title: Differentiation of Mycobacterium tuberculosis complex from non-tubercular mycobacteria by nested multiplex PCR targeting IS6110, MTP40 and 32kD alpha antigen encoding gene fragments.
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 fragments IS6110, MTP40 and 32kD α-antigen encoding genes specific for Mycobacterium 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 for M. 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.
  
  
  CONCLUSIONS
  Nested multiplex PCR using three gene primers is a rapid, reliable and highly sensitive and specific diagnostic technique for the detection and differentiation of M. 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.
authors.affiliation: Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, 221 005, Varanasi, India.
authors.forename: Pallavi
authors.initials: P
authors.lastname: Sinha
authors.affiliation: Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, 221 005, Varanasi, India.
authors.forename: Anamika
authors.initials: A
authors.lastname: Gupta
authors.affiliation: Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, 221 005, Varanasi, India.
authors.forename: Pradyot
authors.initials: P
authors.lastname: Prakash
authors.affiliation: Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, 221 005, Varanasi, India. shampa_anupurba@yahoo.co.in.
authors.forename: Shampa
authors.initials: S
authors.lastname: Anupurba
authors.affiliation: Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, 221 005, Varanasi, India.
authors.forename: Rajneesh
authors.initials: R
authors.lastname: Tripathi
authors.affiliation: Departmrnt of TB and Respiratory Diseases, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India.
authors.forename: G N
authors.initials: GN
authors.lastname: Srivastava
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000942:Antigens, Bacterial / Q000235:genetics / Q000302:isolation & purification; D001992:Bronchoalveolar Lavage Fluid; D002648:Child; D002675:Child, Preschool; D017931:DNA Primers; D005260:Female; D006801:Humans; D007194:India; D008297:Male; D008875:Middle Aged; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D012680:Sensitivity and Specificity; D014397:Tuberculosis, Pulmonary / Q000382:microbiology*; D055815:Young Adult
chemical_list: D000942:Antigens, Bacterial; D017931:DNA Primers
keywords: Composite reference standard; Mycobacterium tuberculosis complex; Nested multiplex PCR; Non-tubercular mycobacteria