Rapid detection of ethambutol-resistant in clinical specimens by real-time polymerase chain reaction hybridisation probe method.

Background: Early diagnosis of drug resistance (DR) to ethambutol (EMB) in tuberculosis (TB) remains a challenge. Simple and reliable method (s) are needed for rapid detection of DR Mycobacterium tuberculosis (MTB) in clinical specimens.

Objectives: The aim of this study was to design fluorescence resonance energy transfer hybridisation probe-based real-time polymerase chain reaction (PCR) method for the early detection of EMB-resistant MTB direct from clinical sputa.

Materials And Methods: Primers and probes were designed against 306 codon of embB gene which is commonly associated with EMB resistance. A comparative study was done between Lowenstein-Jenson (L-J) proportion and hybridisation probe-based real-time PCR method for susceptibility testing. DNA sequencing was used in nine representative isolates to validate the efficiency of real-time PCR method to detect emb306 mutation of MTB.

Results: A total of 52 clinical sputum samples and corresponding culture isolates (from category II pulmonary TB cases) were included in this study. Out of 52 MTB isolates, 32 and 20 were resistant and susceptible to EMB, respectively, as determined by L-J proportion method. Real-time PCR showed 95% specificity, 75% sensitivity and 82.69% accuracy when compared with L-J proportion method. A 100% of concordance was observed by validating the real-time PCR results with DNA sequencing.

Conclusions: Our real-time PCR hybridisation probe method promises for rapid detection of EMB-resistant MTB directly from clinical specimens. However, future studies and modifications of method by incorporating other potential loci along with targeted mutation (emb306) are still required to increase the sensitivity of method.