Rapid diagnosis of tuberculous pleural effusion using polymerase chain reaction.

Between October 1998 and September 1999, 98 patients with symptomatic exudative lymphocytic pleural effusion were enrolled in our study to evaluate the diagnostic sensitivity of polymerase chain reaction (PCR) assay. The mean age was 53.3 years ranging from 18 to 78 years. There were 61 men and 37 women. Pleural fluid was sent for gram staining, AFB staining, aerobic culture, culture of Mycobacterium tuberculosis on LJ media, and cytology. Additional fluid was used for a PCR-assay of the 16 S-23 S rRNA gene spacer sequences and for a nested PCR of the 16 S rRNA gene as a blind control. In cases of free-flow pleural tapping, histopathological analysis was done on three pleural biopsies. Overall etiologies comprised malignancy 53.1%, tuberculosis 36.7%, lymphoma 2.0% and chronic nonspecific inflammation 8.2%. The sensitivity and specificity of AFB-staining were 6% and 79%, respectively; while cultures on LJ media were 17% and 100%, respectively. The sensitivity of the PCR-assay was 50% (95% CI: 40 to 60%) and the specificity was 61% (95 CI: 52 to 71%). When PCR was nested, the sensitivity was 72% (95% CI: 63 to 81%) and specificity was 53% (95% CI: 43 to 63%). Two thirds (26 of 36) of tuberculous pleural effusion cases underwent pleural biopsy, and 62% were diagnosed by histopathology. There were no complications from thoracocentesis or pleural biopsy in any of the patients. We concluded that PCR assay was more sensitive than AFB staining and mycobacteria culture for diagnosis tuberculous pleural effusion but its specificity was quite low.

p53 status and human papillomavirus infection in Thai women with cervical carcinoma.

Loss of p53 function has been implicated in a wide variety of human malignacies. Many studies suggest that in cervical carcinoma p53 function is inactivated either by gene mutation or by complex formation with E6 oncoprotein product of high-risk human papillomavirus (HPV). The aim of this study was to determine the status of HPV infection and p53 gene mutation as well as their correlation in cervical carcinomas. Formalin-fixed paraffin-embedded tissues of 12 cervicitis, 21 cervical intraepithelial neoplasia grade 3 (CIN 3) and 17 squamous cell carcinomas were determined for the presence of HPV using polymerase chain reaction (PCR) amplification and dot blot hybridization. The status of p53 mutations in exons 5-8 was evaluated by polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) and confirmed by direct nucleotide sequencing. HPV infections were detected in all CIN 3 and squamous cell carcinomas (100%). Mutations of p53 were present in 3 of 38 HPV-positive samples: one with an ATG-->TTG transversion (Met-->Leu) in codon 237 of exon 7; and the others with a TGC-->TGG transversion (Cys-->Trp) in codon 242 of exon 7, and a CGT-->CCT transversion (Arg-->Pro) in codon 273 of exon 8, respectively. Our findings show that the frequency of p53 mutation is low in primary cervical carcinoma and that the p53 gene mutation and HPV infection are not mutually exclusive events in the development of cervical cancer. Thus, other genetic events independent of p53 inactivation may also significantly contribute to the carcinogenesis of the uterine cervix.

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.

Use of PCR-PIRA for screening of a point mutation at codon 12 in K-ras oncogene obtained from paraffin embedded tissue sections.

Among various methods which have been developed for facilitating the screening of point mutations in human genomic DNA, PCR-Primer Introduced Restriction Analysis (PCR-PIRA) is of particular interest due to its practicality and short procedure allowing detection of point mutations by simple restriction enzyme digestion directly after PCR amplification. However, one limitation of PCR-PIRA method is the absence of restriction sites in the region of detection, thus creation of the recognition site in primers has been introduced. Detection of a point mutation at codon 12 in K-ras oncogene by BstNI requires one base change in the primer sequence so that only the normal but not mutant PCR product will be digested by the enzyme. However, false positive results generated from undigested normal DNA sequence are always obtained. This effect is compounded when it is used to analyse mixed cell populations in paraffin embedded section of cancer cells. Assay of a mutant band generated from normal DNA by densitometric quantitation enabled the determination of background values and thereby eliminated false positive results. Samples with higher ratios between mutant and normal bands than the background one after the first PCR-PIRA would be subjected to the second PCR-PIRA in order to confirm the results. Screening of such mutations in cervical carcinomas from paraffin embedded sections using the above criteria should reduce misinterpretation of PCR-PIRA results.

Characterization of Pseudomonas pseudomallei antigens by SDS-polyacrylamide gel electrophoresis and western blot.

Immunological characterization of various Pseudomonas pseudomallei preparations was carried out by SDS-PAGE and Western blot using sera from infected humans and from patients with other bacterial infections. Somatic (SOM) and partially purified cell extracts (PCE) gave more complex SDS-PAGE patterns: M(r) ranged from 86 to 12.7 and 48 to 10 kDa, respectively. The culture-filtrated antigens (CF) from 3 different kinds of synthetic media consisted of fairly simple profiles with common bands M(r) of 40, 26 and 16 kDa. PCE and CF reacted specifically with infected human sera; SOM did not. The components with M(r) of 40 kDa in CF reacted consistently with all infected sera but failed to react with sera infected with Escherichia coli, Enterobacter spp., Klebsiella pneumoniae, Proteus mirabilis, Salmonella spp., Staphylococcus aureus, Streptococcus spp., Pseudomonas aeruginosa and P. stutzeri. This peptide was demonstrated to be a major component in CF thus suggesting its potential for development of immunodiagnostic methods for melioidosis.

Prevalence of toxoplasma antibodies in blood donors and pregnant women in Khon Kaen Province.

Serum samples from blood donors and pregnant women in Khon Kaen were examined for antibodies to Toxoplasma by an indirect hemagglutination and indirect fluorescent antibody techniques. It was found that 6.4 per cent of the blood donors were positive by the indirect hemagglutination and 6.2 per cent by indirect fluorescent antibody tests. The seroprevalence in pregnant women were 12.0 per cent by indirect hemagglutination and 4.7 per cent by indirect fluorescent antibody tests. The frequency distribution curves of indirect hemagglutination titers were unimodal in both the groups studied. From the basis of these findings, it was concluded that toxoplasmosis is not endemic in Khon Kaen and the transmission occurs at a very low level.