Serological study for sexually transmitted diseases in patients attending STD clinics in Calcutta

Introduction

Sexually transmitted agents are gaining importance due to their link with the transmission of AIDS. The incidence of sexually transmitted diseases is on the increase. This has been attributed to various factors, including cultural, social, behavioural, economic and microbiological components.[1]

The incidence of specific diseases varies markedly and recent advances have greatly facilitated the identification of their causative agents.[2],[3] A significant proportion of infected individuals harbour two or more STD agents.[2] The types of infection and causative agents are influenced by a number of factors like sexual activity, contraceptive use, genital tract instrumentation, child birth and prior infections.[3]

In the present work, serological study has been undertaken to ascertain the prevalence of syphilis, hepatitis B, chlamydia and HIV infection among patients attending different STD clinics in Calcutta.

Materials and Methods

A total of 457 blood samples were collected from different STD clinics of Calcutta for serological study. Five millilitres of venous blood was collected in a sterile container from each patient using disposable syringe. The serum was separated and preserved at - 20°C in sterile plastic container after adding 0.1% sodium azide. (i) VDRL. Qalitative and quantitative VDRL tests were carried out on all sera using VDRL antigen prepared by the Institute of Serology, Calcutta. For qualitative test, 0.5 ml. inactivated serum was pipetted in one well of a cavity slide and one drop (1/60 ml.) of antigen emulsion was added to it. The slide was rotated for 4 minutes and observed for presence of clumps, under a microscope with low power objective and 100 x magnification. Sera showing no clump were reported as non reactive. The presence of slight roughness was ignored. Samples showing small clumps were considered weakly reactive. Those with medium sized or large clumps were reported as reactive. Quantitative test was performed on all reactive sera including those showing weak or rough reaction. The results were reported in terms of the highest dilution which gave a frank positive reaction. Both reactive and weakly reactive sera were labelled as positive.

(ii) Treponema pallidum haemagglutination assay (TPHA). The THPA test was carried out by using THPA 200 kits manufactured by Newmarket Laboratories Ltd., United Kingdom, according to manufacturer′s instructions. The test kit is prepared using avian erythrocytes coated with antigen of T pallidum (Nicol′s strain). In the presence of antibodies to T pallidum, the causative agent of syphilis, the sensitized red blood cells were haemagglutinated. The cells were suspended in diluent containing components to eliminate non specific reaction. The serum was reported as positive if it showed haemagglutination in the form of full cell pattern covering the bottom of the well and control cells showed negative button. Sera showing borderline reaction (+) were not considered positive.

(iii) Hepatitis B surface antigen (HBs Ag ): Presence of hepatitis B surface antigen (HBs Ag) was tested by ELISA, using pathozyme HBs Ag kit maufactured by Omega Diagnostics Ltd., U.K., according to manufacturers instructions. Monoclonal antibodies, specific for the eight known HBs Ag subtypes, recognized by the W.H.O., are bound to the surface of the microtitration wells. Undiluted test sera and monoclonal anti HBs Ag antibodies conjugated to Horseradish Peroxidase (HPR) is applied. If HBsAg is present in the sample it binds to the captured viral antigens. If HBs Ag is not present, binding does not take place and unbound material is washed away. On addition of the substrate, stabilised 3,3′, 5, 5′ tetramethyl benzidine (TMB), a colour will develop only in those well in which the HRP, is present indicating the presene of HBs Ag. The reaction is stopped by the addition of sulphuric acid and the absorbance is then measured at 450 nm. Any result with an optical density (OD) greater than the cut off value should be considered positive.

(iv) Chlamydia trachomatis IgG antibodies: They were identified by ELISA test using chlamydia IgG ELISA kits obtained from Diagnostic Automation, Inc. Calabasas, CA, USA, according to manufacturers instructions. When antigens bound to solid phase are brought into contact with a patient′s serum, antigen specific antibody, if present, will bind to the antigen on the solid phase forming antigen-antibody complexes. Excess antibody is removed by washing. This is followed by the addition of goat anti-human IgG globulin conjugated with horseradish peroxidase which then binds to the antibody - antigen complexes. The excess conjugate is removed by washing, followed by the addition of cromogen/substrate tetramethyl benzidine (TMB). If specific anti body to the antigen is present in the patient′s serum, a blue colour develops. When the enzymatic reaction is stopped with IN H2SO4, the contents of the wells turn yellow. The plate is read on microwell plate reader.

(v) HIV antibodies: Serodiagnosis of HIV was done in the department of Virology, School of Tropical Medicine, Calcutta. All the sera tested by ELISA method using kits from INNOTEST HIV - 1 / HIV -2 s.p., INNOGENTICS N.V., Belgium. The ELISA reactve sera were confirmed by western blot test using kits from INNOLIA HIV -1 HIV -2 Ab. INNOGENETICS N.V Belgium. The tests were done according to manufacturer′s instructions.

Results

[Table - 1] shows the positivity of different serological tests among patients attending STD clinics. Positivity was highest with TPHA (18.60°%, followed by chlamydia (15.97%), VDRL (8.98%), HIV (6.35%) and HBsAg (3.72%). [Table - 2] shows the positivity for different serological tests among different age groups. Total 37.20% samples were positive. Maximum infection was seen in 15-30 years age group (20.13%), followed by 30-45 years age group (12.69%) and > 45 years age group (4.38%). Single test, two tests and four tests positivity was highest in 15-30 years age group but three tests positivity was highest in 30-45 years age group. [Table - 3] shows positivity for different number of tests. Positivity was maximum in single test (65.29%) followed by two tests (25.88%), three test (8.24%) and four tests (0.59%). [Table - 4] shows that amongst the single test positive, maximum was with chlamydia (36.94%), followed by TPHA (30.63%) HIV (18.91 %),VDRL (7.21%) and HBs Ag (6.31%). [Table - 5] shows two test posi-tivity.VDRL + TPHA positivity was the highest (45.45%). Other combinations were TPHA+ chlamydia (29.54%), chlamydia + HIV (9.09%), HBsAg + Chlamydia (4.55%), TPHA + HIV (4.55%), HBsAg +HIV (4.55%) and TPHA + HBs Ag (2.27%). [Table - 6] shows the positivity in three tests. VDRL+ TPHA + Chlamydia positivity was maximum (71.43%). Other combinations were TPHA + chalmydia + HBsAg (14.28%) and VDRL + TPHA

+HBsAg (14.28%). Only one sample was positive in 4 tests ie VDRL + TPHA +HBsAg+ chlamydia and it was 15-30 years age group. Out of 457 samples tested , 422 were males and 35 females. Positivity was also more in males (31.73%) than in females (5.47%).

Discussion

A number of factors have been suggested to have contributed to the wide dissemination of STD and among them social factors are found to have a particular significance.[4]

Though the number of such studies in developing countries is small, interesting trends and tendencies, as well as comparisons, can be pointed out. Total 37.20% samples were positive for STD infections, out of which 24.29% were positive in single test, 9.63% were positive in two tests, 3.06% in three tests and 0.22% in four tests. In 1999 Thawani et al[5] isolated STD causative agents among sex workers in Calcutta. They got positivity of 59.07%, out of which 38.82% had single infection, 17.30% double infection and 2.95% showed multiple infections. These figures are higher than our study but like their study the number of single infections are highest, followed by double and multiple infections. In the present study, maximum infection (20.13%) was seen in 15-30 years age group. Similarly Thawani et al[6] in 1997 got highest infection in the age group of 25-29 years. The level of seroprevalence of syphilis reveals community exposure to treponemal infection. This confirms the character of such disease in developing countries. In 1985 in Nairobi, D′ costa et al[7] had found seroprevalence of 41.4% while a similar group[8] studied in Ivory Coast had a rate of 47%. Thawani et al[9] got VDRL positivity 57.47% and 63.16% Geyid et al reported presence of syphilis infection by TPHA test in 37.3% of samples. The present study showed TPHA positivity in 18.60% patients which is lower than the above study. This may be due to the fact that all the other studies have collected samples from sex workers but in the present study samples were from patients attending STD clinics which may or may not be sex workers. Like other studies[5],[6],[10],[12] in the present study TPHA positivity is higher than the VDRL test. This may be due to the fact that the TPHA test is much sensitive than VDRL test and these cases may be having past infection which could be missed in VDRL test. Thawani and Jana[5] got chlamydia infection in 36.08% in Calcutta. In another study[10] IgG anti chlamydial antibodies were detected in 95.7%.In the present study, chlamydial antibody was found in 15.97% patients which is lower than the above two studies. The prevalence of HBs Ag varies widely from 0.5% in develolped to 40% in some developing countries". In the present study HBs Ag was 3.72%. Other studies[5],[10],[12] showed HBs Ag positivity in 6.53%,14.9% and 6.03% cases respectively. Thawani et al[10] showed HIV positivity in 4.6% cases but the present study had HIV in 6.35% cases which is higher than the above study.

Acknowledgement

Authors thankfully acknowledge the technical assistance from Mr. R.C. Mondal, Mr. J.C. Ghosh, Mr. R. Naskar and Mr. Deepak Pal.