Molecular detection of Mycobacterium tuberculosis in formalin-fixed, paraffin-embedded tissues and biopsies of gastrointestinal specimens using real-time polymerase chain reaction system.
OBJECTIVE
We aimed to perform the molecular detection of Mycobacterium tuberculosis in formalin-fixed, paraffin-embedded tissues and biopsies of gastrointestinal specimens using real-time polymerase chain reaction system.


METHODS
The study included three groups: (A) control (n=24), with no previous signs of M. tuberculosis complex (MTBC), (B) patients (n=28) with known TB origin and (C) patients (n=50) with clinical and histopathological signs of TB but who were culture- and acid-fast bacilli (AFB)-negative. The samples were obtained from the Medical and Surgical Gastroenterology Departments of Bhopal Memorial Hospital and Research Centre. We extracted DNA using DNeasy Blood & Tissue kit (QIAGEN, Germany) and performed realtime assay using Roche LightCycler 2.0 with fluorescence resonance energy transfer (FRET) hybridization probes obtained from Roche Molecular Diagnostics (USA) for the specific amplification of the 159 bp region of the mycobacterium genome.


RESULTS
All the samples (n=24) of Group A were found to be negative, while in Group B, 27 out of the 28 cases studied were found to be positive by LightCycler real-time polymerase chain reaction (LC PCR). In Group C, 18 out of the 50 cases studied were found to be positive, showing a positivity of 36%. The overall positive and negative predictive values of the test for clinical TB (Group C) were 100% and 96.9%, respectively.


CONCLUSIONS
Results of our investigation demonstrated that the real-time detection technology using FRET probes has much higher sensitivity for the detection of MTBC DNA in tissue biopsy samples and formalin-fixed paraffin-embedded surgically resected tissues of the gastrointestinal tract.
Mishra
Pradyumna Kumar
PK
Department of Research and Training, Bhopal Memorial Hospital and Research Centre, Bhopal, India. pkm_8bh@yahoo.co.uk
Gorantla
Venkata Raghuram
VR
Bhargava
Arpit
A
Varshney
Subodh
S
Vashistha
Purushottam
P
Maudar
Kewal Krishan
KK
D001706:Biopsy; D003199:Computer Systems; D005557:Formaldehyde; D041981:Gastrointestinal Tract / Q000382:microbiology* / Q000473:pathology; D006801:Humans; D025202:Molecular Diagnostic Techniques; D009169:Mycobacterium tuberculosis / Q000302:isolation & purification*; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods
D005557:Formaldehyde