Development of EIA for detection of Chlamydia trachomatis in genital specimens.

A double antibody sandwich enzyme immunoassay (EIA) for chlamydial antigen detection was developed using a monoclonal antibody against lipopolysaccharide (LPS) of Chlamydia trachomatis as a coating antibody. Polyclonal rabbit antiserum against partially purified antigen from elementary body (EB) antibody and horse-radish peroxidase conjugated goat anti-rabbit antibody were used as the primary and secondary antibody respectively. The developed EIA could detect protein of partially purified EB at the lowest concentration of 250 ng/ml. The assay was evaluated against the cell culture (CC), DNA hybridization assay (PACE2 system: Gen-Probe, San Diego, CA, USA) and a commercial enzyme immunoassay (kEIA) (Bioquest, NSW, Australia). The sensitivity, specificity, positive and negative predictive values of the developed EIA (dEIA) were 87, 96.2, 80, 97.7 for the specimens from females and 90.9, 90.7, 71.4, 97.5 for the specimens from males repectively. Cross reaction was not found with Escherichia coli, Acinetobacter anitratus, beta-Streptococcus group A, Enterobacter spp, Enterococcus, Lactobacillus spp, Neisseria spp, but it was found with Candida albicans and herpes simplex virus type 1. The developed EIA can be applied successfully for both genders, particularly males. The cost per test is less than those for CC, kEIA and PACE2.

An epidemiological survey of female reproductive health status: gynecological complaints and sexually-transmitted diseases.

An epidemiological survey of gynecological and sexually-transmitted diseases was conducted in 4 villages of Narmpong district, Khon Kaen, Thailand. It was focused on the reproductive health status of rural women. A mobile gynecological clinic was set up to collect materials and data including demographic characteristics, physical examination and specimen collection. Vaginal swabs were examined by microscope, Gram staining, pH measurement, KOH test and bacteriological cultivation. Endocervical swabs were examined for Chlamydia trachomatis, herpes simplex virus (HSV) and human papilloma virus (HPV) by polymerase chain reaction. Papanicolaou's test was applied for diagnosis of cytological abnormalities. Blood was tested by RPR and TPHA and urine was tested by LED test. The chief complaint was dysmenorrhea (44.8%). The others ranging from 43.4-3.0% were lower abdominal pain to genital ulcer. Prevalence of C. trachomatis, C. albicans, T. vaginalis, T. pallidum and G. vaginalis were found in 4.6, 10.9, 5.1, 2.7 and 1.0% of 586 women and HSV and HPV were found in 6.4% and 1.4% of 110 women, respectively. The three pathogens. C. trachomatis, C. albicans and T. vaginalis, were frequently found among women in the age of 20-49 years. The number of marriages and sex partners in the past year had an association with C. trachomatis infection while vaginal pH > 4.5, marital status, number of marriages and itching of genitalia had an association with T. vaginalis infection.

The prevalence of Chlamydia trachomatis infection in rural Thai women.

A cross sectional study was designed to investigate the prevalence of Chlamydia trachomatis among different groups of rural women in the northeast Thailand. The presence of chlamydial antigens in endocervical swabs was detected by ELISA. The prevalences of Chlamydia trachomatis were 6.8% (31/485), 5.2% (24/466) and 6.7% (12/179) in women attending antenatal, postpartum and family planning clinics respectively. The average prevalences of C. trachomatis among hospital-based and community-based women were 6.1% (67/1,103) and 3.6% (15/411) respectively. In addition, the prevalences of some pathogens including Candida albicans, Trichomonas vaginalis, Treponema pallidum and Neisseria gonorrhoea among hospital-based and community-based women were 14.2, 2.8, 0.7, 0.2 and 10.9, 5.1, 2.7, 0.0% respectively. It was concluded that C. trachomatis was a problem of woman's reproductive health.

Development and assessment of ELISA for serodiagnosis of HIV-infection.

A home-made ELISA for serodiagnosis of HIV-infection was developed. It made use of the HIV viral lysate to coat on ELISA microplates. The purpose was to establish an ELISA for serodiagnosis of HIV-infection. The newly-developed ELISA, "H-ELISA", was applied to test 792 samples of HIV-positive serum as confirmed by Western blot. All 792 samples were positive by H-ELISA. It was also applied to test 540 samples of normal sera obtained from different laboratories in Srinagarind Hospital. A number 530 normal sera was negative, 8 samples were positive and confirmed by Western blot and 2 samples were false positive. It was concluded that the H-ELISA possessed 100% sensitivity with a false positive rate of 2/532(0.38%). The H-ELISA, the cost/test was less than 5 bahts, appears to be promising for substitution of imported commercial kits.

An assessment and evaluation of methods for diagnosis of chlamydial and gonococcal infections.

Chlamydia trachomatis and Neisseria gonorrhoeae were studied in 350 females and 140 males attending the sexually transmitted disease clinic and AIDS Center, Khon Kaen zone 6 and the Division of Obstetrics and Gynecology, Khon Kaen Hospital. Chlamydia trachomatis infection was diagnosed by cell culture (CC), enzyme immunoassay (EIA) (Bioquest, NSW, Australia) and nucleic acid hybridization (PACE2 system: Gen-Probe, San Diego, Calif). It was found that the sensitivity, specificity and positive and negative predictive values in females were 95.7, 100.0, 100.0, 99.7% by the cell culture; 91.3, 99.1, 87.5, 99.4% by the EIA; and 78.3, 99.7, 94.7, 98.5% by the PACE2 respectively. Values of the same parameters in males were 83.3, 100.0, 100.0, 98.5% by the cell culture; 75.0, 99.2, 90.0, 97.7% by the EIA and 91.7, 100.0, 100.0, 99.2% by PACE2 respectively. The methods for detection of Neisseria gonorrhoeae infection were conventional culture, PACE2 test and the direct examination (Gram's stain). In females, the sensitivity, specificity and positive and negative predictive values of the conventional culture were 85.7, 100.0, 100.0, 99.7% and those of the PACE2 were 85.7, 99.1, 66.7, 99.7% respectively. In males, the values of the same parameters were 81.8, 100.0, 100.0, 100.0% by the conventional culture, 95.5, 100.0, 100.0 and 99.2% by the PACE2. The prevalence of chlamydial infection in females was 6.6% (23/350) and that in males was 8.6% (12/140). The prevalence of gonococcal infection in females was 2.0% (7/350) and in males was 15.7% (22/140). The co-infection of Chlamydia trachomatis and Neisseria gonorrhoeae in females was 0.9% (3/350) and no co-infection was found in males. It is concluded that cell culture is an appropriate method for detection of Chlamydia trachomatis in both genders, particularly in females. PACE 2 test is the best method for such detection in symptomatic males while EIA is a good method in females, particularly in symptomatic females. For gonococcal detection, PACE2 test is a sensitive, specific and alternative method to the conventional culture. It can be appropriately applied for the diagnosis of gonococcal infection, particularly in males.

Anti-HIV antibody titer: an alternative supplementary test for diagnosis of HIV-1 infection.

The diagnosis of HIV infection is based on screening of HIV antibodies and confirmed by a more specific supplementary test. The most common confirmation test is Western blot, which is expensive, time consuming and subject to technical skill. The present study was carried out to evaluate whether the anti-HIV-1 antibody titer is valid as a supplementary test for diagnosis of HIV-1 infection. Anti-HIV-1 antibody titers of 2,414 anti-HIV-1 positive sera determined by the particle agglutination (PA) method were analysed in comparison with the Western blot analysis. The Western blot negative result was found in 11 of 2,414 (0.46%) anti-HIV-1 positive sera, these sera also gave negative anti-HIV by ELISA. The PA titers of these sera were found in the range of 16 to 64. Seventeen samples (0.70%) with anti-HIV-1 in the titer range of 16 to 256 showed indeterminate Western blot analysis. The rest, 2,386 of these 2,414 sera (98.84%), were shown to be positive by Western blot. However, all of the 2,356 sera with antibody titers > or = 512 (97.6%) demonstrated positive Western blot results. Five cases among the 17 (29.4%) indeterminate sera were examples of early seroconversion of HIV infection, which were confirmed in follow up specimens. The results suggest that only the samples with antibody titers < 512 are required to be confirmed for HIV infection by Western blot. It is possible that early seroconversion may be inferred from anti-HIV titers. Therefore, in order to reduce time and cost, the PA anti-HIV titer can be used as an alternative supplementary test for diagnosis of HIV-1 infection in most positive screened anti-HIV samples. Western blot is needed for testing in only a few cases.

Epidemiological assessment of anti-HIV 1 antibodies in Thailand.

Anti-HIV 1 antibodies were detected in 4 groups of subjects (peoples attending hospitals or medical clinics for anti-HIV investigation, blood donors, women in massage parlours and thalassemia patients) in the north, northeast and central Thailand. A total number of 1,726 blood samples were initially tested with ELISA. The ELISA reactive samples were confirmed by the Western blot analysis. Using ELISA as a screening test, the highest incidence (9.09%) of anti-HIV 1 antibodies was found in thalassemic children (4 of 44). Six (0.72%) and 4 (1.02%) samples in the first, second and third groups had a repeatedly reactive ELISA respectively. The Western blot analysis confirmed that 7 cases (3 thalassemia and 4 subjects in the first group) had antibodies to HIV 1. Two cases with reactive Western blot test were Westerners while the rest were symptomatic and asymptomatic Thais. The HIV infection has spread to thalassemia patients probably via blood transfusion.

A rapid detection of herpes simplex virus by ELISA in asymptomatic pregnant women and neonates.

Herpes simplex virus (HSV) was detected by the enzyme-linked immunosorbent assay (ELISA). The assay system employed rabbit anti-HSV-2-coated microplates to detect HSV in clinical specimens and the same reagent labelled with peroxidase as a conjugate. The HSV type 2 obtained from vero cell culture and normal cell lysate (NCL) were used as positive and negative reference antigens respectively. HSV was detected in 40 (9.93%) of vaginal swabs obtained from 403 pregnant women just before the deliveries and in 39 (9.68%) fluid samples collected immediately after birth from the mouths of 403 newborns. HSV was detected in five pairs of mother-newborn under investigation. There was no correlation between the incidence of HSV in mothers and newborns (p greater than 0.05).