Sanjay Singh Negi

Targeted detection of 65 kDa heat shock protein gene in endometrial biopsies for reliable diagnosis of genital tuberculosis.

OBJECTIVE To evaluate the clinical utility of PCR compared with other available diagnostic modalities in prompt diagnosis of female genital tuberculosis causing infertility. STUDY DESIGN Prospective case-controlled trial. Premenstrual endometrial biopsy specimens were collected from 150 infertile women of reproductive age group suspected of having genital tuberculosis. All patients underwent diagnostic endoscopy (laparoscopy and hysteroscopy) and the samples obtained were subjected to microscopy, culture by the BACTEC 460 TB System, histopathology and polymerase chain reaction (PCR) for detection of 165 bp region of 65 kDa gene of Mycobacterium tuberculosis. The results were correlated with the laparoscopic findings. RESULTS While the laparoscopy/hysteroscopy findings were indicative of tuberculosis in 12.6% of cases, 14.6% of the specimens showed evidence of 65 kDa gene of M. tuberculosis and only 3.33%, 1.33% and 0.66% were positive by culture, smear and histopathology, respectively. CONCLUSION Since laparoscopy, hysteroscopy other endoscopic procedures are associated with operative risks and may cause flaring of infection, and other conventional laboratory tests including histopathology have poor sensitivity, PCR-based detection of 65 kDa gene of M. tuberculosis in endometrial biopsy specimens could be a promising molecular diagnostic technique compared to conventional methods of diagnosis.

Correlation of partial env gene sequences with disease progression parameters in HIV-positive pregnant women from India

Ever since the beginning of the epidemic of HIV, one of the poignant aspects of HIV infection is transmission of the virus from mother to child. It is not known whether pregnancy accelerates the progression of HIV infection from a clinically asymptomatic stage to a progressive clinical phase. Present study was carried out to understand disease progression in pregnant women from India. We studied co-receptor utilization (the major determinant of HIV disease progression), N-glycosylation sites, and sequence variability. Blood samples were collected from 25 HIV sero-positive patients, eleven from the antenatal risk group (experimental group), nine from heterosexual male, and five from heterosexual female risk group (control group). Partial env gene was amplified by PCR and sequenced. BLAST search and phylogenetic analysis were used to determine the subtype. The deduced amino acid sequence of the V3 region was used to predict co-receptor, determine sequence variability and N-glycosylation site. The experimental group comprising the antenatal risk group did not exhibit any difference in terms of co-receptor, N-glycosylation, and sequence variability when compared with the control, non-pregnant group. Pregnancy does not seem to accelerate the clinical course of HIV infection. The female body during the gestation phase possibly acquires certain strategies to impede or at least alleviate the disease progression during the crucial immune-compromised pregnancy phase, which would otherwise adversely affect the mother as well as the fetus during the infection.

Characterization of rpo B Gene for Detection of Rifampicin Drug Resistance by SSCP and Sequence Analysis

Purpose: Because of the emergence of multidrug-resistant tuberculosis in recent times, the rapid detection of resistance to the first-line anti-tuberculosis drug rifampicin was felt worldwide. Accordingly, this study was conducted to evaluate the diagnostic potential of polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) for checking its utility as a rapid screening test for determination of rifampicin drug resistance. Materials and Methods: A total of 34 isolates of Mycobacterium tuberculosis ( M. tuberculosis ) (22 rifampicin resistant, 11 rifampicin sensitive and one control H37Rv) strains were analysed by PCR-SSCP and DNA sequencing within the 157-bp region of the rpo B gene (Ala 500 -Val 550 ). Results: Rifampicin resistance was detected successfully by PCR-SSCP in 20/22(90.90%) of rifampicin-resistant strains showing a total of nine different mutations in seven codon positions: codon 513 (CAA→CCA), 516 (GAC→GTC), 507 (GGC→GAC), 526 (CAC→GAC, TAC), 531 (TCG→TTG, TGG), 522 (TCG→TGG) and 533 (GTG→CCG). Two rifampicin-resistant strains showed an identical PCR-SSCP pattern with the wild type H37Rv; 77.27% rifampicin-resistant strains showed a single point mutation and 9.09% had no mutation. Three rifampicin-resistant strains showed characteristic double mutations at codon positions 526 and 531. Sensitivity and specificity were calculated as 90.90% and 100%. Conclusions: Rifampicin-resistant genotypes were mainly found in codon positions 516, 526 and 531. PCR-SSCP seems to be an efficacious method of predicting rifampicin resistance and substantially reduces the time required for susceptibility testing from 4 to 6 weeks to a few weeks.