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Background: The gastrointestinal (GI) tract is the sixth commonest extra-pulmonary site to be affected by Mycobacterium tuberculosis complex (MTBC) infections, yet it is often underrated. This form of TB has an insidious course like any other chronic infectious disease without any specific laboratory, radiological or clinical signs and symptoms. Any kind of delay in prompt initiation, leads to treatment failure or develops antibiotic resistance. Due to this non-specificity and difficulties associated with its diagnosis, development of a rapid and more specific investigative method is an urgent need of hour.Objective: To identify the culture negative MTBC infections in endoscopic biopsies and formalin-fixed, paraffin-embedded tissues of gastrointestinal tract using a fluorescence resonance energy transfer (FRET) hybridization probe based real-time polymerase chain reaction (PCR) approach and characterization of its host immunological features.Materials and methods: The present study included three groups (A) control (n=24) with no previous signs of Mycobacterium tuberculosis complex (B) patients (n=28) with known TB origin (C) patients (n=50) with clinical and histo-pathological signs of TB but was culture and AFB negative. Real time PCR assay using Roche Light Cycler (LC) 2.0 with FRET probes was performed for the specific amplification of 159 bp region of mycobacterium genome. Later host immune characterization of the PCR positive samples was performed through immuno-phenotyping and Th1/Th2 cytokine profiling using flow cytometry. Students’ t-test was employed for statistical analysis using SPSS software and p value of ≤ 0.05 was considered to be significant.Results: All the samples (n=24) of group A were found to be negative while in group B 27 out of 28 reported positive by LC PCR. In group C out of total 50 cases studied 18 were found to be positive showing a positivity of 36 %. The sensitivity and specificity of the test was 97.87 % and 100 %. The positive predictive value was 100 % and the negative predictive value was 98 %. On immune characterization of the LC PCR positive group C cases a depleted CD4+ count and increased levels of IFN-g and TNF-a were observed. Conclusion: Application of FRET probe based real-time PCR based diagnosis offer’s a better approach for rapid and specific identification of culture negative tuberculosis infections in the gastro-intestinal tract. In addition, understanding host immunological response against such infections might provide specific therapeutic strategies for prevention and treatment of the infection in future.