Detection of serum antitrichomonal antibodies in urogenital trichomoniasis by immunofluorescence.R Bhatt, D Pandit, L Deodhar
Dept of Microbiology, LTMM College, Sion, Bombay.
Trichomonas vaginalis is a frequently encountered genital pathogen in both males and females. In females, vaginitis due to this parasite is one of the most common manifestation. The indirect fluorescent technique (IFA) test was carried out to detect antitrichomonal antibodies in 370 female patients using whole cell antigen. Seventy one (19.18%) gave positive reaction for either of the class IgG, IgM and IgA antibodies. The level of the IgG class antibodies was found to be higher i.e. 58 (81.69%) than IgM 11 (15.27%) antibodies, which may be suggestive of past infection or a prolonged manifestation by the organisms.
Keywords: Antibodies, Anti-Idiotypic, blood,Evaluation Studies, Female, Fluorescent Antibody Technique, standards,Hospitals, University, Human, Immunoglobulin A, immunology,Immunoglobulin G, immunology,Immunoglobulin M, immunology,India, epidemiology,Mass Screening, Outpatient Clinics, Hospital, Sensitivity and Specificity, Trichomonas Vaginitis, blood,epidemiology,immunology,
Trichomonas vaginalis, a flagellated, frequently encountered genital pathogen is responsible for trichomoniasis in both males and females. In women, the characteristic of trichomonial vaginitis, is a thin to a green frothy discharge.
Besides widely used microscopy and cultural technique to detect T Vaginalis, detection of antibodies to the parasite may help in diagnosis and detection of the asymptomatic carrier or a person with a past history of a trichomonas infection. Immunologic investigation of T vaginalis did not come into the picture until the mid 1940's, when the use of antibiotics allowed the establishment of axenic cultures of this species.
The present study was carried out to detect the antitrichomonal antibody level in serum, from female patients by an indirect fluorescent antibody (IFA) technique.
Three hundred and seventy female patients attending the gynecological outpatients department, with complaints of vaginal discharge, burning micturition and dysuria, were screened for the presence of Tvaginalis and circulating antibodies in their sera. T vaginalis was isolated and maintained in All Culture (AC) medium supplemented with 10% inactivated horse serum, penicillin G (1000 ug/ml) and amphotericin B (250 ug/ml). The cultures were maintained axenically by subculturing every 48 - 72 hrs.
Preparation of Antigen:
T vaginalis cells in an exponential growth phase (24 hrs culture) were washed twice with 0.01 M phosphate buffer solution (PBS) (pH 7.2). The pellet was suspended in 10% formalin in PBS for 10 min. The cells were washed twice with PBS and resuspended in PBS to get 1 x l06 organisms per ml and stored at-20?C.
Pooled sera from patients from whom T vaginalis was isolated represented positive control sera, whereas 100 sera obtained from children below 8 yrs age group represented negative control sera.
Standard indirect fluorescent antibody (IFA) technique was used. A drop of formalin fixed antigen was allowed to air dry in 0.5mm diameter wells on a glass slide. A serial two fold dilution of sera ranging from 1:2 to 1:64 was made in PBS and was layered on antigen coated wells which was allowed to react for 30 min at 37?C followed by three washings, each lasting for 15 min. The slides were air dried and reincubated for 30 min at 37?C with antihuman fluorescein labelled lgG, lgM and lgA (Behring) at 1/16 dilution. Slides were again washed three times as described previously and air dried. Finally, a drop of mounting fluid (9 parts glycerol + 1 part PBS pH 7.2) was layered and the preparation was examined under fluorescent microscope with high pressure HBO 200 mercury lamp.
The positive slides when examined under fluorescent microscope, showed bright apple green fluorescence and the reaction was considered positive when at least 50% of the cells showed bright fluorescence [Figure - 1]. Out of 370 sera screened, antitrichomonal antibodies were detected in 71 cases (19.18%), whereas the parasites were isolated in 51 (13.78%) cases and smear was positive in 45 (12.16%)cases. All the control sera showed negative results [Figure - 2]. Of 71 (19.18%) cases positive by IFA, 58 (81.69%) were positive for IgG antibodies, whereas IgM class antibodies and lgA class antibodies were found in 11 (15.27%) and 2 (2.77%) cases respectively. [Table - 1].
Of 71 IFA positive cases, 50 (70.42%) cases correlated with culture technique, whereas 21 cases failed to show any correlation. these 21 cases were culture negative but IFA positive. Only one case of the 370 was I FA negative but culture positive [Table - 2].
McEntegart et al4 in 1952 identified T vaginalis by the direct immunofluorescence reaction and succeeded in differentiating T vaginalis from T foetus.
Of 370 patients screened, 71 (19.18%) gave positive fluorescence for one of the antibody class, whereas culture was recorded positive in 51 (13.78%) cases only. Correlation of 70.42% was observed between culture and IFA techniques. Of 51 (13.78%) culture positive cases, 50 (98.03%) correlated with IFA technique, whereas only one case showed culture positive but IFA negative results, which is in close accordance with Kramar and Kucera 2, who reported 100% correlation between both the techniques.
Of total 71 positive fluorescence cases, 81.69% of cases were positive for IgG class antibodies whereas IgM and lgA class antibodies were observed in 15.27% and 2.77% of cases respectively. Detection of high titre IgG antibodies ( ? 1:16) among 65% of culture positive cases suggests either active or prolonged infection with T vaginalis, where as failure to detect high titre of IgG antibodies in culture negative cases suggests past infection. Ackers et al5 and Su KE6 have also reported the presence of IgG antibodies in serum because of systemic response to released antigen.
The authors wish to thank Hoechst (Behring), for providing antihuman fluorescein labelled antibodies for the present work.
[Figure - 1], [Figure - 2][Table - 1], [Table - 2]