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.

Vaginal fluid pH as a screening test for vaginitis.

OBJECTIVE: To assess how effective the pH test can detect infectious vaginitis. METHODS: Ambulatory gynecological patients attending the gynecological out-patient department of Srinagarind Hospital from May 1 to July 31, 1997 were assessed for vaginitis by history, overall physical examination and vaginal examination. Specimens were collected for microbiological examination and measurement of pH level. RESULTS: Among 422 women recruited, a vaginal fluid pH level greater than 4.5 was found in 149 (35.3%) cases. The vaginal fluid pH as a screening test for infectious vaginitis showed a sensitivity of 49.7% (95% C.I.: 42.6-56.9). When using vaginal fluid pH combined with clinical symptoms and signs to screen for vaginitis the sensitivity was 67.5% (95% C.I.: 60.4-73.9). If a pH test was used to screen for BV, its sensitivity was 73.4% (95% C.I.; 60.7-83.3). Using a pH test in combination with clinical symptoms and signs of vaginitis to screen for BV, its sensitivity was 81.3% (95% C.I.: 69.2-89.5). CONCLUSION: Vaginal fluid pH combined with clinical symptoms and signs had a considerably high sensitivity to screen for BV.

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.

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.

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 study on contamination of water and diarrheal diseases in a rural community in northeast Thailand.

An epidemiological and bacteriological study of diarrheal diseases as well as the relation between domestic-use water and passage of enteric pathogens with stools of the villagers was performed in the northeastern rural area of Thailand on 4 different occasions during the 3 years from 1992-1994. The questionnaire study indicated that 93% of the residents used rain water stored in containers for drinking and other domestic uses, and that 28% of them filtrated and/or boiled the water before drinking it. About 90% of drinking water samples collected from the same residents showed positive responses for the preliminary test ofEscherichia coli contamination. Furthermore, a precise bacteriological survey indicated that more than half of the drinking water samples collected from containers storing rain water were contaminated with various enteropathogenic bacteria, includingE. coli, Vibrio spp. andShigella spp. The new serotype ofV. cholerae O139, synonym 'Bengal cholerae,' which could be isolated from different specimens, is replacing the traditional species ofVibrio. Forty-one per cent of the villagers carried enteropathogenic bacteria in their stools. Most of them were asymptomatic carriers and only a few of them showed clinical features of diarrhea. A few concrete propositions were discussed to supply safe water in these areas where drinkable underground water is not available.

Socioeconomic and health programme effects upon the behavioral management of diarrhoeal disease in northeast Thailand.

This study investigates the effects of socioeconomic and health programme factors on preventive and curative health behaviors and assesses the impact of preventive health behaviors on the incidence of diarrhoea among children under five years of age. Methodological approaches included focus groups to uncover local definitions of diarrhoeal disease, a baseline survey which collected data on maternal preventive health behaviors for 1,364 children, and a monitoring system which collected data on the incidence of diarrhoea and on maternal curative behaviors among the same group of children. Results indicate that socioeconomic status and exposure to health programmes showed significant relationships with selected maternal preventive behaviors. Children whose mothers washed their hands before breastfeeding, gave their child food immediately after cooking and warmed foods each time before meals had significantly lower proportions with diarrhoea than children of mothers who did not practice these behaviors, and 70 percent of the children with diarrhoea were exposed to high risk of severe dehydration and related health complications. Implications of these findings for health programmes are discussed.

Nutritional status and the incidence and duration of diarrhoeal disease among children in northeast Thailand.

In Thailand, diarrhoeal diseases are among major community health problems. The relationship between nutrition status and the incidence and duration of diarrhoea was studied in a sample of 1,117 mothers and 1,364 children under the age of 5 years. The findings are reported and analysed.

Improved ELISA with immunoabsorbent-purified mycobacterial antigen for serodiagnosis of tuberculosis.

An antigen was purified from Mycobacterium tuberculosis H37Ra culture filtrate by immunoabsorbent affinity chromatography with CNBr-activated sepharose 4B column coupled with pooled gamma-globulin fraction from patients with active tuberculosis. The column was washed extensively with PBS and eluted with 3M sodium thiocyanate. Peak fractions were pooled and used as coating antigen in an ELISA. Sera from 86 normal subjects and 54 patients with active tuberculosis were tested against the immunoabsorbed antigen by ELISA with biotin-conjugated anti-human globulin and avidin-peroxidase reagents. At a selected "cut-off" dilution of 320, 49 (91%) of 54 sera from active cases and 8 (9.3%) of 86 sera from normal subjects gave positive test results--a sensitivity and specificity each of 91%, compared with our previous results of sensitivity 75% and specificity 83% with PPD as antigen.

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).

Antibodies against double-stranded RNA in patients with rheumatoid arthritis, osteoarthrosis and Paget's disease of bone.

Using a liquid phase radioimmunoassay to detect antibodies to 3H-labelled double-stranded RNA the premise that rheumatoid arthritis and Paget's disease of the bone may be associated with a chronic virus disease was examined. About 33% of patients with rheumatoid arthritis had antibody levels above the normal range and 11% had antibody levels below the normal range of controls (blood bank donors). The low binding activities were attributed to the action of a nuclease that degraded the dsRNA. Some patients with Paget's disease of bone had higher binding activities than the normal range and similar binding activity was also found in patients with osteoarthrosis. The increase in antibodies to double-stranded RNA did not correlate with increasing age.