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Treponema pallidum western blot: Comparison with the FTA-ABS test as a confirmatory test for syphilis
Diagnostic Microbiology and Infectious Disease 39 (2001) 9 –14
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Treponema pallidum western blot: Comparison with the FTA-ABS test as a confirmatory test for syphilis Josephine L. Backhouse*, Serge I. Nesteroff Centre for Infectious Diseases and Microbiology Laboratory Services, Institute of Clinical Pathology and Medical Research (ICPMR), Westmead, NSW 2145, Australia Received 10 April 2000; accepted 23 October 2000
Abstract We developed a Treponema pallidum Western blot and compared the results with Treponema pallidum particle agglutination (TPPA) and fluorescent treponemal antibody absorption (FTA-ABS) tests. The Western blot was deemed reactive if the serum reacted with at least three major antigenic bands (TpN47, TpN44.5, TpN17, TpN15). The sensitivities of the Western blot, TPPA and FTA-ABS, were all 100% and the specificities of the Western blot, TPPA and FTA-ABS were 100%, 100% and 94.5% respectively. In 52 problem sera, reactive in only one treponemal test, the agreement between the Western blot and TPPA (61.5%) was significantly better than between Western blot and FTA-ABS (38.5%). The individual sensitivities and specificities of TpN47, TpN44.5, TpN17, TpN15 were 100%, 100%, 96%, 100% and 20%, 96%, 100%, 100% respectively. We conclude that the Western blot is a useful additional confirmatory test or alternative to the FTA-ABS and that a more sensitive and specific criterion for the Western blot would be reactivity with TpN15 and two of the three other major antigens. © 2001 Elsevier Science Inc. All rights reserved.
1. Introduction In the last decade many laboratories, particularly in western Europe and Australia, have moved to screening for syphilis with a specific treponemal test—the Treponema pallidum haemagglutination (TPHA), Treponema pallidum particle agglutination (TPPA) or one of several commercially available enzyme immunoassays (EIAs). This eliminates the problems of prozone, biological false positives (BFP) and low sensitivity in latent syphilis associated with screening for syphilis with a nontreponemal test, such as the Rapid Plasma Reagin (RPR) or Venereal Disease Research Laboratory (VDRL) test. However, other problems have arisen, the most common of which is a reactive or equivocal TPHA, TPPA or EIA test in an asymptomatic patient with no previous history of syphilis. A reactive TPHA, TPPA or EIA requires confirmation by another treponemal test with high sensitivity and specificity. The fluorescent treponemal antibody absorption (FTAABS) test is currently the standard confirmatory test for
syphilis although, except in very early infection, the TPHA is more sensitive and specific (Luger, 1988). Poor specificity of the FTA-ABS has been well documented. False positive reactions can occur in Lyme disease (Hunter et al., 1986), systemic lupus erythematosus and other autoimmune diseases, pregnancy, in the elderly (Larsen et al., 1995) and in the normal population (Luger, 1988). Furthermore, false cross reactivity occurs between EIAs and the FTA-ABS (Chronas et al., 1992) and therefore an alternative treponemal test is needed for confirmation. The Western blot has been evaluated as an alternative confirmatory test (Byrne et al., 1992; Hensel et al., 1985) and in diagnostic problem areas, especially congenital syphilis (Bromberg et al., 1993; Lewis et al., 1990; Meyer et al., 1994; Sanchez et al., 1989). In this study we examined the sensitivity and specificity of the Western blot and its application as a confirmatory test for syphilis. 2. Materials and methods 2.1. Sera
* Corresponding author. Tel.: ⫹61-2-9845-6232; fax: ⫹61-2-98938659. E-mail address: [email protected]. (J.L. Backhouse).
A total of 278 sera were tested including, 98 reactive sera from patients with documented treponemal infection (Table
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J.L. Backhouse and S. I. Nesteroff / Diagnostic Microbiology and Infectious Disease 39 (2001) 9 –14
Table 1 Reactivity of the VDRL, TPPA, FTA-ABS tests and the Western blot in sera from 98 individuals with a history of treponemal infection Category
Status
Primary
untreated treated untreated treated untreated untreated treated untreated untreated treatment not required untreated treated untreated
Secondary Early latent Late latent Neurological Reinfection Passive transfer HIV positive Yaws
No. sera tested
No. sera reactive VDRL
TPPA
FTA-ABS
Western blot ⱖ3 bands
11 10 10 10 6 12 6 4 5 4
11 6 10 5 6 9 1 4 5 0
11 10 10 10 6 12 6 4 5 4
11 10 10 10 6 12 6 4 5 4
11 10 10 10 6 12 6 4 5 4
2 8 10
2 0 8
2 8 10
2 8 10
2* 8* 10
* four sera reacted only with three bands (1 of 2 untreated and 3 of 8 treated)
1); a specificity panel of 128 sera from normal subjects or those with infections that may cause false positive results (Table 2); a panel of 52 problem sera from asymptomatic individuals with no history of treponemal infection, but one reactive treponemal test result (Table 3). All, with the exception of the normal blood donor specificity panel (Australian Red Cross, Sydney, Australia), were from our institute and stored in aliquots at ⫺20°C. 2.2. Serologic methods All sera were examined by the VDRL test (Behringwerke, Marburg, Germany), the FTA-ABS test (sorbent from bioMerieux, Marcy-l’Etoile, France; IgG conjugate
Table 2 Reactivity of the TPPA, FTA-ABS and the Western blot in sera from 128 individuals with no evidence of syphilis Category
Blood donors Antenatal BFPa Leptospirosis Lyme disease Chlamydia Auto immune disease Viral infections: -HIV -HSV -Hepatitis B a
No. sera tested
37 9 12 11 31 2 12
5 4 5
2.3. T. pallidum antigen preparation T. pallidum subsp pallidum, (T. pallidum) Nichols strain, was propagated by testicular passage in adult male New
Number of tests/bands reactive VDRL
0 0 12 0 0 0 0
0 0 0
BFP, biological false positives
TPPA
0 0 0 0 0 0 0
0 0 0
FTA-ABS
0 0 0 0 6 0 0
0 0 1
Western blot bands ⱖ3
2
1
0 0 0 0 0 0 0
1 0 2 0 0 0 0
33 6 9 6 28 2 8
0 0 0
0 0 2
from Behringwerke, Marburg, Germany) using standard methods (Larsen et al., 1990) and the TPPA test (Fujirebio, Tokyo, Japan). For the FTA-ABS, sera were screened at 1:5 dilution in sorbent and for the TPPA diluted to 1:40 in absorbing diluent and read at a final dilution of 1:80 following the addition of sensitised particles. All sera in which results were equivocal, in either TPPA or FTA-ABS tests were retested. The FTA-ABS test was scored according to the level of immunofluorescence, ranging from 4⫹ to 1⫹ (minimally reactive). An initial 1⫹ reading was considered equivocal, if the result was the same on repeat, it was recorded as minimally reactive. Sera giving an equivocal reading in the TPPA test at the recommended final dilution of 1:80 were retested at 1:40, if reactive at this dilution they were recorded as reactive.
0 4 2
Table 3 The Western blot results for 52 asymptomatic individuals with no history of syphilis and low levels of antibody in one of the treponemal (TPPA or FTA-ABS) tests No sera tested
No. tests/bands reactive VDRL
13 12 27
0 3 3 a
TPPA
0 0 27
FTA-ABS minimally reactive
FTA-ABS
13 12a 0
Western blot bands ⱖ3
2
1
3 2 12
2 2 0
7 6 13
J.L. Backhouse and S. I. Nesteroff / Diagnostic Microbiology and Infectious Disease 39 (2001) 9 –14
11
Zealand White rabbits (Communicable Disease Center, 1964) which had been screened by the VDRL, TPPA and FTA-ABS tests for cross reacting T. paraluiscuniculi antibodies and found to be nonreactive. The rabbits were held at 18 –20°C and provided with antibiotic free food and water. The treponemes were harvested 8 to 10 days post inoculation, purified by Percoll density gradient centrifugation (Lukehart et al., 1982), resuspended in phosphate buffered saline (PBS) pH 7.3, sonicated and stored in aliquots at ⫺70°C. The protein concentration of the sonicate was estimated to be 1 mg/ml (BCA Protein Assay, Pierce, Rockford, Il., USA).
2.4. Western blot For sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) aliquots of T. pallidum sonicate were adjusted to contain 0.25 mg/ml protein and boiled for 5 minutes in a ⫻5 reducing sample buffer containing 10% SDS, 0.3125 M Tris (pH 6.8), 0.125% bromphenol blue, 50% glycerol and 11% 2-mercaptoethanol. The sample was allowed to cool and spun at 13,800 g for 5 minutes before 1.6 g protein (approx 1 ⫻ 107 T. pallidum) was applied to each lane of a 4% stacking and 12% separating polyacrylamide gel (Ready-gel, Bio-Rad Laboratories, Richmond, Calif., USA). The gel was electrophoresed using a discontinuous buffer system (Laemmli, 1970) in a mini gel system, 100 volts constant power. Low molecular weight standards and prestained low molecular weight markers (Bio-Rad Laboratories, Richmond, Calif., USA) were run with each gel to determine efficacy of electrophoretic transfer. The separated proteins were transferred to nitrocellulose paper in a mini trans blot system (Bio-Rad Laboratories, Richmond, Calif., USA), at 100 volts constant for 45 minutes (Towbin, et al., 1979). Following transfer, the membrane was blocked in a 5% solution of nonfat powdered milk in PBS for one hour, rinsed three times in PBS and cut into 5 mm ⫻ 75 mm strips. The strips were used immediately or allowed to dry and stored at ⫺20°C. All the following stages were performed at room temperature with rocking. Strips for immunoblotting were placed in individual troughs in blotting trays and incubated overnight with sera at a dilution of 1:100 in PBS-Tween 20 (PBS-Tw). The strips were washed three times with PBS-Tw (five minutes each) and then incubated for two hours in affinity isolated horse radish peroxidase-conjugated sheep antihuman IgG (␥ chain specific) or anti-human IgM ( chain specific) (Silenus, Amrad Biotech, Boronia, VIC, Australia) at dilutions of 1:800 and 1:200 respectively. The strips were washed as described previously and developed with a 4-chloro-1 napthol peroxidase substrate system (Kirkegaard & Perry, Gaithersburg, MD, USA) for 15 minutes. Reactive sera were those which produced visible bands corresponding to antigenic bands, TpN47, Tp 44.5, TpN17, TpN15 (3, 17, 18). A low positive control serum, from a
Fig. 1. Examples of T. pallidum Western blot reactivity. Lane a, molecular weight markers; lane b, reactive syphilis control (⫹); lane c, nonreactive syphilis control (⫺); lane d, primary syphilis (⫹); lane e, treated primary syphilis (⫹); lane f, latent syphilis (⫹); lane g, treated latent syphilis (⫹); lane h, treated syphilis with HIV infection (⫹); lane i, yaws (⫹); lane j, hepatitis B positive, reactivity with two of the major antigens (⫺); lane k, Lyme disease positive, reacting with one major antigen (⫺); lanes l (⫹) and m (⫺), problem sera only reactive in the TPPA test; lanes n (⫹) and o (⫺) problem sera only reactive in the FTA-ABS test. (⫹ reactive; ⫺ nonreactive).
patient with syphilis, which was VDRL nonreactive, TPPA reactive and FTA-ABS reactive (2⫹ immunofluorescence) and a negative control serum from a donor with no history of syphilis, were included in each run. McNemar’s test was used to compare results in the same group of sera and the Z test of proportions used to compare agreement between various tests.
3. Results 3.1. Sensitivity of the Western blot The sensitivity of the Western blot was initially compared with that of the TPPA and the FTA-ABS using the low positive control. The serum control was serially diluted in nonreactive serum in doubling dilutions from 1:2 to 1:1024. Endpoint dilutions of reactivity for each test were: TPPA 1:32, FTA-ABS 1:8 (minimally reactive) and Western blot 1:256. The sensitivities of the TPPA, FTA-ABS and Western blot were compared using a panel of 98 sera from patients with known treponemal infection (Table 1). Fig. 1 shows examples of the reactivity of the Western blot. The four major antigens were visualised in all but eleven sera from patients with syphilis which showed three bands. These sera were retested and, on repeat, the four major antigens were detected in another seven sera. The remaining sera which reacted with only three bands were all from HIV antibody positive patients. Individual antigenic bands TpN47, TpN17 and TpN15 were detected with 100% and TpN44.5 with 96% of sera. To determine whether IgM antibodies against the four
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J.L. Backhouse and S. I. Nesteroff / Diagnostic Microbiology and Infectious Disease 39 (2001) 9 –14
major antigens were present in a group of sera from patients with untreated primary syphilis, the sera were run in parallel in the Western blot with both IgG and IgM conjugates. All sera demonstrated IgG and IgM reactivity against the four major antigens. The intensity of bands with IgM conjugate was less than that with IgG conjugate and varied between sera particularly for bands TpN15 and TpN17. 3.2. Specificity of the Western blot The specificity of the Western blot was compared with that of the TPPA and the FTA-ABS tests in a panel of 128 sera from individuals without evidence of treponemal infection (Table 2). Specificities of both TPPA and Western blot were 100%. There was a statistically significant difference between the results of both TPPA and Western blot and those of the FTA-ABS test, in which 7 of 128 (5.5%; 95% CI 1.5%–9.4%) sera gave false positive results (P ⫽ 0.02). Five sera (3.9%) in this panel reacted with two major antigens, TpN47 and TpN44.5, in the Western blot (ie. a nonreactive result), (e.g. Fig. 1, lanes j and k). These sera included two BFP, two positive for hepatitis B surface antigen, one of which was also minimally reactive in the FTA-ABS test, and one from a blood donor. Fine bands of heavy, moderate and faint levels of reactivity were seen in 19, 13 and 66 sera respectively with the 47kDa antigen only. 3.3. Problem sera The results of the Western blot were also compared with those of the TPPA and FTA-ABS in a panel of 52 problem sera from asymptomatic people with no history of treponemal infection, reactive in only one treponemal test. Thirteen sera were reactive and twelve minimally reactive in the FTA-ABS test and 27 reactive only in the TPPA test (Table 3). To exclude early primary syphilis this group was also tested with combined antihuman IgM and IgG conjugates with no change in the number of bands detected. There was significantly better agreement between the TPPA and the Western blot (32/52; 61.5%) than between the FTA-ABS and the Western blot (20/52; 38.5%) (P ⬍ 0.001). Four of the 25 sera (16%) that were reactive or minimally reactive in the FTA-ABS test only reacted with two of the four major antigens (e.g. Fig. 1, lane k). Reactivity with the 47kDa antigen was only seen in 13/25 (52%) sera reactive in the FTA-ABS only and 13/27 (48%) sera reactive in the TPPA only.
4. Discussion The presence of antibodies to the major antigens of T. pallidum TpN47, TpN44.5, TpN17 and TpN15 is accepted as evidence of past or present treponemal infection (Byrne
et al., 1992; Norris et al., 1987; Norris et al., 1993). In this study in which reactivity to three or four major antigens was the diagnostic criterion, the sensitivity of the Western blot and agreement with the TPPA and FTA-ABS tests were 100%. The sensitivity was not significantly different from that (93.8%) reported previously in clinically defined specimens (P ⫽ 0.09) (Byrne et al., 1992). The specificity of 100% was the same as that of the TPPA and better than that of the FTA-ABS which was 94.5%. These figures are comparable to the specificity of 100% for the Western blot and 92.0% for the double staining FTA-ABS reported by Byrne et al (1992). False positive results in the treponemal tests are often transient (Larsen et al., 1995). However, falsely reactive FTA-ABS has been documented in several conditions including Lyme disease and autoimmune diseases, with an overall specificity of 92–99% (Larsen et al., 1995). Therefore, in this study extra weight was given to sera from subjects with pathogenic spirochaetal infections and autoimmune diseases, to challenge the Western blot. The only false positive reactions were seen with the FTA-ABS in the Lyme disease panel. In the panel of sera from patients with syphilis and yaws, all but four were reactive with the four major antigens, but the intensity of the bands diminished with treatment and time. The four patients who reacted with only three of the four antigenic bands were all HIV positive and three had been treated for syphilis, 12–24 years previously. In the group of problem sera with only one of the treponemal tests reactive, the Western blot supported the possibility of past treponemal infection in five of the FTA-ABS reactive sera and in 12 of the TPPA reactive sera, since all 17 sera reacted with the four major antigens. The assay further suggested that the remaining problem sera were falsely reactive, with only one major antigenic band TpN47 giving low to moderate levels of intensity. By the time clinical signs of syphilis develop most patients have both antitreponemal IgG and IgM (Baker-Zander et al., 1985). In our study all patients with primary syphilis had strongly reactive IgG antibodies against the four major antigens. The FTA-ABS test is more sensitive in primary syphilis (70% to 100%) than the TPHA (69% to 90%) (Larsen et al., 1995). However, the TPPA is more sensitive than the TPHA in early infection in agreement with the FTA-ABS test (Deguchi et al., 1994). Therefore, to exclude early primary syphilis, the problem group was examined for both IgG and IgM antitreponemal antibodies. There was no difference in the number of bands or the level of reactivity when tested for either antitreponemal IgG only or for both antitreponemal IgG and IgM. Inclusion of an IgM conjugate in the assay was not considered necessary as in early primary syphilis reactivity occurs with both IgG and IgM, with no consistent difference between the two immunoglobulins (Baker-Zander et al., 1985). Levels of reactivity in the bands between the IgG and IgM differ between patients, but re-
J.L. Backhouse and S. I. Nesteroff / Diagnostic Microbiology and Infectious Disease 39 (2001) 9 –14
activity with the IgG is generally stronger (Baker-Zander et al., 1985). A small number of sera tested in this study reacted with two of the four major antigens whereas Byrne et al (1992) recorded none in this category. This discrepancy may be related to differences in antigen preparation (Percoll-purified sonicate vs whole cell lysate) or the amount used 107 vs 105 (Byrne et al., 1992; Norris et al., 1993). Finally, we also used a wider range of sera in the specificity panel as well as problem sera, which could also explain the number of sera reacting with two of the major bands. Byrne et al (1992) suggested that reactivity with one or two antigens should be interpreted as negative and equivocal respectively. Of the nine sera in this study which reacted with only two major antigens, four were from subjects with no evidence of syphilis and nonreactive in both treponemal tests and five were from the problem group which were reactive or minimally reactive in the FTA-ABS test only. Overall, 5 of 32 (16%) sera that were reactive or minimally reactive in the FTA-ABS test only, reacted with two major antigens in the Western blot, compared with 4 of 121 (3%) which were nonreactive in both treponemal tests (P ⫽ 0.01) and none of the 27 sera which were reactive in the TPPA only. On reviewing reactivity to the individual major antigens (TpN47, TpN 44.5 TpN 17 and TpN15) the respective sensitivities and specificities were 100%, 100%, 96%, 100% and 20%, 96%, 100%, 100%. These comparative figures show TpN15 to have the best combination of sensitivity and specificity and therefore to be the most accurate indicator of treponemal infection. All nine sera in the study which gave an equivocal Western blot reacted only with the TpN47 and TpN44.5 antigens. Absence of reactivity to the highly sensitive and specific TpN15 antigen suggests these sera were showing nonspecific reactivity to the TpN47 and TpN44.5 antigens. A recent study reassessed the parameters defining a reactive Western blot and found the 17kDa antigen to be the most exact of the four major antigens (George et al., 1998). The absence of this band in the 9 sera giving equivocal Western blot results further supports the interpretation on these sera as nonspecific reactivity. In conclusion the Western blot is a highly sensitive and specific test for syphilis. It is as sensitive and more specific than the FTA-ABS test which makes it a useful additional confirmatory test or an alternative to the FTA-ABS test, particularly with problem sera. Furthermore, these results suggest that the most sensitive and specific criterion for determining present or past treponemal infection would be reactivity to TpN15 and two of the three other major antigens.
Acknowledgment The authors thank Professor GL Gilbert, ICPMR, Westmead, Australia for a critical review of the manuscript.
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References Baker-Zander, S. A., Hook, E. W., III, Bonin, P., Hansfield, H. H., & Lukehart, S. A. (1985). Antigens of Treponema pallidum recognised by IgG and IgM antibodies during syphilis in humans. J Infect Dis 151, 264 –272. Bromberg, K., Rawstron, S., & Tannis, G. (1993). Diagnosis of congenital syphilis by combining Treponema pallidum-specific IgM detection with immunofluorescent antigen detection for T. pallidum. J Infect Dis 168, 238 –242. Byrne, R. E., Laska, S., Bell, M., Larsen, D., Phillips, J., & Todd, J. (1992). Evaluation of a Treponema pallidum Western immunoblot as a confirmatory test for syphilis. J Clin Microbiol 30, 115–122. Chreighton, E. T. (1990). Venereal disease research laboratory slide tests. In A manual of tests for syphilis, 8th ed. Eds Larsen, S. A., Hunter, E. F., Kraus, S. J. American Public Health Association, Washington, D.C., pp. 78 – 89, 130 –139. Chronas, G., Moyes, A., & Young, H. (1992). Syphilis diagnosis: screening by enzyme immunoassay and variation in fluorescent antibody absorbed (FTA-ABS) confirmatory test performance. Med Lab Sci 49, 50 –55. Communicable Disease Centre. (1964). Treponema pallidum immobilisation 200 (TPI-200) test. In Serologic tests for syphilis. PHS publication no. 411, U. S. Department of Health and Human Services, Public Health Service, U. S. Government Printing Office, Washington, D.C., USA, pp 72–74. Deguchi, M., Hosotsubo, H., Yamashita, N., Ohmine, T., & Asari, S. (1994). Evaluation of the gelatin particle agglutination method for determining Treponema pallidum antibody. J Jpn Assoc Infect Dis 68, 1–11. George, R., Pope, V., Fears, M., Morrill, B., & Larsen, S. (1998). An analysis of some antigen-antibody interactions used as diagnostic indicators in a treponemal Western blot (TWB) test for syphilis. J Clin Lab Immunol 50, 27– 44. Hensel, U., Wellensiek, H.-J., & Bhakdi, S. (1985). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis immunoblotting as a serological tool in the diagnosis of syphilitic infections. J Clin Microbiol 21, 82– 87. Hunter, E. F., Russell, H., Farshy, C. E., Sampson, J. S., & Larsen, S. A. (1986). Evaluation of sera from patients with Lyme disease in the fluorescent treponemal antibody-absorption tests for syphilis. Sex Transm Dis 13, 232–236. Hunter, E. F. (1990). Fluorescent treponemal antibody-absorption test. In A manual of tests for syphilis, 8th ed. Larsen, S. A., Hunter, E. F., Kraus, S. J. American Public Health Association, Washington, D.C. pp 130 – 139. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680 – 685. Larsen, S. A., Steiner, B. M., & Rudolph, A. H. (1995). Laboratory diagnosis and interpretation of tests for syphilis. Clin Microbiol Rev 8, 1–212. Lewis, L. L., Taber, L. H., & Baughn, R. E. (1990). Evaluation of immunoglobulin M Western blot analysis in the diagnosis of congenital syphilis. J Clin Microbiol 28, 296 –302. Luger, A. (1988). Serological diagnosis of syphilis: current methods. In Immunological diagnosis of sexually transmitted diseases. Eds. Young, H., and McMillan, A. Marcel Dekker, New York, pp 213–247. Lukehart, S. A., Baker-Zander, S. A., & Gubish, Jr., E. R. (1982). Identification of Treponema pallidum antigens: comparison with a nonpathogenic treponeme. J Immunol 129, 833– 838. Meyer, M., Eddy, P. T., & Baughn, R. E. (1994). Analysis of Western blotting (immunoblotting) technique in diagnosis of congenital syphilis. J Clin Microbiol 32, 629 – 633. Norris, S. J., Aldereete, J. F., Axelsen, N. H., Bailey, M., Baker-Zander, S. A., Baseman, J. B., Bassford, P. J., Baughn, R. E., Cockayne, A.,
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Hanff, P. A., Hindersson, P., Larsen, S. A., Lovett, M. A., Lukehart, S. A., Miller, J. N., Moskophidis, M. A., Muller, F., Norgard, M. V., Penn, C. W., Stamm, C. V., van Embden, J. D. A., & Wicker, K. (1987). Identity of Treponema pallidum subsp. pallidum polypeptides: correlation of sodium dodecyl sulphate-polyacrylamide gel electrophoresis results from different laboratories. Electrophoresis 8, 77–92. Norris, S. J., and the Treponema pallidum polypeptide research group. (1993). Polypeptides of Treponema pallidum: Progress toward under-
standing their structural, functional, and immunologic roles. Microbiol Rev 57, 750 –779. Sanchez, P. J., McCracken, Jr., G. H., Wendel, G. D., Olsen, K., Threlkeld, N., & Norgard, M. V. (1989). Molecular analysis of fetal IgM response to Treponema pallidum antigens: Implications for improved serodiagnosis of congenital syphilis. J Infect Dis 159, 508 –517. Towbin, H., Staehlin, T., & Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76, 4350 – 4354.
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Treponema pallidum western blot: Comparison with the FTA-ABS test as a confirmatory test for syphilis Josephine L. Backhouse*, Serge I. Nesteroff Centre for Infectious Diseases and Microbiology Laboratory Services, Institute of Clinical Pathology and Medical Research (ICPMR), Westmead, NSW 2145, Australia Received 10 April 2000; accepted 23 October 2000
Abstract We developed a Treponema pallidum Western blot and compared the results with Treponema pallidum particle agglutination (TPPA) and fluorescent treponemal antibody absorption (FTA-ABS) tests. The Western blot was deemed reactive if the serum reacted with at least three major antigenic bands (TpN47, TpN44.5, TpN17, TpN15). The sensitivities of the Western blot, TPPA and FTA-ABS, were all 100% and the specificities of the Western blot, TPPA and FTA-ABS were 100%, 100% and 94.5% respectively. In 52 problem sera, reactive in only one treponemal test, the agreement between the Western blot and TPPA (61.5%) was significantly better than between Western blot and FTA-ABS (38.5%). The individual sensitivities and specificities of TpN47, TpN44.5, TpN17, TpN15 were 100%, 100%, 96%, 100% and 20%, 96%, 100%, 100% respectively. We conclude that the Western blot is a useful additional confirmatory test or alternative to the FTA-ABS and that a more sensitive and specific criterion for the Western blot would be reactivity with TpN15 and two of the three other major antigens. © 2001 Elsevier Science Inc. All rights reserved.
1. Introduction In the last decade many laboratories, particularly in western Europe and Australia, have moved to screening for syphilis with a specific treponemal test—the Treponema pallidum haemagglutination (TPHA), Treponema pallidum particle agglutination (TPPA) or one of several commercially available enzyme immunoassays (EIAs). This eliminates the problems of prozone, biological false positives (BFP) and low sensitivity in latent syphilis associated with screening for syphilis with a nontreponemal test, such as the Rapid Plasma Reagin (RPR) or Venereal Disease Research Laboratory (VDRL) test. However, other problems have arisen, the most common of which is a reactive or equivocal TPHA, TPPA or EIA test in an asymptomatic patient with no previous history of syphilis. A reactive TPHA, TPPA or EIA requires confirmation by another treponemal test with high sensitivity and specificity. The fluorescent treponemal antibody absorption (FTAABS) test is currently the standard confirmatory test for
syphilis although, except in very early infection, the TPHA is more sensitive and specific (Luger, 1988). Poor specificity of the FTA-ABS has been well documented. False positive reactions can occur in Lyme disease (Hunter et al., 1986), systemic lupus erythematosus and other autoimmune diseases, pregnancy, in the elderly (Larsen et al., 1995) and in the normal population (Luger, 1988). Furthermore, false cross reactivity occurs between EIAs and the FTA-ABS (Chronas et al., 1992) and therefore an alternative treponemal test is needed for confirmation. The Western blot has been evaluated as an alternative confirmatory test (Byrne et al., 1992; Hensel et al., 1985) and in diagnostic problem areas, especially congenital syphilis (Bromberg et al., 1993; Lewis et al., 1990; Meyer et al., 1994; Sanchez et al., 1989). In this study we examined the sensitivity and specificity of the Western blot and its application as a confirmatory test for syphilis. 2. Materials and methods 2.1. Sera
* Corresponding author. Tel.: ⫹61-2-9845-6232; fax: ⫹61-2-98938659. E-mail address: [email protected]. (J.L. Backhouse).
A total of 278 sera were tested including, 98 reactive sera from patients with documented treponemal infection (Table
0732-8893/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 7 3 2 - 8 8 9 3 ( 0 0 ) 0 0 2 1 3 - 3
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J.L. Backhouse and S. I. Nesteroff / Diagnostic Microbiology and Infectious Disease 39 (2001) 9 –14
Table 1 Reactivity of the VDRL, TPPA, FTA-ABS tests and the Western blot in sera from 98 individuals with a history of treponemal infection Category
Status
Primary
untreated treated untreated treated untreated untreated treated untreated untreated treatment not required untreated treated untreated
Secondary Early latent Late latent Neurological Reinfection Passive transfer HIV positive Yaws
No. sera tested
No. sera reactive VDRL
TPPA
FTA-ABS
Western blot ⱖ3 bands
11 10 10 10 6 12 6 4 5 4
11 6 10 5 6 9 1 4 5 0
11 10 10 10 6 12 6 4 5 4
11 10 10 10 6 12 6 4 5 4
11 10 10 10 6 12 6 4 5 4
2 8 10
2 0 8
2 8 10
2 8 10
2* 8* 10
* four sera reacted only with three bands (1 of 2 untreated and 3 of 8 treated)
1); a specificity panel of 128 sera from normal subjects or those with infections that may cause false positive results (Table 2); a panel of 52 problem sera from asymptomatic individuals with no history of treponemal infection, but one reactive treponemal test result (Table 3). All, with the exception of the normal blood donor specificity panel (Australian Red Cross, Sydney, Australia), were from our institute and stored in aliquots at ⫺20°C. 2.2. Serologic methods All sera were examined by the VDRL test (Behringwerke, Marburg, Germany), the FTA-ABS test (sorbent from bioMerieux, Marcy-l’Etoile, France; IgG conjugate
Table 2 Reactivity of the TPPA, FTA-ABS and the Western blot in sera from 128 individuals with no evidence of syphilis Category
Blood donors Antenatal BFPa Leptospirosis Lyme disease Chlamydia Auto immune disease Viral infections: -HIV -HSV -Hepatitis B a
No. sera tested
37 9 12 11 31 2 12
5 4 5
2.3. T. pallidum antigen preparation T. pallidum subsp pallidum, (T. pallidum) Nichols strain, was propagated by testicular passage in adult male New
Number of tests/bands reactive VDRL
0 0 12 0 0 0 0
0 0 0
BFP, biological false positives
TPPA
0 0 0 0 0 0 0
0 0 0
FTA-ABS
0 0 0 0 6 0 0
0 0 1
Western blot bands ⱖ3
2
1
0 0 0 0 0 0 0
1 0 2 0 0 0 0
33 6 9 6 28 2 8
0 0 0
0 0 2
from Behringwerke, Marburg, Germany) using standard methods (Larsen et al., 1990) and the TPPA test (Fujirebio, Tokyo, Japan). For the FTA-ABS, sera were screened at 1:5 dilution in sorbent and for the TPPA diluted to 1:40 in absorbing diluent and read at a final dilution of 1:80 following the addition of sensitised particles. All sera in which results were equivocal, in either TPPA or FTA-ABS tests were retested. The FTA-ABS test was scored according to the level of immunofluorescence, ranging from 4⫹ to 1⫹ (minimally reactive). An initial 1⫹ reading was considered equivocal, if the result was the same on repeat, it was recorded as minimally reactive. Sera giving an equivocal reading in the TPPA test at the recommended final dilution of 1:80 were retested at 1:40, if reactive at this dilution they were recorded as reactive.
0 4 2
Table 3 The Western blot results for 52 asymptomatic individuals with no history of syphilis and low levels of antibody in one of the treponemal (TPPA or FTA-ABS) tests No sera tested
No. tests/bands reactive VDRL
13 12 27
0 3 3 a
TPPA
0 0 27
FTA-ABS minimally reactive
FTA-ABS
13 12a 0
Western blot bands ⱖ3
2
1
3 2 12
2 2 0
7 6 13
J.L. Backhouse and S. I. Nesteroff / Diagnostic Microbiology and Infectious Disease 39 (2001) 9 –14
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Zealand White rabbits (Communicable Disease Center, 1964) which had been screened by the VDRL, TPPA and FTA-ABS tests for cross reacting T. paraluiscuniculi antibodies and found to be nonreactive. The rabbits were held at 18 –20°C and provided with antibiotic free food and water. The treponemes were harvested 8 to 10 days post inoculation, purified by Percoll density gradient centrifugation (Lukehart et al., 1982), resuspended in phosphate buffered saline (PBS) pH 7.3, sonicated and stored in aliquots at ⫺70°C. The protein concentration of the sonicate was estimated to be 1 mg/ml (BCA Protein Assay, Pierce, Rockford, Il., USA).
2.4. Western blot For sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) aliquots of T. pallidum sonicate were adjusted to contain 0.25 mg/ml protein and boiled for 5 minutes in a ⫻5 reducing sample buffer containing 10% SDS, 0.3125 M Tris (pH 6.8), 0.125% bromphenol blue, 50% glycerol and 11% 2-mercaptoethanol. The sample was allowed to cool and spun at 13,800 g for 5 minutes before 1.6 g protein (approx 1 ⫻ 107 T. pallidum) was applied to each lane of a 4% stacking and 12% separating polyacrylamide gel (Ready-gel, Bio-Rad Laboratories, Richmond, Calif., USA). The gel was electrophoresed using a discontinuous buffer system (Laemmli, 1970) in a mini gel system, 100 volts constant power. Low molecular weight standards and prestained low molecular weight markers (Bio-Rad Laboratories, Richmond, Calif., USA) were run with each gel to determine efficacy of electrophoretic transfer. The separated proteins were transferred to nitrocellulose paper in a mini trans blot system (Bio-Rad Laboratories, Richmond, Calif., USA), at 100 volts constant for 45 minutes (Towbin, et al., 1979). Following transfer, the membrane was blocked in a 5% solution of nonfat powdered milk in PBS for one hour, rinsed three times in PBS and cut into 5 mm ⫻ 75 mm strips. The strips were used immediately or allowed to dry and stored at ⫺20°C. All the following stages were performed at room temperature with rocking. Strips for immunoblotting were placed in individual troughs in blotting trays and incubated overnight with sera at a dilution of 1:100 in PBS-Tween 20 (PBS-Tw). The strips were washed three times with PBS-Tw (five minutes each) and then incubated for two hours in affinity isolated horse radish peroxidase-conjugated sheep antihuman IgG (␥ chain specific) or anti-human IgM ( chain specific) (Silenus, Amrad Biotech, Boronia, VIC, Australia) at dilutions of 1:800 and 1:200 respectively. The strips were washed as described previously and developed with a 4-chloro-1 napthol peroxidase substrate system (Kirkegaard & Perry, Gaithersburg, MD, USA) for 15 minutes. Reactive sera were those which produced visible bands corresponding to antigenic bands, TpN47, Tp 44.5, TpN17, TpN15 (3, 17, 18). A low positive control serum, from a
Fig. 1. Examples of T. pallidum Western blot reactivity. Lane a, molecular weight markers; lane b, reactive syphilis control (⫹); lane c, nonreactive syphilis control (⫺); lane d, primary syphilis (⫹); lane e, treated primary syphilis (⫹); lane f, latent syphilis (⫹); lane g, treated latent syphilis (⫹); lane h, treated syphilis with HIV infection (⫹); lane i, yaws (⫹); lane j, hepatitis B positive, reactivity with two of the major antigens (⫺); lane k, Lyme disease positive, reacting with one major antigen (⫺); lanes l (⫹) and m (⫺), problem sera only reactive in the TPPA test; lanes n (⫹) and o (⫺) problem sera only reactive in the FTA-ABS test. (⫹ reactive; ⫺ nonreactive).
patient with syphilis, which was VDRL nonreactive, TPPA reactive and FTA-ABS reactive (2⫹ immunofluorescence) and a negative control serum from a donor with no history of syphilis, were included in each run. McNemar’s test was used to compare results in the same group of sera and the Z test of proportions used to compare agreement between various tests.
3. Results 3.1. Sensitivity of the Western blot The sensitivity of the Western blot was initially compared with that of the TPPA and the FTA-ABS using the low positive control. The serum control was serially diluted in nonreactive serum in doubling dilutions from 1:2 to 1:1024. Endpoint dilutions of reactivity for each test were: TPPA 1:32, FTA-ABS 1:8 (minimally reactive) and Western blot 1:256. The sensitivities of the TPPA, FTA-ABS and Western blot were compared using a panel of 98 sera from patients with known treponemal infection (Table 1). Fig. 1 shows examples of the reactivity of the Western blot. The four major antigens were visualised in all but eleven sera from patients with syphilis which showed three bands. These sera were retested and, on repeat, the four major antigens were detected in another seven sera. The remaining sera which reacted with only three bands were all from HIV antibody positive patients. Individual antigenic bands TpN47, TpN17 and TpN15 were detected with 100% and TpN44.5 with 96% of sera. To determine whether IgM antibodies against the four
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J.L. Backhouse and S. I. Nesteroff / Diagnostic Microbiology and Infectious Disease 39 (2001) 9 –14
major antigens were present in a group of sera from patients with untreated primary syphilis, the sera were run in parallel in the Western blot with both IgG and IgM conjugates. All sera demonstrated IgG and IgM reactivity against the four major antigens. The intensity of bands with IgM conjugate was less than that with IgG conjugate and varied between sera particularly for bands TpN15 and TpN17. 3.2. Specificity of the Western blot The specificity of the Western blot was compared with that of the TPPA and the FTA-ABS tests in a panel of 128 sera from individuals without evidence of treponemal infection (Table 2). Specificities of both TPPA and Western blot were 100%. There was a statistically significant difference between the results of both TPPA and Western blot and those of the FTA-ABS test, in which 7 of 128 (5.5%; 95% CI 1.5%–9.4%) sera gave false positive results (P ⫽ 0.02). Five sera (3.9%) in this panel reacted with two major antigens, TpN47 and TpN44.5, in the Western blot (ie. a nonreactive result), (e.g. Fig. 1, lanes j and k). These sera included two BFP, two positive for hepatitis B surface antigen, one of which was also minimally reactive in the FTA-ABS test, and one from a blood donor. Fine bands of heavy, moderate and faint levels of reactivity were seen in 19, 13 and 66 sera respectively with the 47kDa antigen only. 3.3. Problem sera The results of the Western blot were also compared with those of the TPPA and FTA-ABS in a panel of 52 problem sera from asymptomatic people with no history of treponemal infection, reactive in only one treponemal test. Thirteen sera were reactive and twelve minimally reactive in the FTA-ABS test and 27 reactive only in the TPPA test (Table 3). To exclude early primary syphilis this group was also tested with combined antihuman IgM and IgG conjugates with no change in the number of bands detected. There was significantly better agreement between the TPPA and the Western blot (32/52; 61.5%) than between the FTA-ABS and the Western blot (20/52; 38.5%) (P ⬍ 0.001). Four of the 25 sera (16%) that were reactive or minimally reactive in the FTA-ABS test only reacted with two of the four major antigens (e.g. Fig. 1, lane k). Reactivity with the 47kDa antigen was only seen in 13/25 (52%) sera reactive in the FTA-ABS only and 13/27 (48%) sera reactive in the TPPA only.
4. Discussion The presence of antibodies to the major antigens of T. pallidum TpN47, TpN44.5, TpN17 and TpN15 is accepted as evidence of past or present treponemal infection (Byrne
et al., 1992; Norris et al., 1987; Norris et al., 1993). In this study in which reactivity to three or four major antigens was the diagnostic criterion, the sensitivity of the Western blot and agreement with the TPPA and FTA-ABS tests were 100%. The sensitivity was not significantly different from that (93.8%) reported previously in clinically defined specimens (P ⫽ 0.09) (Byrne et al., 1992). The specificity of 100% was the same as that of the TPPA and better than that of the FTA-ABS which was 94.5%. These figures are comparable to the specificity of 100% for the Western blot and 92.0% for the double staining FTA-ABS reported by Byrne et al (1992). False positive results in the treponemal tests are often transient (Larsen et al., 1995). However, falsely reactive FTA-ABS has been documented in several conditions including Lyme disease and autoimmune diseases, with an overall specificity of 92–99% (Larsen et al., 1995). Therefore, in this study extra weight was given to sera from subjects with pathogenic spirochaetal infections and autoimmune diseases, to challenge the Western blot. The only false positive reactions were seen with the FTA-ABS in the Lyme disease panel. In the panel of sera from patients with syphilis and yaws, all but four were reactive with the four major antigens, but the intensity of the bands diminished with treatment and time. The four patients who reacted with only three of the four antigenic bands were all HIV positive and three had been treated for syphilis, 12–24 years previously. In the group of problem sera with only one of the treponemal tests reactive, the Western blot supported the possibility of past treponemal infection in five of the FTA-ABS reactive sera and in 12 of the TPPA reactive sera, since all 17 sera reacted with the four major antigens. The assay further suggested that the remaining problem sera were falsely reactive, with only one major antigenic band TpN47 giving low to moderate levels of intensity. By the time clinical signs of syphilis develop most patients have both antitreponemal IgG and IgM (Baker-Zander et al., 1985). In our study all patients with primary syphilis had strongly reactive IgG antibodies against the four major antigens. The FTA-ABS test is more sensitive in primary syphilis (70% to 100%) than the TPHA (69% to 90%) (Larsen et al., 1995). However, the TPPA is more sensitive than the TPHA in early infection in agreement with the FTA-ABS test (Deguchi et al., 1994). Therefore, to exclude early primary syphilis, the problem group was examined for both IgG and IgM antitreponemal antibodies. There was no difference in the number of bands or the level of reactivity when tested for either antitreponemal IgG only or for both antitreponemal IgG and IgM. Inclusion of an IgM conjugate in the assay was not considered necessary as in early primary syphilis reactivity occurs with both IgG and IgM, with no consistent difference between the two immunoglobulins (Baker-Zander et al., 1985). Levels of reactivity in the bands between the IgG and IgM differ between patients, but re-
J.L. Backhouse and S. I. Nesteroff / Diagnostic Microbiology and Infectious Disease 39 (2001) 9 –14
activity with the IgG is generally stronger (Baker-Zander et al., 1985). A small number of sera tested in this study reacted with two of the four major antigens whereas Byrne et al (1992) recorded none in this category. This discrepancy may be related to differences in antigen preparation (Percoll-purified sonicate vs whole cell lysate) or the amount used 107 vs 105 (Byrne et al., 1992; Norris et al., 1993). Finally, we also used a wider range of sera in the specificity panel as well as problem sera, which could also explain the number of sera reacting with two of the major bands. Byrne et al (1992) suggested that reactivity with one or two antigens should be interpreted as negative and equivocal respectively. Of the nine sera in this study which reacted with only two major antigens, four were from subjects with no evidence of syphilis and nonreactive in both treponemal tests and five were from the problem group which were reactive or minimally reactive in the FTA-ABS test only. Overall, 5 of 32 (16%) sera that were reactive or minimally reactive in the FTA-ABS test only, reacted with two major antigens in the Western blot, compared with 4 of 121 (3%) which were nonreactive in both treponemal tests (P ⫽ 0.01) and none of the 27 sera which were reactive in the TPPA only. On reviewing reactivity to the individual major antigens (TpN47, TpN 44.5 TpN 17 and TpN15) the respective sensitivities and specificities were 100%, 100%, 96%, 100% and 20%, 96%, 100%, 100%. These comparative figures show TpN15 to have the best combination of sensitivity and specificity and therefore to be the most accurate indicator of treponemal infection. All nine sera in the study which gave an equivocal Western blot reacted only with the TpN47 and TpN44.5 antigens. Absence of reactivity to the highly sensitive and specific TpN15 antigen suggests these sera were showing nonspecific reactivity to the TpN47 and TpN44.5 antigens. A recent study reassessed the parameters defining a reactive Western blot and found the 17kDa antigen to be the most exact of the four major antigens (George et al., 1998). The absence of this band in the 9 sera giving equivocal Western blot results further supports the interpretation on these sera as nonspecific reactivity. In conclusion the Western blot is a highly sensitive and specific test for syphilis. It is as sensitive and more specific than the FTA-ABS test which makes it a useful additional confirmatory test or an alternative to the FTA-ABS test, particularly with problem sera. Furthermore, these results suggest that the most sensitive and specific criterion for determining present or past treponemal infection would be reactivity to TpN15 and two of the three other major antigens.
Acknowledgment The authors thank Professor GL Gilbert, ICPMR, Westmead, Australia for a critical review of the manuscript.
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