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Makeshkumar V.,SRM University | Madhavan R.,SRM University | Narayanan S.,National Institute for Research in Tuberculosis ICMR
Indian Journal of Medical Research

Background & objectives: Diagnosis of extrapulmonary tuberculosis (EPTB) is difficult using conventional diagnostic methods. This study was conducted to evaluate the use of polymerase chain reaction (PCR) in diagnosis of definitive and probable extrapulmonary tuberculosis patients, and to assess the performance of insertion sequence (IS) 6110 based PCR assay as compared to conventional culture by Lowenstein-Jensen (LJ) method for the diagnosis of EPTB. Methods: A total of 178 non repeated clinical specimens were collected from clinically suspected extrapulmonary tuberculosis patients. The specimens included 59 ascitic fluid, 54 pleural fluid, 25 cerebrospinal fluid (CSF), 12 fine needle aspiration (FNA), 8 urine, 7 pus, 6 synovial fluid, 2 skin tissue, one pericardial fluid, one liver abscess, one pancreatic cyst fluid, one omental biopsy and one semen sample. All these clinical samples were subjected to Ziehl-Neelsen staining (ZN) for acid fast bacilli (AFB) and culture on LJ medium. PCR was performed by targeting 123bp fragment of insertion sequence IS6110 of Mycobacterium tuberculosis (MTB). Results: Of the 178 specimens, 10 (5.61%) were ZN smear positive for AFB, six (3.37%) were L-J culture positive from 10 AFB smear positive cases and 48 (26.96%) were PCR IS 6110 positive for M. tuberculosis. Interpretation & conclusions: PCR using IS6110 primer was able to pick up more EPTB patients compared to conventional L-J culture method for detection of M. tuberculosis. False positive PCR IS6110 in three CSF samples may be due to latent TB infection which was limitation in this study. Source

Devasundaram S.,National Institute for Research in Tuberculosis ICMR | Raja A.,National Institute for Research in Tuberculosis ICMR
Infection, Genetics and Evolution

Tuberculosis continues to be a major public health problem in many parts of the world, despite intensified efforts taken to control the disease. The remarkable success of M. tuberculosis as a pathogen is largely due to its ability to persist within the host for long periods. To develop the effective intervention strategies, understanding the biology of persistence is highly required. Accumulating evidences showed oxygen deprivation (hypoxia) as a potential stimulus for triggering the transition of M. tuberculosis to a non-replicating persistent state analogous to latency in vivo. To date, in vitro hypoxia experimental models used the laboratory adapted isolate H37Rv and very little is known about the behavior of clinical isolates that are involved during disease outbreaks. Hence, we compared the transcription profiles of H37Rv and two south Indian clinical isolates (S7 and S10) under hypoxia to find differences in gene expression pattern. The main objective of this current work is to find "differentially regulated genes" (genes that are down regulated in H37Rv but upregulated in both the clinical isolates) under hypoxia. Microarray results showed, a total of 502 genes were down regulated in H37Rv under hypoxia and 10 out of 502 genes were upregulated in both the clinical isolates. Thus, giving less importance to down regulated genes based on H37Rv model strain might exclude the true representative gene candidates in clinical isolates. Our study suggests the use of most prevalent clinical isolates for in vitro experimental model to minimize the variation in understanding the adaptation mechanisms of the strains. © 2016 Elsevier B.V. Source

Parasa V.R.R.,National Institute for Research in Tuberculosis ICMR | Sikhamani R.,Government Hospital of Thoracic Medicine | Raja A.,National Institute for Research in Tuberculosis ICMR

Background: NK cells express several specialized receptors through which they recognize and discriminate virally-infected/tumor cells efficiently from healthy cells and kill them. This ability to lyse is regulated by an array of inhibitory or activating receptors. The present study investigated the frequency of various NK receptors expressed by NK cell subsets from HIV-infected TB patients. The effect of IL-15+IL-12 stimulation on the expression of NK receptors was also studied. Methodology/Principal Findings: The study included 15 individuals each from normal healthy subjects, pulmonary tuberculosis patients, HIV-infected individuals and patients with HIV and tuberculosis co-infection. The expression of NK cell receptors was analyzed on two NK cell subsets within the peripheral blood: CD16+CD3- and CD56+CD3- using flow cytometry. The expression of inhibitory receptors (CD158a, CD158b, KIRp70, CD85j and NKG2A) on NK subsets was increased in HIV, when compared to NHS. But the response in HIV-TB was not uniform. Stimulation with IL-15+IL-12 dropped (p<0.05) the expression of CD85j and NKG2A in HIV. The basal expression of natural cytotoxicity receptors (NKp30 and NKp46) on NK cell subsets was lowered (p<0.05) in HIV and HIV-TB as compared to NHS. However, the expression of NKp44 and NKG2D was elevated in HIV. Enhanced NKp46 and NKG2D expression was observed in HIV with IL-15+IL-12 stimulation. The coreceptor NKp80 was found to be expressed in higher numbers on NK subsets from HIV compared to NHS, which elevated with IL-15+IL-12 stimulation. The expression of NK receptors and response to stimulation was primarily on CD56+CD3- subset. Conclusions/Significance: IL-15+IL-12 has an immunomodulatory effect on NK cell subsets from HIV-infected individuals viz down-regulation of iNKRs, elevation of activatory receptors NKp46 and NKG2D, and induction of coreceptor NKp80. IL-15+IL-12 is not likely to be of value when co-infected with TB probably due to the influence of tuberculosis. © 2012 Parasa et al. Source

Pukazhvanthen P.,National Institute for Research in Tuberculosis ICMR | Anbarasu D.,National Institute for Research in Tuberculosis ICMR | Kabeer Basirudeen S.A.,National Institute for Research in Tuberculosis ICMR | Raja A.,National Institute for Research in Tuberculosis ICMR | Singh M.,Lionex Diagnostics and Therapeutics GmbH

Serodiagnostic potential of four recombinant proteins (38 kDa[Rv0934], MPT64[Rv1980c], Adk[Rv0733], and BfrB[Rv3874]) was evaluated in Healthy control subjects (HCS), Healthy household contacts (HHC), Pulmonary tuberculosis patients (PTB), and Human immuno deficiency virus & Tuberculosis co-infected patients (HIV-TB). All the antigens tested individually for the detection of serum IgG by indirect ELISA. All the four antigens have a significantly higher antibody response in PTB compared to healthy controls (P < 0.05). The sensitivity of individual antigens ranged from 20% to 52.5% for the prefixed specificity of 95%. When results of all 4 antigens were combined the sensitivity was increased to 75% and specificity was reduced 89% in HCS. In smear- and culture-positive (S+C+) PTB, four antigen combination gives maximum sensitivity (89.6%) with 89% specificity. In smear negative culture negative (S-C+) PTB, three antigen combination (38 kDa with MPT64 and BfrB) gives maximum sensitivity (69.5%) and specificity (91.6%). In HIV-TB, 4 antigen combinations give the maximum sensitivity of 51.2% with 89% specificity. Combining serology (Four antigen combination) with smear was able to increase the sensitivity from 70% to 92.5% in culture positive PTB. So, we propose that this serology test can be used as adjunct test along with smear for rapid diagnosis of PTB. © 2014 Elsevier Ltd. Source

Syed Ahamed Kabeer B.,National Institute for Research in Tuberculosis ICMR | Paramasivam P.,National Institute for Research in Tuberculosis ICMR | Raja A.,National Institute for Research in Tuberculosis ICMR
Journal of Infection

Objective: This study aimed to compare the levels of TB-antigen specific Interferon gamma (IFN-γ) and IFN-γ inducible protein (IP)-10 in culture of whole blood samples from healthy controls (HC) and healthy household contacts (HHC). Methodology: A total of 386 study subjects, which included 186 HC and 200 HHC, were recruited. QuantiFERON-TB Gold in-tube (QFT-IT) assay was employed to measure IFN-γ levels. IP-10 levels were measured in the supernatants collected from QFT-IT tubes. Tuberculin skin test was also performed. Results: The levels of TB antigen specific IFN-γ and IP-10 were significantly higher in HHC compared to HC. There was no significant difference observed between positivity of QFT-IT and IP-10 in HC and HHC. The positivity of TST was significantly lower in subjects <17 year, when compared to IP-10 (p< 0.005). The reduced cut-off point 0.22. IU/ml significantly increased the positivity of QFT-IT among children with high risk for latent TB infection (LTBI). Conclusions: Measurement of TB antigen specific IFN-γ and IP-10 can be potential markers for the detection of LTBI. © 2012 The British Infection Association. Source

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