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Lakshmipathy D.,Larsen and Toubro Microbiology Research Center | Kulandai L.T.,Larsen and Toubro Microbiology Research Center | Ramasubban G.,Larsen and Toubro Microbiology Research Center | Hajib Narahari Rao M.,Larsen and Toubro Microbiology Research Center | And 2 more authors.
International Journal of Mycobacteriology | Year: 2015

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 icl2, 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 icl2, hspx, and rRNAP1 genes were indigenously designed using the Primer-BLAST software, and optimized for sensitivity and specificity. The icl2, 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 icl2, 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. © 2015 Asian African Society for Mycobacteriology. Source


Aarthi P.,Larsen and Toubro Microbiology Research Center | Bagyalakshmi R.,Larsen and Toubro Microbiology Research Center | Mohan K.R.,Kanchi Kamakoti Childs Trust Hospital | Krishna M.,Kanchi Kamakoti Childs Trust Hospital | And 3 more authors.
Indian Journal of Medical Microbiology | Year: 2013

Purpose: To detect and identify the aetiological agent in the peripheral blood from the cases of neonatal sepsis. Materials and Methods: Four neonates from geographically different regions of South India presented with signs of neonatal sepsis and all the routine clinical and laboratory investigations were performed. Blood culture by Bac T Alert 3D was negative. To establish the aetiology, polymerase chain reaction (PCR) for eubacterial genome and subsequent amplification with Gram positive and Gram negative primers were performed followed by deoxyribonucleic acid (DNA) sequencing. Results: PCR for the detection of eubacterial genome was positive in all the four neonates and further amplification with designed Gram positive and Gram negative primers revealed the presence of Gram negative bacteria. The amplicons were identified as Orientia tsutsugamushi in three neonates and Coxiella burnetti in the other neonate. Multalin analysis was done to further characterise the strain variation among the three strains. Conclusion: PCR-based DNA sequencing is a rapid and reliable diagnostic tool to identify the aetiological agents of neonatal sepsis. This is the first case series of emerging Rickettsial neonatal sepsis in India. Source


Therese K.L.,Larsen and Toubro Microbiology Research Center | Gayathri R.,Larsen and Toubro Microbiology Research Center | Dhanurekha L.,Larsen and Toubro Microbiology Research Center | Sridhar R.,Stanley Medical College | And 2 more authors.
Indian Journal of Medical Microbiology | Year: 2013

Background: Early diagnosis of tuberculosis is critical for its effective management and prevention. Several gene amplification methods are used in the detection of tubercle bacilli from clinical specimens. MPB64 gene and IS6110 region have been identified as potential gene targets for the specific detection of Mycobacterium tuberculosis from direct clinical specimens. Objective: The present study was conducted to evaluate the diagnostic utility of simultaneous application of two nested polymerase chain reaction (nPCRs) targeting MPB64 and IS6110 region for the detection of M. tuberculosis genome. Materials and Methods: A total of 100 and 354 clinical specimens from the control group and clinically suspected tuberculosis patients, respectively, were included in the study. nPCRs targeting MPB64 and IS6110 region were performed. Results and Conclusion: All of the 100 clinical specimens from the control group were negative for both nPCRs. Out of the 354 clinical specimens, 339 were positive for both culture and nPCRs, 10 and 5 were positive for culture, and nPCR targeting IS6110 and MPB64 regions, respectively. To conclude, nPCRs targeting MPB64 and IS6110 region are reliable and specific targets when applied simultaneously on clinical specimens to attain 100% sensitivity for the detection of M. tuberculosis genome. Source


Janani M.K.,Larsen and Toubro Microbiology Research Center | Malathi J.,Larsen and Toubro Microbiology Research Center | Appaswamy A.,Kanchi Kamakoti CHILDS Trust Hospital KKCTH | Singha N.R.,Larsen and Toubro Microbiology Research Center | Madhavan H.N.,Larsen and Toubro Microbiology Research Center
Journal of Infection in Developing Countries | Year: 2015

Introduction: Infectious mononucleosis (IM) caused by the Epstein-Barr virus (EBV) is commonly diagnosed by detection of antibodies in the patient’s sera. Differentiation of acute from chronic and differential diagnosis of EBV-induced IM from IM-like syndrome caused by human cytomegalovirus (CMV) is important. The objective of this study was to standardize and use polymerase chain reaction (PCR) for diagnosis of EBV and evaluate it against enzyme-linked immunosorbent assay (ELISA). Methodology: ELISA for detection of IgM and IgG antibodies to viral capsid antigen (VCA) and PCR targeting the VCA and EBNA1 gene of EBV and mtrII gene of CMV were performed on180 peripheral blood samples collected from 180 patients with suspected IM. The analytical sensitivity of PCR was evaluated against that of ELISA. Results: Using the standard serological profile as the reference, the EBV-VCA gene was detected in 41 (95%) of 45 samples collected from patients with early primary infections, in 41 (54%) of 75 with recent primary infections, and in7 (17%) of 39 with past infections. The result of VCA PCR was statistically significant in virus detection during early or primary stage of infection. Nineteen (49%) EBV-seropositive samples were positive for CMV by PCR. All control samples tested negative for both VCA and EBNA1by PCR. Conclusions: VCA PCR is sensitive for the detection of EBV DNA in the early or primary stage of infection and can be considered a reliable method to rule out the cross-reactivity and differential diagnosis of EBV-induced IM from IM-like syndrome. © 2015 Janani et al. Source


Harishankar A.,Larsen and Toubro Microbiology Research Center | Jambulingam M.,Larsen and Toubro Microbiology Research Center | Gowrishankar R.,SASTRA University | Venkatachalam A.,Larsen and Toubro Microbiology Research Center | And 4 more authors.
Virology Journal | Year: 2012

Background: HSV-1 genome is a mosaic of recombinants. Clinical Herpes simplex virus -1 (HSV1) isolates were already genotyped as A, B and C types based on nucleotide variations at Unique Short (US) 4 (gG) and US 7 (gI) regions through phylogeny. Analysis of Glycoprotein C (gC) exon present on the Unique Long (UL) region had also revealed the existence of different genotypes. Glycoprotein C is mainly involved in initial viral attachment to heparan sulphate on host cell surface facilitating the virus's binding and penetration into cell. As the amount of heparan sulphate on the host cell surface varies according to the cell type, it is plausible that different genotypes bind differentially to cell types. Hence, this study was framed to determine the existence of novel genotypes/sub genotypes in the US or UL regions which could associate with clinical entities. Results: All the twenty five isolates analyzed in this study were of genotype A as per their gG gene sequences. In case of gI gene, 16 out of 25 were found to be type A and the remaining nine were type B putative intergenic recombinants. Intragenic recombinations were also encountered in both the US genes, with gG possessing novel subgenotypes, arbitrarily designated A1 and A2. The 9 type B isolates of gI genes also branched out into 2 clades due to genetic variations. Glycoprotein C of UL region had two distinct genotypic clades and , whose topological distribution was significantly different from that of the US region. Neither the US nor UL regions, however, showed any preference among the genotypes to a specific anatomic site of infection. Even the non synonymous variations identified in the functional domain of gC, were not confined to a particular genotype/clinical entity. Conclusion: The analyses of the US and UL regions of the HSV-1 genome showed the existence of variegated genotypes in these two regions. In contrary to the documented literature, in which Asian strains were concluded as more conserved than European ones, our study showed the existence of a higher degree of variability among Indian strains. However, the identified novel genotypes and subgenotypes were not found associated with clinical entities. © 2012 Harishankar et al; licensee BioMed Central Ltd. Source

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