Nested reverse transcriptase-polymerase chain reactions targeting the messenger RNA of icl[2], hspx, and rRNAP1 genes to detect viable Mycobacterium tuberculosis directly from clinical specimens

There is an urgent need for a rapid and reliable test to detect actively multiplying Mycobacterium tuberculosis directly from clinical specimens for an early initiation of the appropriate antituberculous treatment. This study was aimed at the optimization and application of nested reverse transcriptase-PCR [nRT-PCR] targeting the messenger RNA of the icl[2], hspx, and rRNAP1 genes directly from sputum specimens, and their evaluation against the culture by the BACTEC MicroMGIT mycobacterial culture system. 203 Sputum samples from clinically suspected tuberculosis patients and 30 control specimens [clinically proven viral or bacterial infections other than tuberculosis] were included in this study. The mycobacterial culture was performed by the BACTEC MicroMGIT system following the manufacturer's instructions. The primers for nRT-PCRs targeting icl[2], hspx, and rRNAP1 genes were indigenously designed using the Primer-BLAST software, and optimized for sensitivity and specificity. The icl[2], hspx, and rRNAP1 genes were able to pick up 63.9%, 67.2%, and 58.75%, respectively, of culture-negative sputum specimens collected from clinically suspected tuberculosis patients. However, three [1.4%] were negative for nRT-PCR, but M. tuberculosis culture positive. All the 30 controls were negative for culture by the BACTEC MicroMGIT method and all three nRT-PCR. The novel nRT-PCRs targeting icl[2], hspx, and rRNAP1 genes developed in this study are rapid and reliable diagnostic tools to detect viable M. tuberculosis directly from sputum specimens. However, further study by including a larger number of sputum specimens needs to be carried out to ascertain the diagnostic utility of the novel nRT-PCRs optimized in the study

Application of real time polymerase chain reaction targeting kex 1 gene & its comparison with the conventional methods for rapid detection of Pneumocystis jirovecii in clinical specimens.

Background & objectives: As there are no standard laboratory techniques for the rapid detection of Pneumocystis jirovecii in India, this study was undertaken to evaluate and establish an optimal and rapid technique for the detection of P. jirovecii by comparing three different techniques - staining technique, application of a real time polymerase chain reaction (RT-PCR) targeting kex 1 gene and application of nested PCR targeting mitochondrial large subunit (mtLSU) gene for rapid detection of P. jirovecii in HIV positive patients. Methods: One hundred and fifty sputum specimens from HIV positive (n = 75) and HIV negative (n = 75) patients were subjected to three different techniques -KOH/Calcoflour and Grocott methanamine silver staining (GMS), RT-PCR targeting kex1 gene, PCR targeting mtLSU region followed by DNA sequencing and BLAST analysis. Results: Among the 75 HIV positive patients, P. jirovecii was detected in 19 (25.33%) patients by the staining techniques, and in 23 (30.65%) patients each by PCR targeting mtLSU region and by RT- PCR targeting kex1 gene of P. jirovecii. PCR based DNA sequencing targeting mtLSU region revealed 97-100 per cent sequence homology with P. jirovecii sequences in GenBank. Interpretation & conclusions: Of the three techniques for detection of P. jirovecii evaluated in this study, false negativity was found to be more in staining technique and it also required high technical expertise to interpret the result. Both nested PCR and RT-PCR were reliable and equally sensitive, in rapid detection of P. jirovecii, but RT-PCR technique also generated the copy numbers for knowing the severity of infection.