Real-Time PCR and High-Resolution Melt Analysis for Rapid Detection of Mycobacterium leprae Drug Resistance Mutations and Strain Types

Abstract

c Drug resistance surveillance and strain typing of Mycobacterium leprae are necessary to investigate ongoing transmission of leprosy in regions of endemicity. To enable wider implementation of these molecular analyses, novel real-time PCR-highresolution melt (RT-PCR-HRM) assays without allele-specific primers or probes and post-PCR sample handling were developed. For the detection of mutations within drug resistance-determining regions (DRDRs) of folP1, rpoB, and gyrA, targets for dapsone, rifampin, and fluoroquinolones, real-time PCR-HRM assays were developed. Wild-type and drug-resistant mouse footpadderived strains that included three folP1, two rpoB, and one gyrA mutation types in a reference panel were tested. RT-PCR-HRM correctly distinguished the wild type from the mutant strains. In addition, RT-PCR-HRM analyses aided in recognizing samples with mixed or minor alleles and also a mislabeled sample. When tested in 121 sequence-characterized clinical strains, HRM identified all the folP1 mutants representing two mutation types, including one not within the reference panel. The false positives (<5%) could be attributed to low DNA concentration or PCR inhibition. A second set of RT-PCR-HRM assays for identification of three previously reported single nucleotide polymorphisms (SNPs) that have been used for strain typing were developed and validated in 22 reference and 25 clinical strains. Real-time PCR-HRM is a sensitive, simple, rapid, and high-throughput tool for routine screening known DRDR mutants in new and relapsed cases, SNP typing, and detection of minor mutant alleles in the wild-type background at lower costs than current methods and with the potential for quality control in leprosy investigations. Leprosy is an infectious disease of skin and nerves caused by Mycobacterium leprae. The disease remains endemic in many parts of the world and is now listed as a neglected tropical disease (27) by the World Health Organization. The drug dapsone was introduced in 1950 and was administered in the form of long-term monotherapy for treatment of leprosy; unfortunately, drug resistance emerged during the 1960s and 1970s (4). For this reason, in 1982, the World Health Organization (WHO) formally recommended multidrug therapy (MDT) which includes dapsone, rifampin, and clofazimine for the treatment and control of multibacillary (MB) leprosy (43). Sporadic reports of clinical resistance to dapsone and rifampin started appearing in several countries such as Vietnam, Mexico, India, and Philippines (1,8,13,19,24,25,26). Noncompliance and inadequate therapy may be the causes of the clinical resistance, particularly for MB leprosy. The drug targets and the mutations in the coding genes folP1, rpoB, and gyrA that lead to clinical resistance to dapsone, rifampin, and the fluoroquinolones (used in an alternative leprosy drug regimen), respectively, have been identified and characterized (5,10,14,39). The in vivo drug susceptibility or resistance phenotypes of various mutations seen in clinical strains in patient skin biop...

qPCR-High resolution melt analysis for drug susceptibility testing of Mycobacterium leprae directly from clinical specimens of leprosy patients

“…leprae and have previously shown to generate distinct “V” HRM profiles [12]. Results from the current study support these observations and represent over 80% of the RMP-R mutants described worldwide[16].…”

Mycobacterium leprae in Colombia described by SNP7614 in gyrA, two minisatellites and geography

“…Of 13 samples with the C64 genotype, 9 were available for further analysis. Using the recently developed HRM assay for SNP typing they were found to be of type 1 (Li et al, 2012). …”

“…Secondary and higher level genotyping should then be possible due to the growing number of VNTR and SNP markers (Kimura et al, 2009, Jensen et al, 2011). There are other alternatives for genotyping which are not dependent on DNA sequencing methods such as the high resolution melt-PCR (HRM-PCR); as applied here for the SNP typing of a subset of samples (Li et al, 2012) and SNP typing by PCR-RFLP (Sakamuri et al, 2009). …”

Comparison of Two Assays for Molecular Determination of Rifampin Resistance in Clinical Samples from Patients with Buruli Ulcer Disease

“…In combination with a recently described 16S rRNA-based viability assay (12), molecular drug resistance testing would also allow a reliable differentiation of individuals harboring viable drug-resistant organisms opposed to mycobacterial DNA residues detectable in secondary lesions (4). Furthermore, novel real-time PCR highresolution melt analysis assays without allele-specific primers or probes may constitute a promising tool for screening clinical isolates in future studies (20). …”