5
JOURNAL OF CLINICAL MICROBIOLOcY, Mar. 1994. p. 629-633 0095-1 137/94/$04.00 +() Copyright © 1994. American Society for Microbiology Analysis of Western Blotting (Immunoblotting) Technique in Diagnosis of Congenital Syphilis M. P. MEYER,12* T. EDDY,2 AND R. E. BAUGHN' Departments of Paediatrics and Child Health' and Clinical Science and Immunology, ' University of Cape Towni, Cape Town, South Africa, and the Department of Microbiology and Immuinology, Baylor College of Medicine and the Veterans Affairs Medical Center, Houston, Texas 770303 Received 12 July 1993/Returned for modification 7 September 1993/Accepted 7 December 1993 The diagnosis of congenital syphilis in apparently healthy infants continues to be problematic. Immuno- globulin M antibodies specific for a subset of Treponema pallidum antigens have been detected by Western blotting (immunoblotting). In the present study we investigated the sensitivity and specificity of this method. We tested 26 infants aged 0 to 4 months who fulfilled the accepted criteria for the diagnosis of congenital syphilis. There were 14 symptomatic infants. Sera from 13 of these infants were positive for the 47-kDa treponemal antigen (92% sensitivity). The remaining 12 infants were clinically asymptomatic when tested at birth but subsequently displayed features consistent with the disease. Reactive blots (antibodies to the 47- and/or the 15-kDa antigens) were noted in 10 of the 12 infants (83% sensitivity). Thirty infants whose mothers had syphilis were monitored and shown to be uninfected. Nonreactive blots were seen in sera from 27 infants, while sera from 3 older infants had false-positive tests (90% specificity). The Western blotting technique is sensitive (even in the diagnosis of clinically inapparent cases) and, in the absence of immunoglobulin M rheumatoid factor, is a useful confirmatory test for congenital syphilis. Despite the resurgence of congenital syphilis and increased awareness of the condition, laboratory diagnosis remains dif- ficult (7). Part of the problem arises because the pathogen, Treponema pallidlum subsp. pallidum, is not easily identified. In addition, the standard serological tests are less useful in newborns because of immunoglobulin G (IgG) transfer across the placenta. IgM tests which depend on the infant's response (e.g., the fluorescent treponemal antibody absorption test [FTA-ABS] and investigations such as radiographs of the long bones) have reduced sensitivities in asymptomatic patients (9, 1 1). Consequently, newer tests including Western blotting (immunoblotting) have been developed, and a sensitivity of 92% in diagnosing symptomatic congenital syphilis has been reported (10). However, the majority of newborns who are subsequently proven to have congenital syphilis are asympto- matic at birth, and the sensitivity of the procedure in this group is unknown. Here we report our experience with the test and include results for at-risk infants in whom the diagnosis of congenital syphilis was established at follow-up. MATERIALS AND METHODS Background. The study was performed on infants born in the Peninsula Maternal and Neonatal Service as well as those presenting to the Red Cross War Memorial Children's Hospi- tal in Cape Town, South Africa. During the time of the investigation the hospital policy was similar to that proposed by Kaufman et al. (8) and the World Health Organization (15); i.e., patients with clinical signs compatible with the diagnosis of congenital syphilis were treated, while apparently healthy infants were monitored and serial Venereal Disease Research Laboratory (VDRL) titers were determined. Those with a titer that showed a fourfold rise or whose sera did not become negative by 6 months were considered to be infected and were * Corresponding author. Mailing address: Neonatal Medicine, Old Main Building H46, Groote Schuur Hospital, Observatory 7925, Cape Town, South Africa. treated with procaine penicillin at 50,000 U/kg of body weight per day for 10 days. Informed consent for the study was obtained from the parents, and the protocol was approved by the Ethics and Research Committee of the University of Cape Town. We enrolled infants in the first 4 months of life, and the infants described here were examined clinically at birth and were monitored by one of the authors (M.P.M.). The patients with congenital syphilis fulfilled the criteria suggested by Kaufman et al. (8) and were divided into the following two groups: those with clinical signs (symptomatic) and those who were clinically asymptomatic (regardless of laboratory test results). This approach was followed because in South Africa the availability of diagnostic tests at birth is variable and the usefulness of some tests, e.g., the cerebrospinal fluid VDRL in asymptomatic infants, is debatable (5, 14). Patients. Fourteen infants presented with clinical signs compatible with symptomatic congenital syphilis. Their moth- ers were asymptomatic and had VDRL titers ranging from 1:16 to 1:512; all had a positive T pallidlunm hemagglutination (TPHA) test result. The mothers had not received antenatal therapy for syphilis because of failure to attend antenatal clinics or because of enrollment in an antenatal clinic late in their pregnancies. Congenital syphilis was diagnosed at birth in 12 infants, at age 2 weeks in 1 infant, and at 10 weeks in another infant. The commonest findings were hepatospleno- megaly (14 infants), bone changes (13 infants), edema (8 infants and skin rashes (6 infants). Twelve infants had no physical features of disease at birth. Their mothers were untreated or inadequately treated in the last month of pregnancy. The maternal VDRL titers varied from 1:2 to 1:512, and the TPHA test was positive in all cases. Four of the infected infants had abnormal long-bone X rays and were treated in the first few days of life; 8 infants had at least a fourfold increase in VDRL titer by 4 months of age. Five of the eight infants developed snuffles and organomegaly, in addition to the rise in VDRL titer. These 12 infants have been described in a publication which evaluated the FTA-ABS 629 Vol. 32. No. 3 on March 5, 2020 by guest http://jcm.asm.org/ Downloaded from

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JOURNAL OF CLINICAL MICROBIOLOcY, Mar. 1994. p. 629-6330095-1 137/94/$04.00+()Copyright © 1994. American Society for Microbiology

Analysis of Western Blotting (Immunoblotting) Technique inDiagnosis of Congenital SyphilisM. P. MEYER,12* T. EDDY,2 AND R. E. BAUGHN'

Departments of Paediatrics and Child Health' and Clinical Science and Immunology, ' University of Cape Towni,Cape Town, South Africa, and the Department of Microbiology and Immuinology, Baylor College

of Medicine and the Veterans Affairs Medical Center, Houston, Texas 770303Received 12 July 1993/Returned for modification 7 September 1993/Accepted 7 December 1993

The diagnosis of congenital syphilis in apparently healthy infants continues to be problematic. Immuno-globulin M antibodies specific for a subset of Treponema pallidum antigens have been detected by Westernblotting (immunoblotting). In the present study we investigated the sensitivity and specificity of this method.We tested 26 infants aged 0 to 4 months who fulfilled the accepted criteria for the diagnosis of congenitalsyphilis. There were 14 symptomatic infants. Sera from 13 of these infants were positive for the 47-kDatreponemal antigen (92% sensitivity). The remaining 12 infants were clinically asymptomatic when tested atbirth but subsequently displayed features consistent with the disease. Reactive blots (antibodies to the 47-and/or the 15-kDa antigens) were noted in 10 of the 12 infants (83% sensitivity). Thirty infants whose mothershad syphilis were monitored and shown to be uninfected. Nonreactive blots were seen in sera from 27 infants,while sera from 3 older infants had false-positive tests (90% specificity). The Western blotting technique issensitive (even in the diagnosis of clinically inapparent cases) and, in the absence of immunoglobulin Mrheumatoid factor, is a useful confirmatory test for congenital syphilis.

Despite the resurgence of congenital syphilis and increasedawareness of the condition, laboratory diagnosis remains dif-ficult (7). Part of the problem arises because the pathogen,Treponema pallidlum subsp. pallidum, is not easily identified. Inaddition, the standard serological tests are less useful innewborns because of immunoglobulin G (IgG) transfer acrossthe placenta. IgM tests which depend on the infant's response(e.g., the fluorescent treponemal antibody absorption test[FTA-ABS] and investigations such as radiographs of the longbones) have reduced sensitivities in asymptomatic patients (9,1 1). Consequently, newer tests including Western blotting(immunoblotting) have been developed, and a sensitivity of92% in diagnosing symptomatic congenital syphilis has beenreported (10). However, the majority of newborns who aresubsequently proven to have congenital syphilis are asympto-matic at birth, and the sensitivity of the procedure in this groupis unknown. Here we report our experience with the test andinclude results for at-risk infants in whom the diagnosis ofcongenital syphilis was established at follow-up.

MATERIALS AND METHODS

Background. The study was performed on infants born inthe Peninsula Maternal and Neonatal Service as well as thosepresenting to the Red Cross War Memorial Children's Hospi-tal in Cape Town, South Africa. During the time of theinvestigation the hospital policy was similar to that proposed byKaufman et al. (8) and the World Health Organization (15);i.e., patients with clinical signs compatible with the diagnosis ofcongenital syphilis were treated, while apparently healthyinfants were monitored and serial Venereal Disease ResearchLaboratory (VDRL) titers were determined. Those with a titerthat showed a fourfold rise or whose sera did not becomenegative by 6 months were considered to be infected and were

* Corresponding author. Mailing address: Neonatal Medicine, OldMain Building H46, Groote Schuur Hospital, Observatory 7925, CapeTown, South Africa.

treated with procaine penicillin at 50,000 U/kg of body weightper day for 10 days. Informed consent for the study wasobtained from the parents, and the protocol was approved bythe Ethics and Research Committee of the University of CapeTown.We enrolled infants in the first 4 months of life, and the

infants described here were examined clinically at birth andwere monitored by one of the authors (M.P.M.). The patientswith congenital syphilis fulfilled the criteria suggested byKaufman et al. (8) and were divided into the following twogroups: those with clinical signs (symptomatic) and those whowere clinically asymptomatic (regardless of laboratory testresults). This approach was followed because in South Africathe availability of diagnostic tests at birth is variable and theusefulness of some tests, e.g., the cerebrospinal fluid VDRL inasymptomatic infants, is debatable (5, 14).

Patients. Fourteen infants presented with clinical signscompatible with symptomatic congenital syphilis. Their moth-ers were asymptomatic and had VDRL titers ranging from 1:16to 1:512; all had a positive T pallidlunm hemagglutination(TPHA) test result. The mothers had not received antenataltherapy for syphilis because of failure to attend antenatalclinics or because of enrollment in an antenatal clinic late intheir pregnancies. Congenital syphilis was diagnosed at birth in12 infants, at age 2 weeks in 1 infant, and at 10 weeks inanother infant. The commonest findings were hepatospleno-megaly (14 infants), bone changes (13 infants), edema (8infants and skin rashes (6 infants).Twelve infants had no physical features of disease at birth.

Their mothers were untreated or inadequately treated in thelast month of pregnancy. The maternal VDRL titers variedfrom 1:2 to 1:512, and the TPHA test was positive in all cases.Four of the infected infants had abnormal long-bone X raysand were treated in the first few days of life; 8 infants had atleast a fourfold increase in VDRL titer by 4 months of age.Five of the eight infants developed snuffles and organomegaly,in addition to the rise in VDRL titer. These 12 infants havebeen described in a publication which evaluated the FTA-ABS

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(IgM) test (12). Sera which were obtained from the infants atbirth were divided into aliquots and were stored at - 80°C untiluse; repeated freezing and thawing was avoided.

Thirty infants were uninfected, even though they were bornto seropositive mothers with untreated or inadequately treatedsyphilis. These babies were examined clinically at birth, and aVDRL titer and long-bone X rays were obtained. At follow-up,the VDRL titer became negative and there were no clinicalsigns of congenital syphilis. Twenty-one of the serum speci-mens tested were specimens obtained at birth; the other nineserum specimens were from infants aged 6 weeks to 4 months.

In addition, sera from 10 healthy infants who, together withtheir mothers, had negative serology for syphilis (VDRL andTPHA tests) were subjected to Western blotting. Pooled serumfrom 10 adults with secondary or early latent syphilis was usedas a positive control.Western blots. A suspension of T pallidum antigens was

subjected to sodium dodecyl sulfate-polyacrylamide gel elec-trophoresis by using a 4% stacking gel and a 12% separatinggel as described previously (1). Antigens and low-molecular-weight standards (Bio-Rad) were then electrophoreticallytransferred to nitrocellulose paper. Strips of nitrocellulosewere cut and blocked for 1 h with 5% fetal calf serum. Testserum (1:100) in Tris-buffered saline (TSB; pH 7.4) with 0.05%Tween 20 (TSB-T) was reacted with the strips for 16 h at roomtemperature.

After three washes with TSB-T, a mouse anti-human IgMmonoclonal antibody (p. chain specific; kindly provided by J.Conradie, Natal Blood Transfusion Service, Durban, SouthAfrica) diluted 1:1,000 in TSB was added. After washing, abiotinylated rabbit anti-mouse antibody (1:1,000 in TSB) andthen streptavidin-biotin horseradish peroxidase complex(Strept ABComplex HRP; Dako, Glostrup, Denmark) wereadded. Diaminobenzene (25 mg/ml) with 3% cobalt chlorideand hydrogen peroxide was used to develop the blots.The molecular weights of the T. pallidum antigens were

determined as described previously (10); the identification ofantigens was assisted by silver staining of T. pallidum gels andby reacting nitrocellulose strips with pooled serum from adultswith recent syphilis and pooled IgG from rabbits infected withT. pallidum.IgM fractionation. IgM fractions were prepared by passing

100 p.1 of serum over an ion-exchange column (Quick Sep,System II; Isolab). The eluted IgM was reconcentrated to ca.60 I.1 with the Centricon 30 microconcentrator system [Ami-con]). The sample was then subjected to high-pressure liquidchromatography (HPLC) by using a size exclusion column(Biosep SEC S3000 with dimensions of 300 by 7.8 mm;Phenomonex). The mobile phase was 20 mM sodium phos-phate with 0.1 M sodium sulfate, and the flow rate was 0.5mVmin. The IgM fractions were identified by timing theappearance of IgM standards and by a dot blot method. Forthe latter, 2 p.1 of the HPLC fraction was applied to nitrocel-lulose paper. Peroxidase-labelled antibodies to human IgMand IgG (Cappel, Cochranville, Pa.) at dilutions of 1:1,000 and1:2,000, respectively, were added; this was followed by theaddition of substrate (a 3-mg/ml solution of 4-chloro-1-naph-thol in methanol). In this way, IgM fractions free of IgG wereobtained. The IgM level was measured by nephelometry(Beckman), and Western blotting was carried out with thesame IgM concentration as was originally present in the serum.Owing to the small quantities of serum generally available, weprepared IgM fractions only in selected cases. Rheumatoidfactor (RF) latex tests (Orthodiagnostics, Beerse, Belgium)were carried out at a serum dilution of 1:5.

Evaluation. The sensitivity of the Western blotting proce-

110-8447

24-17

a b c dFIG. 1. Reaction profiles in Western blots. Lane a, IgM reactivity

with pooled adult serum; lane b, profile with pooled rabbit IgG; lanesc and d, IgM reactivity in sera from infants with symptomatic congen-ital syphilis.

dure was defined as the percentage of patients with congenitalsyphilis (according to the criteria of Kaufman et al. [8]) with apositive blot. The specificity was defined as the percentage ofuninfected infants with a nonreactive test in relation to thetotal number of unaffected infants (9). In the present study theinfants of seropositive mothers who did not develop congenitalsyphilis were regarded as true negatives (9).

RESULTS

Symptomatic patients. The reaction profiles of pooled adultserum, pooled rabbit IgG, and sera from symptomatic infantsare compared in Fig. 1. The infant sera demonstrated reactivitywith treponemal antigens with molecular masses of between110 and 15 kDa (Fig. 1, lanes c and d; Fig. 2, lanes a, b, and c);on average, each serum specimen reacted with seven antigens(Table 1). The commonest responses were directed against 47-and 45-kDa antigens (13 of 14 infants) the 72-kDa protein (9infants), a 39-kDa antigen (8 infants), and a 42-kDa compo-nent (7 infants).

Clinically asymptomatic patients. Sera from the 12 infants

1 1 0 - I

8447 ....47 ......._

N-

24-17-'

a b c d e f gFIG. 2. IgM reactivity in Western blots. Lanes a to c, sera from

infants with symptomatic congenital syphilis; lanes d to f, sera frominfants with asymptomatic congenital syphilis; lane g, serum from an

uninfected infant of a seropositive mother.

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IgM WESTERN BLOTS IN CONGENITAL SYPHILIS 631

TABLE 1. IgM reactivity to T. pallidum antigens in whole,unfractionated infant sera

No. of patientsMolecular mass

(kDa) Symptomatic Asymptomatic Uninfected, at risk(n = 14) (n = 12) (n = 30)

110 3 0 084 6 6 672 9 0 161 4 5 1059 0 0 156 0 0 151 1 0 147 13 8 345 13 8 242 7 2 539 8 0 837 6 1 033 4 0 030 4 1 028 5 0 424 5 1 417 4 0 015 4 3 0

in the clinically asymptomatic group demonstrated IgM reac-tivity against fewer treponemal antigens (average, three perpatient). Sera from eight individuals had an IgM response tothe 47- and 45-kDa antigens (Fig. 2, lanes d and e), whereassera from five individuals reacted with the 84-kDa protein. Insera from two patients, there was reactivity with the 15-kDaantigen without a response to the 47- or 45-kDa protein, whilean additional two individuals showed no seroreactivity to the47- or 45-kDa antigens or lower-molecular-mass proteins.None of the serum specimens reacted with the 72-kDa antigen.

Uninfected infants of seropositive mothers. Sera from theuninfected 30 infants of seropositive mothers showed reactivityagainst a number of treponemal antigens (Fig. 2, lane g), themost frequent being 84 kDa (six infants), 39 kDa (eightinfants), 42 kDa (five infants), and 28 or 24 kDa (six infants).Of the 21 neonates, sera from 16 had no IgM reactivity or a

single faint band only; sera from none of the neonates had a

positive blot for the 47- or 45-kDa protein. In contrast, serafrom all nine older infants (1 to 4 months of age) showed a

response to one or more treponemal antigens, and sera fromthree infants reacted with the 47-kDa antigen (Table 1). Serafrom two of these infants were RF latex test positive.

Healthy infants. The serum samples from the newborns withnegative serologies for syphilis showed no reaction with trepo-nemal antigens when Western blotting was carried out.

Definition of positive and negative blots. Overall, reactivityto a number of antigens (those of 72, 47, 45, 37, 33, 30, 17, and15 kDa) was more frequent (P < 0.05) in patients withcongenital syphilis than in uninfected infants of seropositivemothers (Table 1). We therefore regarded blots with reactivi-ties to any of these antigens as positive. Responses to theremaining antigens, including the proteins of 84, 42, 39, 28, and24 kDa, were nonspecific and were observed in both patientsand uninfected infants and, for the purposes of the study, wereregarded as a negative test result. By applying this categoriza-tion, the sensitivity of Western blotting in the diagnosis ofcongenital syphilis was 92% in symptomatic patients, 83% inthose without physical signs, and 75% in the subgroup withnormal long-bone X rays. The specificity was 90%.IgM fractionation. IgM fractions were tested in three symp-

110 -m84 .*

47

2417

a b c d e f 9FIG. 3. IgM reactivity in Western blots. Lane a, sera from infants

with symptomatic congenital syphilis; lanes b and c, sera from infantswith symptomatic congenital syphilis before (lane b) and after (lane c)IgM fractionation; lanes d and e, sera from infants with asymptomaticcongenital syphilis before (lane d) and after (lane e) IgM fractionation;lanes f and g, serum from an uninfected infant of a seropositive motherbefore (lane f) and after (lane g) IgM fractionation.

tomatic infants whose sera were RF latex test positive. One ofthe three was a patient whose whole serum showed noresponse to the 47- and 45-kDa proteins and had a negativeblot with whole serum (Fig. 3, lane b). In this individual,reactivity to the 47- and 33-kDa antigens was identified whenthe IgM separated by chromatography was tested (Fig. 3, lanec). Results for the other two symptomatic infants showedminor differences when the patterns obtained before and afterIgM fractionation were compared (Table 2).Three clinically asymptomatic infants whose IgM fractions

were tested had no IgM response to the 47- or 45-kDa protein.There were few changes in the blots after the procedure, andthe sensitivity of the procedure was not improved (Fig. 3, lanesd and e). The IgM reaction to the 15-kDa antigen was nolonger evident in one patient (Table 2).IgM fractions were also prepared on three serum specimens

from uninfected seropositive patients whose sera had false-

TABLE 2. IgM reactivity to T. pallidunm antigens before and afterIgM fractionation

Antigen (kDa)Patient group

Whole serum IgM fraction

SymptomaticPatient 1 110, 72, 47, 45, 42, 39, 110, 84, 72, 69, 59, 47,

37, 28 45, 42, 28Patient 2 84, 72, 47, 45, 39, 37, 72, 47, 45, 39, 37, 33,

33, 24, 17, 15 24, 17, 15Patient 3 110, 84 110, 61, 47, 33

Clinically inapparentPatient 4 84, 61 84Patient 5 84, 61, 15 84Patient 6 84 84

Uninfected, at riskPatient 7 47, 39 No reactivityPatient 8 110, 84, 47, 45, 42, 39, 110, 84

35, 33, 24, 17, 15Patient 9 72, 61, 47, 45, 42, 39 72, 61, 59

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632 MEYER ET AL.

positive blots and whose whole serum reacted with the 47-kDaantigen. When the blots were repeat tested, fewer antigenswere detected and no response to the 47-kDa protein wasidentified (Fig. 3, lanes f and g).

DISCUSSION

We investigated Western blotting as a test for the diagnosisof congenital syphilis in patients with symptomatic disease,clinically inapparent syphilis, and two groups of uninfectedinfants. Practically, it is important that tests for congenitalsyphilis be able to distinguish asymptomatic infants of seropos-itive mothers who are infected from those who are not (9). Wewere able to investigate this question by monitoring at-riskinfants without abnormal physical features.

Overall, our findings obtained by Western blotting withwhole sera confirm and extend those of previous studies (6, 10,13). The 47- and 45-kDa antigens appear to be the majorimmunogens in both symptomatic and asymptomatic patientswith congenital syphilis. The pattern of reactivity seen in thesera of patients presenting with clinical signs of the disease wassimilar to that described by other authors. Sanchez et al. (13)reported that IgM responses to treponemal antigens withmolecular masses of 72, 47, 45, 42, 37, 17, and 15 kDa wereuseful for distinguishing those infants with syphilis.We found, in addition, that reactivity to the 33- and 30-kDa

proteins also detects infants congenitally infected with T.pallidum. Conversely, an IgM response to the 42-kDa antigenwas nonspecific, being present in the sera of 17% of uninfectedinfants aged 1 to 4 months. The sensitivity and specificity of theWestern blotting procedure were not increased by consideringreactivity to antigens other than those with molecular massesof 47 and 15 kDa.

In the sera of infants with clinically inapparent disease, wenoted IgM responses not only to the 47- and 45-kDa antigensbut also to the 15-kDa protein (in the absence of RF). Becausetwo of the three asymptomatic infants whose sera reacted withthe 15-kDa antigen had no IgM response to the 47- or 45-kDaantigen, we suggest that the former response may occur earlyin the disease. On the other hand, none of the asymptomaticinfants demonstrated reactivity with the 72-kDa antigen, indi-cating that such a response may occur relatively late in thedisease.We noted a sensitivity of 75% for the demonstration of IgM

by Western blotting in asymptomatic infants with normallong-bone X rays, and this is similar to the figure of 65%obtained by FTA-ABS to detect IgM in patients with delayed-onset disease (9). These results should be compared with asensitivity of 14% reported for Western blots of sera fromasymptomatic infants in the recent study of Bromberg et al.(3). The presence of positive immunofluorescence for T.pallidum on a nasal swab obtained soon after birth wasregarded as presumptive evidence of congenital syphilis. How-ever, it has not been established that all infants with such animmunofluorescence response develop systemic infection withT. pallidum.

False-positive blots were found with sera from three unin-fected infants aged I to 4 months, and two of the threefalse-positive blots were RF latex test positive. Removal of IgGeliminated the reactivity to the 47-kDa antigen, suggesting thatRFs, either apparent or hidden, were responsible for theobserved pattern. IgM reactivities to a number of othertreponemal antigens were also noted, which is similar to thefindings of Dobson et al. (6). In the latter study, five of eightnewborns whose mothers were serofast (i.e., had repeatedunchanging positive RPR of low titers in the light of adequate

treatment for previous infection) or had a biologic false-positive nontreponemal test were seroreactive with antigens of190, 83, 71, 45, and 41 kDa. Sanchez et al. (13) also presentedresults for uninfected newborns of seropositive mothers. NoIgM response was present, but only IgM fractions were testedand the authors pointed out that their method may have beenrelatively insensitive because sera from mothers of infectedinfants showed no IgM reactivity. Previous reports have notpresented results of Western blots of sera from older unin-fected infants, but our findings are in keeping with thepresence of cross-reactive epitopes on nonpathogenic trepo-nemes. The molecular masses of the shared antigens whichmost frequently elicit an antibody response in adults arebetween 80 and 90 kDa and 30 and 40 kDa (2, 4).The testing of IgM fractions only did not markedly affect the

results for infants with congenital syphilis. This finding is inagreement with those of earlier studies (6, 13). One infant wasfound to have reactivity to the 47- and 33-kDa antigens afterthe procedure; competitive inhibition between IgM and IgGantibodies for binding sites may have been responsible for this.Among the asymptomatic infants, the IgM response to the15-kDa antigen was no longer apparent in the serum of oneinfant after fractionation. This patient was IgM RF negative,and the reason for the altered result is unclear, although IgMmay have been lost during fractionation. Sanchez et al. (13)noted greater clarity with the blotting technique when the IgMfraction was tested, although no infants with delayed-onsetdisease were investigated.We were able to compare the results obtained by Western

blotting and FTA-ABS (IgM). The latter was performed onwhole serum without the removal of IgG (this significantlyreduces sensitivity [12]). The fluorescence test was positive forsera from all symptomatic infants and was reactive with serafrom 10 of 12 of the clinically asymptomatic infants. Of the twoinfants with negative tests, one also had a nonreactive blot,whereas the other had antibody to the 15-kDa antigen. Thenumber of false-positive results was the same by both tests(10%). In the present study, the performances of the two testswere similar, although the Western blotting method may bemore objective and easier to standardize.We found that the Western blotting procedure had a high

degree of sensitivity, even with sera from infants with clinicallyinapparent disease. Nevertheless, not all patients with syphiliswere identified, so that a negative pattern of reactivity cannotexclude the diagnosis. Furthermore, whereas a reactive blot isa useful indicator of congenital syphilis, false-positive resultswere noted in infants older than 1 month. The possibility ofsimplifying the procedure to a dot blot method or enzyme-linked immunosorbent assay as a confirmatory test is attractive,and the most useful antigens appear to be those with molecularmasses of 47 and 15 kDa.

ACKNOWLEDGMENTS

Funding was obtained from the Medical Research Council of SouthAfrica and from the U.S. Department of Veterans Affairs.

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2. Baughn, R. E., and D. M. Musher. 1985. Radioimmunoassays forthe detection of antibodies to treponemal polypeptide antigens inserum. J. Clin. Microbiol. 21:922-929.

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IgM WESTERN BLOTS IN CONGENITAL SYPHILIS 633

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immunoglobulin M Western blot analysis in the diagnosis ofcongenital syphilis. J. Clin. Microbiol. 28:296-302.

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analysis of the outcome of pregnancy in relation to treatment in943 cases. JAMA 102:503-510.

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