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 ~  Abstract
 ~  Materials and me...
 ~  Results
 ~  Discussion
 ~  Acknowledgements
 ~  References

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ORIGINAL ARTICLE
Year : 2003  |  Volume : 21  |  Issue : 3  |  Page : 179-183
 

Serodiagnosis of syphilis in a community: An evaluatory study


Department of Microbiology, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Chennai - 600 113, India

Correspondence Address:
Department of Microbiology, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Chennai - 600 113, India

 ~ Abstract 

PURPOSE: To analyse the prevalence of syphilis in the apparently healthy population and to provide data for implementation of the joint STD/HIV control programme, a population based study was undertaken by using 'probability proportional to size' cluster survey method in three randomly chosen districts of Tamil Nadu, India namely Dindigul, Ramnad and Tanjore. METHODS: Blood samples were collected from adults (n=1873) aged 15-45 years, from the selected households enrolled in this study. The sera were tested parallelly by rapid plasma reagin (RPR) and Treponema pallidum haemagglutination (TPHA) tests. Reactive samples by RPR and/or TPHA were later analysed by fluorescent treponemal antibody absorption (FTA-ABS) test. RESULTS: The prevalence of syphilis in the community of Tamil Nadu as per RPR positivity was 2.7% (50/1873) as against 0.7% by TPHA (13/1873). FTA-ABS positivity was observed in only 12 out of 48 (25%) RPR/TPHA reactive samples tested. By taking the positivity by two of the three tests, the community prevalence of acute ongoing syphilis in Tamil Nadu was determined as 1.1% (20/1873). CONCLUSIONS: The results confirmed that no single serological test for syphilis can act as the marker of ongoing acute infection in an apparently healthy population. The study suggests that for specific diagnosis of ongoing syphilis, the FTA-ABS test may be performed along with RPR and TPHA.

How to cite this article:
Rajendran P, Thyagarajan S P, Pramod N P, Joyee A G, Murugavel K G, Balakrishnan P, Hari R, Jeyaseelan L, Kurien T, STD study group. Serodiagnosis of syphilis in a community: An evaluatory study. Indian J Med Microbiol 2003;21:179-83


How to cite this URL:
Rajendran P, Thyagarajan S P, Pramod N P, Joyee A G, Murugavel K G, Balakrishnan P, Hari R, Jeyaseelan L, Kurien T, STD study group. Serodiagnosis of syphilis in a community: An evaluatory study. Indian J Med Microbiol [serial online] 2003 [cited 2019 Sep 23];21:179-83. Available from: http://www.ijmm.org/text.asp?2003/21/3/179/8011


Emerging epidemic of aquired immuodeficiency syndrome / human immunodeficiency virus (AIDS/HIV) diseases in India has made sexually transmitted disease (STD) control as one of the strategies imperative and probably the most important one to decrease HIV transmission in the community. Baseline information on the prevalence of classical STDs in India are inadequate.[1],[2] Among various STDs, prevalence pattern of syphilis in the community is pivotal and has been conventionally assessed by routine serological method in both symptomatic and asymptomatic populations. The basic question, often raised from seroepidemiological angle, is whether any one of the standard serological tests for syphilis can become acceptable in terms of sensitivity and specificity at all stages of infection in the study population. The present study was conducted to assess the community prevalence of syphilis in Tamil Nadu and to answer questions related to the choice of the diagnostic test for acute ongoing syphilis in the community and the relative merits of the confirmatory treponemal antibody tests.

 ~ Materials and methods Top

Community prevalence of syphilis in the apparently healthy population of Tamil Nadu was carried out as a part of the AIDS Prevention and Control (APAC) project, “Community prevalence of STDs in Tamil Nadu”, a collaborative study with participation of Department of Microbiology, Dr.ALM PGIBMS, Chennai; Clinical Epidemiology Unit, Christian Medical College and Hospital (CMCH), Vellore and Meenakshi Mission Hospital & Research Center, Madurai, with financial support from US-AID.
A representative population from the state of Tamil Nadu based on 1991 census data was chosen using the 'probability proportional to size' (PPS) cluster survey technique.[3] From the randomly selected three districts, Dindigul, Ramnad and Tanjore, 30 clusters from each district were selected using the PPS method. The clusters were villages and wards from the rural and urban areas respectively based on the census data. Adults, both men and women aged 15-45 years in the selected households from each of the clusters were enrolled. Totally 90 medical camps (1 camp/cluster) were conducted in the selected districts to enroll the participants and to collect the specimens. Ethical clearance for the study was obtained from the Institutional Review Board of CMCH, Vellore. The details of enrolment, data and specimen collection procedures were as described previously.[4]
Serum and plasma samples were parallelly obtained from the blood samples collected from 1873 study subjects (Dindigul= 636; Ramnad= 657 and Tanjore = 580). All plasma samples were screened by rapid plasma reagin (RPR) test and all sera were subjected for Treponema pallidum haemagglutination (TPHA) test. RPR test was performed using Zydes- Pathline kit (Cadila Healthcare Ltd, Ahmedabad). TPHA was conducted using Wellcosyph-HA kit from Murex diagnostics, Germany. The tests were conducted as per the manufacturer's instructions. All reactive samples were repeated for confirmation and subjected for quantitative titration. Forty-eight out of 57 samples which were positive by either RPR or TPHA or both were later analysed by fluorescent treponemal antibody absorption test (FTA-ABS). FTA-ABS test could not be performed on the remaining nine samples because of insufficient sera for this repeat study. The FTA-ABS test was performed with antigen coated slides obtained from Sanofi Pasteur using the FITC conjugate from Sero-Tech, UK. Briefly, the sera were first incubated at 56°C to eliminate complement. To 200 µL of the test serum sample, 50 µL of Reiter's absorbate (1:5 dilution) was added, mixed and allowed to absorb for 5 min. 20 µL of each absorbed test serum was added on to the antigen coated immunofluorescent slides (Syphilam; Sanofi Pasteur, France) and incubated in a humid chamber for 30 min at 37°C. Positive and negative controls were included in every run of the test. The slides were washed with phosphate buffered saline and were blot dried using filter paper. To each well, 20 µL of 1:100 diluted fluorescent conjugate (Serotech, UK) was added and again incubated for 30 min at 37°C. The unbound conjugate was washed away using PBS and the slides were air dried. The slides were examined under a fluorescent microscope (Optiphot-2; Nikon Corp, Tokyo, Japan). All reactive samples were repeated for confirmation and subjected for quantitative titration.
Data analysis was performed by using statistical softwares SPSS version 8.0. for windows and SUDAAN.[5] Adjustments were made for three levels of cluster effect (district, cluster and households). The 95% CIs for prevalences were estimated incorporating the cluster adjustment.

 ~ Results Top

The study subjects included 799 males and 1074 females. The overall positivity by RPR was 2.7% (50/1873) while TPHA positivity was 0.7%. There were 57 (3%) study subjects who were positive either by RPR or TPHA or both. The quantitative titer of positivity ranged from 2 to 128. The break up of overall positivity by RPR/TPHA in relation to gender is shown in [Table - 1].
The analysis of genitourinary symptoms in males and females vs. syphilis positivity by RPR and / or TPHA is presented in [Table - 2] and [Table - 3]. It could be seen that 20/27 (74.1%) males who were positive for syphilis serology did not exhibit any symptoms, while only 7/30 (23.3%) females did not have genitourinary symptoms. The common symptoms expressed among the seropositives were discharge, dysuria and itching. Interestingly, while the 12 FTA-ABS positive cases were similarly analysed, 3/4 males did not report any symptom and all the eight females had genitourinary symptoms.
The analysis of the seropositivity pattern by FTA-ABS in RPR and/or TPHA positive samples is presented in [Table - 4].
The quantitative titration has shown that FTA-ABS positivity ranged from 5 to 20 titer. It could be seen that all the three tests were reactive in only 3/48 samples (6.3%) and RPR only was positive in 58.3% (28/48) cases. The analysis revealed that a) no serological test for syphilis can act as a marker of ongoing disease b) RPR can act as a screening test only detecting past exposure as well besides non-specific positivity c) FTA-ABS emerged as a test of equal sensitivity with TPHA, each detecting 12/48 cases; However, in terms of specific diagnosis of ongoing syphilis infection, FTA-ABS missed 8/12 TPHA positive cases and TPHA missed 8/12 FTA-ABS positive cases. Therefore, positivity by any two of the serological tests were taken to determine the community prevalence of acute ongoing syphilis in Tamil Nadu and was observed as 1.1% (20/1873).

 ~ Discussion Top

Diagnosis of syphilis in clinical settings is usually facilitated conjointly by symptoms and serological tests of the non-treponemal antibody type, like VDRL and RPR tests. Atypical presentations of clinical cases necessitate almost always confirmatory treponemal antibody tests like TPHA. Reports from several countries world wide like Saudi Arabia,[6] Copenhagn,[7] Sweden[8] and India[9] confirm such practice as routine in hospitals receiving STD patients.[10] The situation is much more complicated while documenting the prevalence rate of syphilis in a community to draw baseline for instituting preventive measures. The reasons are: (i) population studies on apparently healthy adults are limited (ii) the non-treponemal antibody tests like RPR and VDRL which detect only the reaginic antibodies do not conclusively prove the active stage of the disease and the actual need for anti-syphilitic treatment in the absence of suggestive clinical symptoms. (iii) the occurrence of biological false positivity due to physiological conditions and certain infections.[11] (iv) biological false negative results in late and latent syphilis.[11] (v) Even, TPHA as a treponemal antibody test does not satisfy the requirements since it is insufficient for excluding syphilis in the elderly because of immunological impairment seen in aged persons[12] and it lacks the sensitivity in sera from patients with primary syphilis.[13]
The observations made in the present PPS survey in Tamil Nadu are to be analysed under the backdrop of the above concepts. The RPR positivity rate of 2.7% and TPHA positivity of 0.7% in the community as observed by us is comparable to that of Thaker et al,[9] from Nagpur, India. However, comparison of these tests does not yield reliable interpretation for confirming the diagnosis of ongoing syphilitic disease. Similar observations were earlier made by Larsen et al,[13] and Garner et al.[14] A recent recommendation by the public health laboratory services (PHLS) syphilis serology working group[15] has suggested that VDRL/ RPR/ TPHA positive sera are to be confirmed by FTA-ABS test. Accordingly FTA-ABS test was performed on RPR/ TPHA positive samples. Surprisingly, it was observed that FTA-ABS was negative in 8/12 TPHA positive cases. Similar observation has been made by Wong et al,[16] from Singpore reporting 25.4% FTA-ABS negativity in TPHA positive cases. Therefore, positivity by any two tests of the three tests adopted seems to confirm the diagnosis of acute syphilis in the community where typical symptoms are seldom observed.
The analysis of atypical symptoms vis-ŕ-vis serological positivity for syphilis, in the present study, projects that a) atypical syphilis is prevalent in the community of Tamil Nadu b) males in the community seem to be suffering more often asymptomatically from syphilis due to inadequate treatment while females seem to be lacking the treatment seeking attitude and c) there is an urgent need in the community to effectively implement partner notification and effective treatment of the infected spouse.

 ~ Acknowledgements Top

We gratefully acknowledge the financial support obtained for the project from United States Agency for International Development (USAID) through AIDS Prevention And Control (APAC) project of Voluntary Health Services (VHS), Chennai, India. The help obtained from Dr.(Air Com.) KP Das (Director, Administration, MMHRC) and Dr.VN Rajasekaran (Medical Director, MMHRC) during the survey is gratefully acknowledged. The Clinical Epidemiology Unit at CMC, Vellore expresses gratitude for help and encouragement provided by the International Clinical Epidemiology Network (INCLEN Inc). 

 ~ References Top

1.Kantharaj K. Special Report. Baseline survey on STD in Tamil Nadu, India (WHO project of Tamil Nadu State AIDS Control Society). 1992:1-8.  Back to cited text no. 1    
2.Merten TE, Smith GD, Kanthraj K, Mugritchian D, Radhakrishnan KM. Observations of sexually transmitted disease contributions in India. Public Health 1998;112:123-128.  Back to cited text no. 2    
3.Sullivan KM, Houston R, Gorstein J, Cervinskas J, eds. Monitoring universal salt iodization programs: cluster surveys. PAMM/M/ICCIDD. 1995. ISBN 0-660-16000-5 Sponsored by UNISEF, PAMM, ICCIDD & WHO.  Back to cited text no. 3    
4.Thomas K, Thyagarajan SP, Jeyaseelan L, et al. Community prevalence of sexually transmitted diseases and human immunodeficiency virus infection in Tamil Nadu, India: a probability proportional to size cluster survey. Natl Med J India 2002;15:135-40.  Back to cited text no. 4    
5.Shah BV, Barnwell BG, Bieler GS. SUDAAN software for the statistical analysis of correlated data. Users manual Release 7.5. Research Triangle Institute, Research Triangle Park NC 27709, USA. 1997.  Back to cited text no. 5    
6.Hussain A. serological tests for syphilis in Saudi Arabia. Genitourin Med 1986;62:293-297.  Back to cited text no. 6    
7.Perdersen NS, Drum O, Mouritsen S. Enzyme linked immunosorbent assay for detection of antibodies to the veneral disease research laboratory (VDRL) antigen in syphilis. J Clin Microbiol 1987;25:1711-1716.  Back to cited text no. 7    
8.Lowhagen OIB. Syphilis test procedures and therapeutic strategies. Semin Dermatol 1990;9:152-159.  Back to cited text no. 8    
9.Thaker YS, Chande Cmahullzy AD, Sauji AM. Serprevalence and syphilis by TPHA test. Indian J Pathol Microbiol 1996;39:135-138.  Back to cited text no. 9    
10.Wiis J Sheller JP. Treponemal infection among children in Ramorswa, Botswana, A serological study. Ugeskr Laeger 1995;157:4134-4136  Back to cited text no. 10    
11.Smikle MF, James OB, Prabhaker P. Biological tube positive serological tests for syphilis in Jamaican population. Genitourin Med 1990;66:76-78.  Back to cited text no. 11    
12.Kanda T, Shinohora H, Suzuki T, Murata K. Depressed CD4/CD8 ratio in TPHA negative patients with syphilis. Microbiol Immnol 1992;36:317-320.  Back to cited text no. 12    
13.Larsen SA, Hambie EA, Pettit DE, Perryman MW, Kraus SJ. Specificity, sensitivity and reproducibility among the fluorescent Treponemal pallidum antibodies and hemaagglutination treponemal test for syphilis. J Clin Microbiol 1981;14:441-445.  Back to cited text no. 13    
14.Garner MF, Backhouse JL, Daskalopoulos G, Walsh JL. The Treponema pallidum haemagglutination (TPHA) test in biological false positive and leprosy sera. J Clin Pathol 1973;26:258-160.  Back to cited text no. 14    
15.Egglestone SI, Turner AJL for the PHLS Syphilis Serology Working Group. Serological diagnosis of syphilis. Commun Dis Public Health 2000;3:158-62.  Back to cited text no. 15    
16.Wong SS, Teo DL, Chan RK. Confirmatory serological testing of blood donors positive on TPHA screening in Singapore. Int J STD AIDS 1997;8:760-763.  Back to cited text no. 16    
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