Differentiation of Mycobacterium tuberculosis complex from non-tubercular mycobacteria by nested multiplex PCR targeting IS6110, MTP40 and 32kD alpha antigen encoding gene fragments. Issue 1 (December 2016)

• Record Type: Journal Article
• Authors: Sinha, Pallavi; Gupta, Anamika; Prakash, Pradyot; Anupurba, Shampa; Tripathi, Rajneesh; Srivastava, G.
• Is Part Of: BMC infectious diseases. Volume 16:Issue 1(2016)
• Page Start: 1
• Page End: 10
• Publication Date: 2016-12
• Subjects: Mycobacterium tuberculosis complex -- Non-tubercular mycobacteria -- Nested multiplex PCR -- Composite reference standard; Communicable diseases -- Periodicals; Sexually Transmitted Diseases -- Periodicals; 616.905
• DOI: 10.1186/s12879-016-1450-1
• Languages: English
• ISSNs: 1471-2334

Abstract

Abstract Background Control of the global burden of tuberculosis is obstructed due to lack of simple, rapid and cost effective diagnostic techniques that can be used in resource poor-settings. To facilitate the early diagnosis of TB directly from clinical specimens, we have standardized and validated the use of nested multiplex PCR, targeting gene fragmentsIS6110, MTP40 and32kD α-antigen encoding genes specific forMycobacterium tuberculosis complex and non-tubercular mycobacteria (NTM), in comparison to smear microscopy, solid culture and single step multiplex PCR. The results were evaluated in comparison to a composite reference standard (CRS) comprising of microbiological results (smear and culture), clinical, radiological and cytopathological findings, clinical treatment and response to anti-tubercular therapy. Methods The nested multiplex PCR (nMPCR) assay was evaluated to test its utility in 600 (535 pulmonary and 65 extra-pulmonary specimens) clinically suspected TB cases. All specimens were processed for smear, culture, single step multiplex PCR and nested multiplex PCR testing. Results Out of 535 screened pulmonary and 65 extra-pulmonary specimens, 329 (61.5 %) and 19 (29.2 %) cases were culture positive forM. tuberculosis . Based on CRS, 450 patients had "clinical TB" (definitive-TB, probable-TB and possible-TB). Remaining 150 were confirmed "non-TB" cases. For culture, the sensitivity was low, 79.3 % for pulmonary and 54.3 % for extra-pulmonary cases. The sensitivity and specificity results for nMPCR test were evaluated taken composite reference standard as a gold standard. The sensitivity of the nMPCR assay was 97.1 % for pulmonary and 91.4 % for extra-pulmonary TB cases with specificity of 100 % and 93.3 % respectively. Conclusion Nested multiplex PCR using three gene primers is a rapid, reliable and highly sensitive and specific diagnostic technique for the detection and differentiation ofM. tuberculosis complex from NTM genome and will be useful in diagnosing paucibacillary samples. Nested multiplex PCR assay was found to be better than single step multiplex PCR for assessing the diagnosis of TB.