|Year : 2013 | Volume
| Issue : 3 | Page : 275-279
Assessment of reactivity of three treponemal tests in non-treponemal non-reactive cases from sexually transmitted diseases clinic, antenatal clinic, integrated counselling and testing centre, other different outdoor patient departments/indoor patients of a tertiary care centre and peripheral health clinic attendees
M Bala, V Singh, S Muralidhar, V Ramesh
Apex Regional STD Teaching, Training and Research Centre, Vardhman Mahavir Medical College and Safdarjung Hospital, New Delhi, India
|Date of Submission||18-Feb-2013|
|Date of Acceptance||20-May-2013|
|Date of Web Publication||25-Jul-2013|
Apex Regional STD Teaching, Training and Research Centre, Vardhman Mahavir Medical College and Safdarjung Hospital, New Delhi
Source of Support: NACO and ICMR,, Conflict of Interest: None
In India, many state reference centres for sexually transmitted infections perform only a single screening assay for syphilis diagnosis. In this study, Treponema pallidum haemagglutination (TPHA) was performed on 1115 Venereal Disease Research Laboratory (VDRL)/rapid plasma regain (RPR) non-reactive and 107 reactive sera out of 10,489 tested by VDRL/RPR according to the National AIDS Control Organisation syphilis testing protocol. A total of 47 Specimens reactive in TPHA and non-reactive with VDRL test were subjected to fluorescent treponemal antibody absorption and enzyme-immunoassay. Seroprevalence considering both VDRL and TPHA positivity was highest (4.4%) in sexually transmitted diseases clinic attendees than in other subject groups. Positivity by two treponemal tests in 24 (2.2%) cases non-reactive by VDRL/RPR was representative of the fully treated patients or latent or late syphilis cases. The findings highlight that a suitable treponemal confirmatory test should be performed in all the diagnostic laboratories.
Keywords: Fluorescent treponemal antibody absorption, non-treponemal tests, syphilis, Treponema pallidum haemagglutination, treponemal tests
|How to cite this article:|
Bala M, Singh V, Muralidhar S, Ramesh V. Assessment of reactivity of three treponemal tests in non-treponemal non-reactive cases from sexually transmitted diseases clinic, antenatal clinic, integrated counselling and testing centre, other different outdoor patient departments/indoor patients of a tertiary care centre and peripheral health clinic attendees. Indian J Med Microbiol 2013;31:275-9
|How to cite this URL:|
Bala M, Singh V, Muralidhar S, Ramesh V. Assessment of reactivity of three treponemal tests in non-treponemal non-reactive cases from sexually transmitted diseases clinic, antenatal clinic, integrated counselling and testing centre, other different outdoor patient departments/indoor patients of a tertiary care centre and peripheral health clinic attendees. Indian J Med Microbiol [serial online] 2013 [cited 2020 Aug 11];31:275-9. Available from: http://www.ijmm.org/text.asp?2013/31/3/275/115638
| ~ Introduction|| |
The accuracy of diagnostic tests is imperative for successful control of sexually transmitted infections (STIs). Syphilis is one of the most common STIs in India.  Antibody detection by non-treponemal/cardiolipin and treponemal tests is the mainstay for diagnosing syphilis and for monitoring the success of subsequent antibiotic treatment. , The most widely used cardiolipin and specific tests are respectively the Venereal Disease Research Laboratory (VDRL) test/rapid plasma regain (RPR) test and the Treponema pallidum haemagglutination (TPHA) test. Both are suitable for large-scale use. Where necessary, the fluorescent treponemal antibody absorption (FTA-Abs) test is used for verification of treponemal infection. Cardiolipin tests are not truly specific for syphilis. Their value lies in their low-cost and in the ease with which they are titred. Specific tests are less likely to give false-positive results in diseases not caused by treponemes, although the tests may give positive results in patients with non-venereal treponematoses, lepromatous leprosy and infectious mononucleosis and in a few healthy people.
TPHA test is simple to perform and reproducible and has specificity and sensitivity comparable with the FTA-Abs test. The problem of the specificity of the TPHA is of great practical significance since it has been widely used as a confirmatory test in India because of its high sensitivity and specificity in accordance with the recommendations of the National AIDS Control Organisation (NACO). Even United States Centers for Disease Control (CDC) continues to recommend that non-treponemal tests be used to screen for syphilis and that treponemal testing be used to confirm syphilis as the cause of non-treponemal reactivity.  However, if reverse sequence screening is used as is the practice in some clinical laboratories and blood banks, CDC recommends that a specimen with reactive enzyme-immunoassay (EIA)/chemiluminescence immunoassays results be tested with a quantitative non-treponemal test.  The results of the TPHA and other treponemal tests have been assessed by many workers.  In some reports certain reservations were expressed regarding the specificity of the TPHA test in pregnant women. This study was undertaken to assess the reactivity the TPHA test in comparison to FTA-Abs and Treponema pallidum-enzyme-immunoassay (TP-EIA) and to determine its value as a confirmatory test for syphilis in non-treponemal non-reactive and reactive sera in a laboratory serving different subject groups.
| ~ Materials and Methods|| |
Source of samples
Sera included in the study were from both normal individuals and patients with a variety of illnesses and conditions. A total of 10,489 sera, derived from the following sources of a tertiary care super specialty hospital of North India were examined over a 4 month period (April 2011-July 2011).
Sexually transmitted diseases (STD) clinic attendees - 844 (567 males, 277 females); antenatal clinic (ANC) attendees - 5203; husbands of antenatal cases - 822; peripheral health centres (PHCs) attendees - 1103 (4 males, 1099 females); integrated counselling and testing centre (ICTC) attendees - 2040 (1189 males, 851 females); patients referred to this centre for VDRL testing from different outdoor patient departments OPDs/wards - 1299 (166 males, 1133 females).
In above subject groups, the method for syphilis screening was as per the recommendations of NACO, i.e., VDRL test or the RPR test and confirmation of reactive sera by TPHA. Sera non-reactive with the screening assay were discarded. Sera from ICTC attendees were screened by RPR test (Span Diagnostics Ltd., India) and from all other study groups by VDRL test (using antigen from Serologist to Government of India, Kolkata).
In this study, we performed TPHA (Omega Diagnostic, Scotland, UK.) for detection of anti-treponemal antibodies on 1115 VDRL non-reactive sera to determine the specificity of TPHA. TPHA testing was also carried out for 107 VDRL reactive sera out of 10,489 tested by VDRL according to the national syphilis confirmatory testing protocol [Figure 1].
|Figure 1: Algorithm used for diagnosis of syphilis as per National AIDS Control Organisation, India syphilis testing guidelines. On the left-hand side, Treponema pallidum Haemagglutination (TPHA) test was performed on Venereal Disease Research Laboratory (VDRL)/rapid plasma regain (RPR) reactive samples (both with titre < 1:8 and ≥ 1:8). On the right-hand side, the diagnosis procedure using the TPHA is shown in VDRL/RPR non-reactive cases, including follow-up confirmation using Treponema pallidum-enzyme-immunoassay, fluorescent treponemal antibody absorption tests|
Click here to view
Specimens reactive in TPHA test and non-reactive with VDRL test were subjected to FTA-Abs (Biocientifica S.A., Argentina) and a competitive EIA immunoglobulin (IgG + IgM) (Omega Diagnostic, Scotland, UK) [Figure 1]. All tests were performed and interpreted according to standard procedures following manufacturer's instructions in kit inserts.
Quality control and external quality assurance scheme (EQAS)
The laboratory used its own syphilitic sera and non-reactive sera as controls. This centre participated in syphilis Serology Proficiency Testing conducted by the CDC, Atlanta, Georgia in April, July, October 2012 and April 2013. Under this EQAS, a panel of five sera were received from the reference laboratory. The results of VDRL, RPR, TPHA, TP-EIA and FTA-Abs of these sera were communicated and feedback was received. EQAS results showed almost 100% agreement with the CDC results for VDRL, TPHA, TP-EIA and FTA-Abs. This laboratory has also been conducting EQAS for screening tests for syphilis (VDRL/RPR) of 25 microbiology laboratories of North India since the year 1999  and at national level since 2009 of 30 laboratories.
| ~ Results|| |
The qualitative and quantitative test results obtained by VDRL/RPR are shown in [Figure 1]. Out of 10,489 sera tested for syphilis by non-treponemal VDRL/RPR test, 10382 (99%) were non-reactive and 107 (1%) reactive. VDRL reactivity was similar (0.5%) in both males and females.
In VDRL reactive sera samples, 87 sera were having titre < 1:8 and 20 samples were shown to have titre ≥ 1:8. Both VDRL and TPHA reactivity was observed in 84 cases, indicating active infection in 0.8% and biological false positive reactions in 0.2% [Table 1] and [Figure 1]. Seroprevalence for syphilis considering both VDRL and TPHA positivity was highest (4.4%, 37/844) in STD clinic attendees, followed by 1% (13/1299) in the referrals from other OPDs and wards, 0.8% (16/2040) in ICTC attendees, the least in ANC cases (0.3%, 15/5203) and in PHC subjects (0.3%, 3/1103), which represents community population [Table 1].
|Table 1: Results of TPHA in different subject groups with VDRL/RPR titre <1:8 and ≥1:8 |
Click here to view
The TPHA test was performed for 1115 VDRL non-reactive samples (450 males, 669 females). Out of these 48 (4.3%) sera were positive by TPHA (11 males, 37 females). FTA-Abs and TP-EIA was performed on 47 out of 48 TPHA positive sera as quantity of one sample was not sufficient to perform all three procedures. Of the 48 patients, with positive results in the TPHA test, 12 had positive results to both FTA-Abs and TP-EIA and 12 had positive results for TP-EIA, negative for FTA-Abs [Figure 1]. In 23 patients, the TPHA test gave a positive result, whereas the EIA and the FTA-Abs tests gave negative results. Positivity by all three treponemal tests was highest (2.1%, 6/284) in STD clinic attendees followed by 1.9% (2/102) in PHC attendees, 0.6% (4/677) in ANC attendees and nil in patients from different OPDs/wards and ICTC attendees [Table 2].
|Table 2: Results of TPHA, TP-EIA, FTA-Abs positivity in VDRL/RPR non-reactive cases |
Click here to view
| ~ Discussion|| |
The present study has described the performance of three treponemal tests in VDRL/RPR non-reactive sera, within the context of a National Reference Laboratory for STIs that is accustomed to screening large numbers of a different kind of subject groups for the diagnosis of syphilis. Positive TPHA results in VDRL/RPR non-reactive sera were confirmed with TP-EIA or FTA-Abs. A total of 47 specimens were tested by above three tests and 24 were positive (12 by TPHA + FTA-Abs + TP-EIA and 12 by TPHA + TP-EIA). The instances where sera were negative by VDRL/RPR and positive by either two or three treponemal tests may have been the fully treated patients or latent or late syphilis cases as non-treponemal tests also lack sensitivity in late stage infection. Some of these patients might be even cases of primary syphilis as TP-EIA used in this study detected both IgG and IgM. It has been explained earlier also that a number of highly sensitive and specific EIAs can shorten the seronegative window following infection. , United Kingdom guidelines, considering the sensitivity and specificity of EIAs have proposed that either an EIA alone or a combination of VDRL/RPR tests and Treponema pallidum particle agglutination assay (TPPA)/TPHA can be used for syphilis screening.  Moreover, in the US also, treponemal tests have been introduced for screening in blood banks. 
The positivity of FTA-Abs test was found to be less than TPHA and EIA in our study; although, it has been considered the gold standard treponemal test traditionally.  It has been reported in other studies also that the FTA-Abs test has probably lower sensitivity and specificity than other treponemal tests.  In addition to inherent subjectivity, the FTA-Abs test also requires trained personnel and a dedicated fluorescence microscope. For these reasons, CDC recommends that the FTA-Abs test not be used to confirm discordant treponemal screening results. 
In the present study, positivity of treponemal tests in non-treponemal negative cases was observed to be more in STD clinic attendees than other subject groups. The history and clinical records of these STD clinic attendees were suggestive of syphilis infection indicating more sensitivity and specificity of treponemal tests over VDRL/RPR and the findings are consistent with another report. 
In the current study, the traditional diagnostic algorithm performed well, identifying 0.8% persons with active infection in all the subject groups, maximum (4.4%) being in STD clinic attendees. Biological false positive results due to VDRL/RPR were reported only in 0.2%. These findings are comparable with an earlier study from this centre and some other studies. ,
On the basis of the results presented, we conclude that the treponemal tests are more sensitive and specific than the VDRL/RPR in the detection of latent or late syphilis whether the treated or untreated. In India, many diagnostic laboratories and even state reference centres for STIs perform only a single screening assay, VDRL or RPR for syphilis diagnosis, with financial or staff/skill resources cited as the reason for the inability to undertake additional testing. Therefore, the treponemal test such as TPHA should be more widely applied as a confirmatory test in these laboratories. Inconsistent results obtained by VDRL and TPHA should be verified by another treponemal test.
| ~ Acknowledgment|| |
The authors acknowledge National AIDS Control Organisation (NACO), Ministry of Health and Family Welfare, Government of India, New Delhi and Delhi State AIDS Control Society for financial assistance. The authors are thankful to Indian Council of Medical Research (ICMR) for providing SRF fellowship to Vikram Singh. We are thankful to the Medical Superintendent, VMMC and Safdarjung Hospital for permitting us to carry out this study. Technical support of Mrs. Leelamma Peter and Mrs. Renu Mehta is gratefully acknowledged.
| ~ References|| |
|1.||Ray K, Bala M, Gupta SM, Khunger N, Puri P, Muralidhar S, et al. Changing trends in sexually transmitted infections at a Regional STD Centre in north India. Indian J Med Res 2006;124:559-68. |
|2.||Müller I, Brade V, Hagedorn HJ, Straube E, Schörner C, Frosch M, et al. Is serological testing a reliable tool in laboratory diagnosis of syphilis? Meta-analysis of eight external quality control surveys performed by the German infection serology proficiency testing program. J Clin Microbiol 2006;44:1335-41. |
|3.||Centers for Disease Control and Prevention. Sexually Transmitted Diseases Treatment Guidelines, 2010. MMWR Morb Mortal Wkly Rep 2010;59:27-8. Available from: http://www.cdc.gov/std/treatment/2010. [Last accessed on 2012 Nov 16]. |
|4.||Centers for Disease Control and Prevention (CDC). Discordant results from reverse sequence syphilis screening - Five laboratories, United States, 2006-2010. MMWR Morb Mortal Wkly Rep 2011;60:133-7. |
|5.||Karaca Y, Cöplü N, Gözalan A, Oncül O, Akin L, Esen B. Establishment of an algorithm for serological testing of syphilis identification. Mikrobiyol Bul 2010;44:35-45. |
|6.||Gupta SM, Bala M, Muralidhar S, Ray K. Evaluation of test results of microbiology laboratories of North India for standard tests for syphilis under an external quality assurance scheme. Eur J Clin Microbiol Infect Dis 2009;28:461-8. |
|7.||Cole MJ, Perry KR, Parry JV. Comparative evaluation of 15 serological assays for the detection of syphilis infection. Eur J Clin Microbiol Infect Dis 2007;26:705-13. |
|8.||Maple PA, Ratcliffe D, Smit E. Characterization of Treponema pallidum particle agglutination assay-negative sera following screening by treponemal total antibody enzyme immunoassays. Clin Vaccine Immunol 2010;17:1718-22. |
|9.||Health Protection Agency. Serological Diagnosis of Syphilis. National Standard Method, London, United Kingdom. 2007;V44. Available from: http://www.hpa-standardmethods.org.uk. [Last accessed on 2012 Aug 20]. |
|10.||Larson SA, Pope V, Johnson RE, Kennedy EJ. A Manual of Tests for Syphilis. 9 th ed. Washington DC: American Public Health Association; 1998. |
|11.||Aktas G, Young H, Moyes A, Badur S. Evaluation of the fluorescent treponemal antibody absorption test for detection of antibodies (immunoglobulins G and M) to Treponema pallidum in serologic diagnosis of syphilis. Int J STD AIDS 2007;18:255-60. |
|12.||Chronas G, Moyes A, Young H. Syphilis diagnosis: Screening by enzyme immunoassay and variation in fluorescent treponemal antibody absorbed (FTA-ABS) confirmatory test performance. Med Lab Sci 1992;49:50-5. |
|13.||Young H, Henrichsen C, Robertson DH. Treponema pallidum haemagglutination test as a screening procedure for the diagnosis of syphilis. Br J Vener Dis 1974;50:341-6. |
|14.||Bala M, Toor A, Malhotra M, Kakran M, Muralidhar S, Ramesh V. Evaluation of the usefulness of Treponema pallidum hemagglutination test in the diagnosis of syphilis in weak reactive venereal disease research laboratory sera. Indian J Sex Transm Dis 2012;33:102-6. |
|15.||Geusau A, Kittler H, Hein U, Dangl-Erlach E, Stingl G, Tschachler E. Biological false-positive tests comprise a high proportion of venereal disease research laboratory reactions in an analysis of 300,000 sera. Int J STD AIDS 2005;16:722-6. |
[Table 1], [Table 2]
|This article has been cited by|
||Seroprevalence of syphilis in patients attending a tertiary care hospital in Southern India
| ||Sadia Khan,Godfred Antony Menezes,Rahul Dhodapkar,Belgode Narasimha Harish |
| ||Asian Pacific Journal of Tropical Biomedicine. 2014; 4(12): 995 |
|[Pubmed] | [DOI]|