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Published on: July 15, 2011
Rapid Identification of Drug-Resistant Strains of Mycobacterium leprae through qPCR High-Resolution Melt Analysis
Rahul Sharma1,2, Itu Singh1, Saurabh Gupta2
1Stanley Browne Laboratory, The Leprosy Mission Community Hospital, Nand Nagari, New Delhi, India.
Background:
Rapid detection of drug-resistant Mycobacterium leprae is essential for effective treatment. Real-time polymerase chain reaction high-resolution melt (qPCR-HRM) analysis is a promising tool for this purpose. This study aimed to evaluate the reliability of qPCR-HRM for detecting drug-resistant strains of M. leprae in India.
Patients And Methods:
Skin biopsies or slit-skin scrapings from 48 leprosy patients; relapse ( n = 10), defaulters ( n = 2), reactional ( n = 17), and new cases ( n = 19), were analyzed. Drug resistance testing was conducted using qPCR-HRM and PCR followed by Sanger sequencing, following WHO guidelines.
Results:
Drug resistance was detected in 9/48 (rifampicin), 11/48 (dapsone), and 5/48 (ofloxacin) by both methods. Dual resistance was seen in three patients: two with rifampicin and dapsone resistance, and one with rifampicin and ofloxacin resistance. Among new cases, mutations were detected in 10.5% (rifampicin) and 21.1% (dapsone); among reactional cases, in 23.5% (rifampicin), 11.8% (dapsone), 11.8% (ofloxacin), and 5.9% (rifampicin and ofloxacin); and among relapse cases, in 30% (rifampicin and ofloxacin), 50% (dapsone), and 20% (rifampicin and dapsone). No resistance was observed in defaulters. Sequencing confirmed mutation sites, and qPCR-HRM results were consistent with sequencing data.
Limitations:
A limitation of the study is possible misclassification of samples that amplify beyond a threshold cycle (CT) of 30, which requires confirmation by PCR followed by DNA sequencing. Further, the inability of qPCR-HRM to determine exact mutation positions requires PCR/sequencing confirmation and a small sample size, which may limit generalizability.
Conclusion:
qPCR-HRM is a reliable, rapid, and effective method for detecting drug resistance in leprosy. It provides results comparable to PCR and sequencing, making it suitable for large-scale screening in endemic regions.
