Larsen and Toubro Microbiology Research Center

Chennai, India

Larsen and Toubro Microbiology Research Center

Chennai, India
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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.


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.


PubMed | Institute of Thoracic Medicine, Larsen and Toubro Microbiology Research Center and Stanley Medical College
Type: Evaluation Studies | Journal: International journal of mycobacteriology | Year: 2016

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 manufacturers 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.


Gayathri R.,Larsen and Toubro Microbiology Research Center | Lily Therese K.,Larsen and Toubro Microbiology Research Center | Deepa P.,Larsen and Toubro Microbiology Research Center | Mangai S.,Larsen and Toubro Microbiology Research Center | Madhavan H.N.,Larsen and Toubro Microbiology Research Center
Journal of Postgraduate Medicine | Year: 2010

Background: The rapidly growing mycobacteria (RGM) causing human infections primarily consist of the Mycobacterium fortuitum group, Mycobacterium abscessus and Mycobacterium chelonae. The antibiotic susceptibility testing is important to determine the appropriate therapy as the antibiotics used to treat RGM are different from those used for treating infections caused by slow growers of mycobacteria. Aim: To determine antibiotic susceptibility of RGM using Kirby Bauer method and following Clinical and Laboratory Standards Institute (CLSI) guidelines. Settings and Design: Larsen and Toubro Microbiology Research Centre, Vision Research Foundation, Sankara Nethralaya, Chennai, Retrospective study. Materials and Methods: The antibiotic susceptibility testing was performed following CLSI method for the drugs Amikacin, Azithromycin, Tobramycin, Ceftazidime, Cephotaxime, Cefuroxime, Cefaperazone, Ceftriaxone, Ciprofloxacin, Ofloxacin, Norfloxacin, Gatifloxacin and Moxifloxacin. Results and Conclusions: Out of the 148 RGM isolates 146 (98%) were susceptible to amikacin, 138 (91%) to gatifloxacin, 132 (87%) to moxifloxacin, 122 (76%) to ciprofloxacin and 116 (74%) to Norfloxacin. Majority of the RGM were resistant to Ceftazidime, Cephotaxime and Cefaperazone. All the M. abscessus isolates were resistant to tobramycin. The in vitro antibiotic susceptibility testing by disc diffusion method showed that majority of the RGM were sensitive to Amikacin followed by Gatifloxacin, Moxifloxacin and Ciprofloxacin.


Madhavan H.N.,Larsen and Toubro Microbiology Research Center | Bagyalakshmi R.,Larsen and Toubro Microbiology Research Center | Revathy M.,Larsen and Toubro Microbiology Research Center | Aarthi P.,Larsen and Toubro Microbiology Research Center | Malathi J.,Larsen and Toubro Microbiology Research Center
Indian Journal of Medical Microbiology | Year: 2015

Purpose: To optimise a polymerase chain reaction (PCR) based DNA sequencing technique for genotyping polyoma virus in clinical specimens obtained from renal transplant patients. Materials and Methods: A hundred and thirty (106 peripheral blood and 24 urine) clinical specimens collected from renal transplant patients were included in the study for detecting the presence of DNA of BK virus (BKV), JC virus (JCV) by PCR targeting the viral protein 1 (VP1) gene. PCR based DNA sequencing was performed to determine the genotypes of polyoma virus and subjected to bioinformatics analysis to determine the amino acid sequences and screen for mutations in the VP1 gene. Results: Polyoma virus was detected in 23 (17.69%) specimens of which 19 (82.60%) were positive for BK virus, 3 (13.04%) for JC virus and 1 for both BK and JC virus. PCR based DNA sequencing detected BK virus genotype I in 12 (50%), genotype IV in 8 (33.3%) and JC virus in 4 (16.6%) clinical specimens. BKV genotype I was the predominant genotype (64.2% in peripheral blood and 33.33% in urine) prevalent in south India. Six novel mutations were found-at position 29, 30 to 47 of BKV genotype I; at position 11 and 15 of BKV genotype IV and at position 2 and 30 of JCV. Conclusion: BKV genotype I is the prominent genotype in India and novel mutations detected in the VP1 gene of BKV and JCV are being reported for the first time in literature.


PubMed | Larsen and Toubro Microbiology Research Center
Type: Case Reports | Journal: Indian journal of medical microbiology | Year: 2013

To detect and identify the aetiological agent in the peripheral blood from the cases of neonatal sepsis.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.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.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 .


PubMed | Larsen and Toubro Microbiology Research Center
Type: Comparative Study | Journal: Journal of infection in developing countries | Year: 2015

Infectious mononucleosis (IM) caused by the Epstein-Barr virus (EBV) is commonly diagnosed by detection of antibodies in the patients 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).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.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.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.


PubMed | Larsen and Toubro Microbiology Research Center
Type: Journal Article | Journal: Journal of virological methods | Year: 2012

The main objective of the study is to optimize a subtype specific polymerase chain reaction (PCR) for identification of hepatitis C virus (HCV) subtypes in patients with renal disease. Thirty two peripheral blood specimens obtained from 28 patients (post renal transplant N=14, chronic kidney disease N=14) were subjected to HCV viral load determination followed by genotyping analysis. Based on the mixed genotypes and subtypes (>one subtype) obtained by type specific PCR, specific patterns from Pattern I through Pattern X were assigned. All the 32 peripheral blood specimens revealed mixed HCV subtype patterns (>one subtype). The detection of Pattern I in 12 (42.8%) out of 28 patients was statistically significant (Chi square test, P value<0.001). HCV subtyping assay developed using stringent thermal profile revealed the presence of mixed subtype patterns (>one subtype) which is for the first time being reported in literature.


PubMed | Larsen and Toubro Microbiology Research Center
Type: Journal Article | Journal: Microbiological research | Year: 2013

To develop a novel RNA based assay to determine the Gram reaction and viability of bacteria in clinical specimens.Reverse transcriptase PCR (RT-PCR) targeting 16SrRNA region was optimized using Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa ATCC 27853 by using two novel sets of primers. Sixty clinical specimens consisting of 31 intraocular specimens (19 vitreous fluids and 12 aqueous humor), 11 peripheral blood specimens and 18 other clinical specimens were subjected to standard microbiological culture and RT-PCR to determine the Gram reaction and viability of bacteria. The amplified products were subjected to DNA sequencing to identify the bacterium.The sensitivity of RT-PCR was 0.4fg and the primers amplified bacterial cDNA. RT-PCR detected the presence of bacteria in 60 clinical specimens indicating the presence of viable bacteria. Concordant results were obtained with both primer sets. Seventy five bacterium comprising 52 single (69.3%) and 23 mixed bacteria (30.6%), both Gram positive and Gram negative were detected. These results correlated with the bacterial identity by PCR based DNA sequencing.RT-PCR is a reliable tool to identify the presence of viable bacteria and to precisely determine Gram reaction.


PubMed | Larsen and Toubro Microbiology Research Center
Type: | Journal: Indian journal of medical microbiology | Year: 2015

To optimise a polymerase chain reaction (PCR) based DNA sequencing technique for genotyping polyoma virus in clinical specimens obtained from renal transplant patients.A hundred and thirty (106 peripheral blood and 24 urine) clinical specimens collected from renal transplant patients were included in the study for detecting the presence of DNA of BK virus (BKV), JC virus (JCV) by PCR targeting the viral protein 1 (VP1) gene. PCR based DNA sequencing was performed to determine the genotypes of polyoma virus and subjected to bioinformatics analysis to determine the amino acid sequences and screen for mutations in the VP1 gene.Polyoma virus was detected in 23 (17.69%) specimens of which 19 (82.60%) were positive for BK virus, 3 (13.04%) for JC virus and 1 for both BK and JC virus. PCR based DNA sequencing detected BK virus genotype I in 12 (50%), genotype IV in 8 (33.3%) and JC virus in 4 (16.6%) clinical specimens. BKV genotype I was the predominant genotype (64.2% in peripheral blood and 33.33% in urine) prevalent in south India. Six novel mutations were found--at position 29, 30 to 47 of BKV genotype I; at position 11 and 15 of BKV genotype IV and at position 2 and 30 of JCV.BKV genotype I is the prominent genotype in India and novel mutations detected in the VP1 gene of BKV and JCV are being reported for the first time in literature.

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