Direct identification of Mycobacterium tuberculosis from sputum on Ziehl-Neelsen acid fast stained slides by use of silica-based filter combined with polymerase chain reaction assay.

This paper describes a method for isolation of deoxyribonucleic acid (DNA) from Ziehl-Neelsen stained sputum smears on glass slides; and isolated DNA was used for the IS6110 polymerase chain reaction (PCR)-based identification of M. tuberculosis. A total of 221 samples from newly diagnosed suspected tuberculosis cases were first examined by microscopic examination. For DNA extraction by silica-based filter, a home-made modified spin column gave the efficacy as did the nucleospin tissue reagent kit and therefore was selected for PCR template preparation. The extracted DNA was amplified by the IS6110 PCR using a primer pair that amplifies a 377-bp target, and the product was analyzed by agarose gel electrophoresis with confirmation by Southern blot hybridization. In comparison with culture, PCR with template prepared by the silica based filter showed overall sensitivity and specificity of 91.7 and 100 per cent, respectively. This study used the over one year and less than one year slides samples to study the effect of storage time. In the more than one year storage group, PCR assay gave a sensitivity and specificity of 83.3 and 100 per cent, respectively. In conclusion, the applicability of the PCR directly to DNA extracted from Ziehl-Neelsen stained smears could become a valuable alternative approach for rapid identification of M. tuberculosis, and could be used to evaluate quality of the control of local laboratories in tuberculosis (TB) screening and solve the problem of specimen transportation. In addition, the method could be used in retrospective studies involving a wide range of PCR-based analyses, such as detection of rifampicin resistant gene in multidrug-resistant tuberculosis (MDR-TB) study.

Drug-resistant tuberculosis among prisoners of three prisons in Bangkok and the vicinity.

The purpose of this study was to determine the prevalence of drug-resistant tuberculosis and some factors associated with drug resistance among prisoners of three prisons in Bangkok and the vicinity. Susceptibility testing to four first-line antituberculous drugs was performed on 165 M. tuberculosis strains isolated from prisoners of three prisons including Klongprem Central (KC) prison, Bangkwang Central (BC) prison and the Correctional Institution (CI) for Male Drug Addicts. Of 165 smear positive tuberculosis (TB) cases with drugs susceptibility results, resistance to one or more drugs was 49.7 per cent. Resistance to one, two, three, and four drugs was 20.0, 13.3, 4.2 and 12.1 per cent, respectively. Multidrug resistant tuberculosis (MDR-TB) was 18.8 per cent. Patients classified as primary and acquired drug resistant were 6.7 and 50.0 per cent. The primary drug resistance to one or more drugs among prisoners at KC, BC and CI were 42.5, 36.4 and 53.9 per cent, respectively and MDR-TB were 8.2, 3.0, and 7.7 per cent, respectively. Of several factors analyzed in the present study, only a history of previous TB treatment was significantly associated with drug resistance (p < 0.05). In conclusion, the results indicate the high prevalence of drug-resistant tuberculosis and the seriousness of the TB problem in prisons. The public health sector and prison authorities should work in close collaboration and co-ordination to continue improving TB case detection. Directly Observed Treatment Short course (DOTS) is highly recommended. Moreover, discharged prisoners with tuberculosis should be appropriately referred to hospitals or TB control centers.

Comparison of microplate hybridization with gel electrophoresis and dot blot hybridization for the rapid detection of Mycobacterium tuberculosis PCR products.

A microplate ELISA hybridization assay has been developed for the detection of the IS6110 PCR products of M. tuberculosis from sputum specimens. In this study, its efficacy was evaluated by comparison with agarose gel electrophoresis (AGE) and dot blot hybridization (DBH), with culture results as the 'gold standard'. The assay was used with 190 sputum samples: the PCR results detected by ELISA and AGE showed close agreement, with sensitivity, specificity and accuracy of 90%, 100% and 96% respectively. The same values for DBH were 92%, 98% and 96% respectively. The validities of these methods were not statistically significantly different (p>0.05). The agreement rates of PCR product detection by AGE comparing with DBH and ELISA were 0.964 and 0.964 respectively, while that of DBH and ELISA was 1.0 by Kappa analysis. The overall agreement was not statistically significantly different (p>0.05). Use of DBH or ELISA hybridization increased the sensitivity of detection by AGE 10-fold from 10 pg to 1 pg of purified DNA per reaction; ie from about 30 to about 3 organisms. The amount of PCR product detected by ELISA was only one half of that detected by the other methods; the total assay time of ELISA following the PCR was 4 hours. In conclusion, the microplate hybridization assay may replace AGE and DBH for the detection of the PCR products of M. tuberculosis because of its sensitivity, specificity and accuracy. Additional advantages of the microplate assay over AGE and DBH include rapidity, ease of use, greater safety, cost effectiveness and greater objectivity in the reading of results; the technique is suitable for use in epidemiological studies for the analysis of a large number of samples.