria wasobserved (data not shown).

Upon addition of the specific rabbit antiserum and anti-rabbit immunoglobulin G–FITC conjugate, the majority of theLeptospira population shifted from region R2 to region R3[Fig. 1B(I)], which reflected stronger FSC and SSC signals.The same events that reached the new SSC and FSC region(region R3) [Fig. 1B(I)] were also the events with higher flu-orescence signals and were observed in region R1 on the SSC-FL1 dot plot [Fig. 1B(II)]. This correlation indicates that thelarger particles observed by the light scatter parameters (FSCand SSC) in region R3 are the agglutinated leptospires; hence,they light up by specific immune staining, as shown in theSSC-FL1 dot plot (region R1). No agglutination was observedby performing the same experiment with the same experimen-tal setup but with nonimmune rabbit serum (data not shown).The correlation between the events located in region R3 and

TABLE 1. Reference serovars used in this study

Species andserovar no. Serovar Origin

L. interrogans1 Icterohaemorrhagiae M20

copenhageniHuman, Denmark, 1935

29 Icterohaemorrhagiae RGA ATCC 436422 Canicola canicola Hond

Utrecht (IV)ATCC 23470

3 Grippothyphosa grippo-thyphosa Moskva (V)

ATCC 23469

5 Sejroe sejroe M-84 Human, Denmark, 19376 Szwajizak szwajizak Human, Australia, 19517 Mini sari Human, Italy, 19419 Hardjo hardjoprajitno Human, Indonesia, 193811 Pomona ATCC 2347812 Bataviae bataviae ATCC 2346813 Rachmati ATCC 2360314 Borgpetersenii javanica ATCC 2347916 Australis ATCC 2360517 Cynopteri canalzonae Panama Canal Zone18 Pyrogenes ATCC 2348019 Ballum castellonis ATCC 2358021 Ballum mus 127 Mus musculus, Denmark,

194323 Tarassovi tarassovi ATCC 2348124 Sejreo bratislava Rome, 1970

L. biflexa10 Semaranga patoc I Surface water, Italy15 Andamana andamana Human, Andamans, 1930

VOL. 42, 2004 SEROLOGICAL TEST FOR LEPTOSPIROSIS BY FLOW CYTOMETRY 1681

on July 22, 2015 by guesthttp://jcm

.asm.org/

Dow

nloaded from

those located in region R1 indicates that light scatter param-eters are sufficient for the analysis of agglutinated Leptospira.

The shifts of the Leptospira signals from region R2 to regionR3 were found to be dependent on the serum concentrationand the incubation time (Fig. 1C and D, respectively). By theFCM technique, it was possible to observe agglutination ofLeptospira in an incubation time shorter than 5 min with aserum dilution of 1:2,000. The titer in the same serum sampleby end-point titration by MAT was 1:3,200 after incubation for1 h, while by the FCM technique agglutination could be ob-served after 35 min of incubation, even when a dilution of1:8,000 was used. Hence, compared to MAT, FCM analysis ismore sensitive and rapid.

FCM analysis of sera from suspected human leptospirosiscases. Twenty-seven serum samples from 18 patients were ex-amined by MAT and FCM analysis. Since a single titer �1:200by MAT is considered positivity for leptospirosis, FCM anal-ysis was performed with a serum dilution of 1:200. The serawere incubated for 60 min with each Leptospira serovar andthen subjected to FCM analysis.

Quantification of the agglutination process, based on thelight scatter parameters, was achieved by calculating Af, as de-scribed in the Materials and Methods section. Calculation ofAf values for negative control sera enabled us to set a thresholdthat distinguished between negative and positive sera. In orderto set the threshold, we measured the Af values for each of the

FIG. 1. Dot plots of L. interrogans serovar icterohaemorrhagiae obtained by FCM. (A) Leptospira in phosphate-buffered saline; B(I) and B(II)light scatter (SSC-FSC) and fluorescence (SSC-FL1) dot plots, respectively, of L. interrogans serovar icterohaemorrhagiae in the presence of rabbitanti-icterohaemorrhagiae serum (30 min, 37°C) and the secondary antibody goat anti-rabbit immunoglobulin G–FITC (20 min, 37°C); (C) lightscatter dot plots of Leptospira in the presence of rabbit anti-icterohaemorrhagiae serum (1:2,000) after 1 min (I), 5 min (II), and 10 min (III) of incubationat 37°C; (D) same as panel C after 35 min of incubation at 37°C with the serum dilution levels of 1:8,000 (I), 1:4,000 (II), and 1:2,000 (III). R1 (green),the region of FITC-stained agglutinated Leptospira; R2 (red), nonagglutinated Leptospira; R3 (pink), agglutinated Leptospira.

1682 YITZHAKI ET AL. J. CLIN. MICROBIOL.

on July 22, 2015 by guesthttp://jcm

.asm.org/

Dow

nloaded from

26 negative serum samples (described in Materials and Meth-ods) against 13 serovars, resulting in a total of 338 tests. Theaverage Af value was calculated to be 1.8 � 1.5, and serumsamples with Af values �9.3 (average plus five times the

standard deviation) were considered negative for leptospi-rosis.

Figure 2A presents the results of a typical FCM analysis ofa serum sample (from patient 2) positive by MAT and analyzed

FIG. 2. (A) Light scatter dot plots of 13 different Leptospira serovars (as presented in the Materials and Methods section and indicated inparentheses in each panel) in the presence of human serum sample 2/II. R2, nonagglutinated Leptospira; R3, agglutinated Leptospira. (B) Af forserum sample 2/II in comparison to that for a negative serum sample.

VOL. 42, 2004 SEROLOGICAL TEST FOR LEPTOSPIROSIS BY FLOW CYTOMETRY 1683

on July 22, 2015 by guesthttp://jcm

.asm.org/

Dow

nloaded from

for 13 different Leptospira serovars. It was concluded from theFSC and SSC results that the serum sample was positive forserovars icterohaemorrhagiae copenhageni (serovar 1 in Fig.2A), sejroe sejroe M84 (serovar 5), szwajizak szwajizak (sero-var 6), and icterohaemorrhagiae RGA (ATCC 43642) (serovar29). All of the same serovars except sejroe sejroe M84 werealso found to be positive by MAT, with titers of 1:400, 1:400,and 1:200, respectively.

Figure 2B shows the Af values for each serovar, as calculatedby equation 1, for a negative serum sample and for the lepto-spirosis-positive patient whose results are provided in Fig. 2A.It can be seen that for the leptospirosis-positive patient the Af

values for four serovars were much higher than the negative Af

values (positive or negative serum with negative results). TheAf values for the sera with positive results for serovars ictero-haemorrhagiae copenhageni (serovar 1 in Fig. 2A), sejroesejroe M84 (serovar 5), szwajizak szwajizak (serovar 6), ictero-haemorrhagiae RGA (ATCC 43642) (serovar 29), were 105,44, 120, and 330, respectively. In this case, the results by bothMAT and FCM indicate that serovar icterohaemorrhagiae isthe predominant serogroup.

Table 2 summarizes the predominant serogroups obtainedby MAT and FCM analyses of the 27 serum samples from 18leptospirosis patients. In addition to exhibiting typical clinicalsymptoms of the disease, the sera of these patients were foundto be positive for leptospires by MAT, which was performedagainst 21 serovars. Moreover, nine of these patients (patients3, 5, 7, 8, 9, 15, 16, 17, and 18) were known to work in areas

where leptospirosis outbreaks had occurred and from whichserovar hardjo had been isolated.

For 15 patients, the highest Af values for the specific sero-group were measured by FCM analysis, and the results thuscorresponded to the highest titers obtained by MAT. For ex-ample, for patients 1, 2, and 10, the highest Af values and MATtiters were for serogroup icterohaemorrhagiae, whereas forpatients 3, 5, 7, 8, 9, 15, 16, 17, and 18 the highest values werefor serogroup hardjo.

In some cases it was possible to identify leptospirosis inpatients by FCM significantly before it was detected accordingto the appropriate titers by MAT. For example, the indicationof positivity for leptospirosis by FCM appeared in the acute-phase sera of patients 2, 6, and 16, whereas by MAT it ap-peared only in the sera obtained later. These results demon-strate that the FCM technique can reliably be used as a tool forthe diagnosis of leptospirosis at an early stage of the diseaseand for the identification of the infecting serogroup.

From Table 2 one can see that the Af value did not alwayscorrespond to the MAT titers. For example, the titer obtainedby MAT was 1:400 for both patients 2 and 3, while the Af valueswere 330 and 40, respectively. Despite these differences, thepredominant serogroup appeared to be the same by bothmethods. In three other patients (patients 4, 11, and 14), al-though the patients were found to be Leptospira positive byboth the MAT and the FCM techniques, the predominantserogroups identified and defined by MAT and FCM weredifferent. The predominant serogroups found by MAT wereicterohaemorrhagiae, icterohaemorrhagiae, and canicola, re-spectively, whereas the predominant serogroups found by FCMwere szwajizak, szwajizak, and ballum, respectively. However,the serogroups that were predominant by MAT also gave pos-itive Af values. These differences can be explained by the dif-ferences in the natures of the two different methods and by thecross-reactivities of the serovars.

DISCUSSION

This paper has demonstrated a new methodology for sero-logical testing for leptospirosis. The FCM technique was usedfor the diagnosis of leptospirosis by monitoring the agglutina-tion of various serovars following incubation with human se-rum. By developing and applying an arithmetic equation tocalculate Af, it was shown that FCM analysis could lead to anobjective quantification of agglutination. This equation takesinto account the changes in the light scatter parameters andthe sizes of the populations of both the agglutinated and thenonagglutinated leptospires. Analysis of sera from 26 patientswith other defined diseases, which were used as negative con-trols and each of which was tested against 13 Leptospira sero-vars, was used to define the threshold Af between negative andpositive patients (Af � 9.3).

By implementation of this methodology, FCM analysis en-abled the detection of the serogroup in all patients, and inthree patients (patients 2, 6, and 16) serogroup detection oc-curred in the acute phase, when the MAT result was stillnegative.

However, the Af value did not always correspond to theMAT titers, and in 3 of 18 patients (patients 4, 11, and 14), thepredominant serogroup determined by MAT differed from

TABLE 2. FCM and MAT results for leptospirosispatients and negative controls

Patient no./serumsample no.

Serogroup byMATa (titer)

Serogroup with highestpositive (Af value)

1/I Icterob (1,600) Ictero (300)2/I Negative Ictero (10)2/II Ictero (400) Ictero (330)3/I Negative Negative3/II Hardjo (400) Hardjo (40)4/I Ictero (800) Szwajizak (1,790), ictero (600)5/I Hardjo (400) Hardjo (650)6/I Negative Ballum (15,000)6/II Ballum (400) Ballum (5,600)7/I Hardjo (400) Hardjo (170)8/I Hardjo (800) Hardjo (24)9/I Hardjo (400) Hardjo (340)10/I Ictero (800) Ictero (330)11/I Negative Negative11/II Ictero (800) Szwajizak (65)11/II Szwajizak (400) Ictero (16)12/I Negative Negative12/II Bataviae (400) Bataviae (590)13/I Negative Negative13/II Australis (200) Australis (12)14/I Canicola (3,200) Ballum (400), canicola (205)14/II Canicola (400) Ballum (7,500) canicola (260)15/I Hardjo (400) Hardjo (215)15/II Hardjo (800) Hardjo (250)16/I Negative Hardjo (40)16/II Hardjo (800) Hardjo (610)17/II Hardjo (3,200) Hardjo (206)18/II Hardjo (200) Hardjo (36)Negative controlsc Negative Negative (�9.3)

a Predominant serogroups.b Ictero, Leptospira icterohaemorrhagiae serogroup.c Twenty-six serum samples; for details, see Materials and Methods.

1684 YITZHAKI ET AL. J. CLIN. MICROBIOL.

on July 22, 2015 by guesthttp://jcm

.asm.org/

Dow

nloaded from

that determined by FCM. This can be explained by the differ-ences in the natures of the two methods: microscopy takes intoaccount only the presence or the absence of aggregates,whereas FCM measures light scatter parameters and the anal-ysis is more precise, as it considers size, shape, and number ofthe aggregates. Moreover, FCM analysis can detect very smallaggregates not visible by light microscopy, as can be found inthe early stages of agglutination. This may lead to positive Af

values and negative MAT results in the early stages of thedisease. Moreover, it was recently shown (19) that serologicalanalysis by MAT could not always predict and identify theinfecting serovar in individual patients. The reasons for thepoor predictive ability of MAT could emanate from the cross-reactivity between serogroups and from the paradoxical reac-tion of an acute-phase or an early-convalescent-phase serumsample. It is possible that analysis by FCM overcomes some ofthese problems as a result of its capability to analyze multipleparameters (FSC, SSC, and fluorescence) and will be able toimprove the specificities and sensitivities of serological tests.

In addition, an intrinsic limitation of MAT is the subjectiveinterpretation of the results and the difficulties in ensuringstandardization between laboratories. The FCM methodologyeliminates these drawbacks due to the accuracy and objectivitystemming from the nature of the FCM analysis. Another ad-vantage of FCM is its rapidity, as the entire procedure, includ-ing incubation time and analysis, is completed in 1.5 h. Fur-thermore, the analysis can be automated and used to performlarge numbers of tests.

In conclusion, FCM uses standard equipment, is available inmany hospitals, and is used mainly for blood counts and oth-er immunological purposes. This method can easily be usedfor the diagnosis of leptospirosis because of its sensitivity andobjectivity and because automated procedures can be appliedto FCM.

REFERENCES

1. Adler, B., A. M. Murphy, S. A. Locarnini, and S. Faine. 1980. Detection ofspecific antileptospiral immunoglobulins M and G in human serum by solid-phase enzyme-linked immunosorbent assay. J. Clin. Microbiol. 11:452–457.

2. Alvarez-Barrientos, A., J. Arroyo, R. Canton, C. Nombela, and M. Sanchez-Perez. 2000. Applications of flow cytometry to clinical microbiology. Clin.Microbiol. Rev. 13:167–195.

3. Appassakij, H., K. Silpapojakul, R. Wansit, and J. Woodtayakorn. 1995.Evaluation of the immunofluorescent antibody test for the diagnosis ofhuman leptospirosis. Am. J. Trop. Med. Hyg. 52:340–343.

4. Bajani, M., D., D. A. Ashford, S. L. Bragg, C. W. Woods, A. Tin, R. A. Spiegel,B. A. Plikaytis, M. Phelan, P. N. Levett, and R. S. Weyant. 2003. Evaluationof four commercially available rapid serologic tests for diagnosis of lepto-spirosis. J. Clin. Microbiol. 41:803–809.

5. Brandao, A. P., E. D. Camargo, E. D. da Silva, M. V. Silva, and R. V. Abrao.1998. Macroscopic agglutination test for rapid diagnosis of human leptospi-rosis. J. Clin. Microbiol. 36:3138–3142.

6. Centers for Disease Control and Prevention. 1997. Case definitions forinfectious conditions under public health surveillance. Morb. Mortal. Wkly.Rep. 46(RR-10):49.

7. Clarke, R. G., and A. C. Pinder. 1998. Improved detection of bacteria by flowcytometry using a combination of antibody and viability markers. J. Appl.Microbiol. 84:577–584.

8. Cole, J. R., Jr., C. R. Sulzer, and A. R. Pursell. 1973. Improved microtech-nique for the leptospiral microscopic agglutination test. Appl. Microbiol.25:976–980.

9. Cui, J. J., G. X. Xiao, T. Z. Chen, G. F. Zhu, T. Sato, M. Seki, S. Kobayashi,

and Y. Arimitsu. 1991. Further evaluation of one-point microcapsule agglu-tination test for the diagnosis of leptospirosis. Epidemiol. Infect. 106:561–565.

10. Cumberland, P. C., C. O. R. Everard, and P. N. Levett. 1999. Assessment ofthe efficacy of the IgM enzyme-linked immunosorbent assay (ELISA) andmicroscopic agglutination test (MAT). Am. J. Trop. Med. Hyg. 61:731–734.

11. Davey, H. M., A. Jones, A. D. Shaw, and D. B. Kell. 1999. Variable selectionand multivariable methods for the identification of microorganisms by flowcytometry. Cytometry 35:162–168.

12. Faine, S. 1982. Guidelines for the control of leptospirosis. World HealthOrganization, Geneva, Switerland.

13. Faine, S., B. Adler, C. Bolin, and P. Perolat. 1999. Leptospira and leptospi-rosis. MediSci, Melbourne, Australia.

14. Galton, M. M., D. K. Powers, A. M. Hall, and R. G. Cornell. 1958. A rapidmicroscopic-slide screening test for the serodiagnosis of leptospirosis. Am. J.Vet. Res. 19:505–512.

15. Kawaoka, Y., M. Naiki, and R. Yanagawa. 1979. Radioimmunoassay systemusing a serovar-specific lipopolysaccharide antigen of Leptospira. J. Clin.Microbiol. 10:313–316.

16. Kmety, E., and H. Dikken. 1993. Classification of the species Leptospirainterrogans and history of its serovars. University Press Groningen, Gro-ningen, The Netherlands.

17. Koch, A. L., B. R. Robertson, and D. K. Button. 1996. Deduction of the cellvolume and mass from forward scatter intensity of bacteria analyzed by flowcytometry. J. Microbiol. Methods 27:49–61.

18. Levett, P. N. 2001. Leptospirosis. Clin. Microbiol. Rev. 14:296–326.19. Levett, P. N. 2003. Usefulness of serologic analysis as a predictor of the

infecting serovar in patients with severe leptospirosis. Clin. Infect. Dis. 36:447–452.

20. Levett, P. N., and C. U. Whittington. 1998. Evaluation of the indirect hem-agglutination assay for diagnosis of acute leptospirosis. J. Clin. Microbiol.36:11–14.

21. Mayers, D. M. 1985. Manual of laboratory methods for the diagnosis ofleptospirosis. Technical note no. 30. Pan American Zoonoses Center, WorldHealth Organization, Geneva Switzerland.

22. Mayers, D. M. 1987. Serodiagnosis of human leptospirosis by counterimmu-noelectrophoresis. J. Clin. Microbiol. 25:897–899.

23. Nebe von Caron, G., P. Stephens, and A. R. Badley. 1999. Bacterial detectionand differentiation by cytometry and fluorescent probes. Proc. R. Microsc.Soc. 34:321–327.

24. Phillips, A. P., and K. L. Martin. 1988. Limitation of flow cytometry for thespecific detection of bacteria in mixed populations. J. Immunol. Methods106:109–117.

25. Pinder, A. C., and R. G. McClelland. 1994. Rapid assay for pathogenicSalmonella organisms by immunofluorescence flow cytometry. J. Microsc.176:17–22.

26. Shapiro, H. M. 1985. Practical flow cytometry. Alan R. Liss, Inc., New York,N.Y.

27. Smits, H. L., Y. V. Ananyina, A. Chereshsky, L. Dancel, A. F. R. F. Lai, H. D.Chee, P. N. Levett, T. Masuzawa, Y. Yanagihara, M. A. Muthusethupathi,E. J. Sanders, D. M. Sasaki, H. Domen, C. Yersin, T. Aye, S. L. Bragg, G. C.Gussenhoven, M. G. Goris, W. J. Terpstra, and R. A. Hartskeerl. 1999.International multicenter evaluation of the clinical utility of a dipstick assayfor detection of Leptospira-specific immunoglobulin M antibodies in humanserum specimens. J. Clin. Microbiol. 37:2904–2909.

28. Stopa, P. J. 2000. The flow cytometry of Bacillus anthracis spores revisited.Cytometry 41:237–244.

29. Terpstra, W. J., G. S. Ligthart, and G. J. Schoone. 1985. ELISA for thedetection of specific IgM and IgG in human leptospirosis. J. Genet. Micro-biol. 131:377–385.

30. Torten, M., E. Shenberg, and J. van der Hooden. 1966. The use of immu-nofluorescence in the diagnosis of human leptospirosis by genus-specificantigen. J. Infect. Dis. 116:537–543.

31. Turner, L. H. 1968. Leptospirosis. II. Serology. Trans. R. Soc. Trop. Med.Hyg. 62:880–889.

32. Vorauer-Uhl, K., A. Wagner, N. Borth, and H. Katinger. 2000. Determina-tion of liposome size distribution by flow cytometry. Cytometry 39:166–171.

33. Wolf, J. W. 1954. The laboratory diagnosis of leptospirosis. Charles CThomas, Publisher, Springfield, Ill.

34. World Health Organization. 1999. Leptospirosis worldwide. Wkly. Epide-miol. Rec. 74:237–242.

35. Zahavy, E., M. Fisher, A. Bromberg, and U. Olshevsky. 2003. Detection offrequency resonance energy transfer pair on double-labeled microsphereand Bacillus anthracis spores by flow cytometry. Appl. Environ. Microbiol.69:2330–2339.

VOL. 42, 2004 SEROLOGICAL TEST FOR LEPTOSPIROSIS BY FLOW CYTOMETRY 1685

on July 22, 2015 by guesthttp://jcm

.asm.org/

Dow

nloaded from