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. 2017 Jun 1;11(6):e0005506.
doi: 10.1371/journal.pntd.0005506. eCollection 2017 Jun.

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

Affiliations

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

Sergio Araujo et al. PLoS Negl Trop Dis. .

Abstract

Background: Real-Time PCR-High Resolution Melting (qPCR-HRM) analysis has been recently described for rapid drug susceptibility testing (DST) of Mycobacterium leprae. The purpose of the current study was to further evaluate the validity, reliability, and accuracy of this assay for M. leprae DST in clinical specimens.

Methodology/principal findings: The specificity and sensitivity for determining the presence and susceptibility of M. leprae to dapsone based on the folP1 drug resistance determining region (DRDR), rifampin (rpoB DRDR) and ofloxacin (gyrA DRDR) was evaluated using 211 clinical specimens from leprosy patients, including 156 multibacillary (MB) and 55 paucibacillary (PB) cases. When comparing the results of qPCR-HRM DST and PCR/direct DNA sequencing, 100% concordance was obtained. The effects of in-house phenol/chloroform extraction versus column-based DNA purification protocols, and that of storage and fixation protocols of specimens for qPCR-HRM DST, were also evaluated. qPCR-HRM results for all DRDR gene assays (folP1, rpoB, and gyrA) were obtained from both MB (154/156; 98.7%) and PB (35/55; 63.3%) patients. All PCR negative specimens were from patients with low numbers of bacilli enumerated by an M. leprae-specific qPCR. We observed that frozen and formalin-fixed paraffin embedded (FFPE) tissues or archival Fite's stained slides were suitable for HRM analysis. Among 20 mycobacterial and other skin bacterial species tested, only M. lepromatosis, highly related to M. leprae, generated amplicons in the qPCR-HRM DST assay for folP1 and rpoB DRDR targets. Both DNA purification protocols tested were efficient in recovering DNA suitable for HRM analysis. However, 3% of clinical specimens purified using the phenol/chloroform DNA purification protocol gave false drug resistant data. DNA obtained from freshly frozen (n = 172), formalin-fixed paraffin embedded (FFPE) tissues (n = 36) or archival Fite's stained slides (n = 3) were suitable for qPCR-HRM DST analysis. The HRM-based assay was also able to identify mixed infections of susceptible and resistant M. leprae. However, to avoid false positives we recommend that clinical specimens be tested for the presence of the M. leprae using the qPCR-RLEP assay prior to being tested in the qPCR-HRM DST and that all specimens demonstrating drug resistant profiles in this assay be subjected to DNA sequencing.

Conclusion/significance: Taken together these results further demonstrate the utility of qPCR-HRM DST as an inexpensive screening tool for large-scale drug resistance surveillance in leprosy.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Comparison of M. leprae qPCR-HRM DST and PCR/DNA sequencing DST results of RMP-resistant M. leprae Br-3.
A) Difference plots graphic display of the post-qPCR HRM analysis of the rpoB drug resistance determining region (DRDR) of M. leprae Br-3. The DNA melting curves obtained in the analysis of mutant strains, deviate from the wild type profile (Thai-53 strain, shown in blue color). B) DNA chromatogram result for the DNA sequence of rpoB DRDR of M. leprae Br-3 sample, showing three independent mutations associated with RMP resistance.
Fig 2
Fig 2. Comparison of M. leprae qPCR-HRM DST difference plots graphic displays of the post-qPCR HRM analyses of the rpoB and folP1 drug resistance determining regions (DRDR) of M. lepromatosis and M. leprae for DDS and RMP susceptibility.
DNA melting curves, obtained from the analysis of strains, deviate from the wild-type profile (Thai-53 strain, shown in dark blue color); A) folP1 DRDR HRM profiles and B) rpoB DRDR HRM profiles.
Fig 3
Fig 3. Comparison of M. leprae qPCR-HRM DST difference plot graphic displays of the post-qPCR HRM analyses of the folP1 from clinical strains.
DDS-resistant strains: T(ACC)53A(GCC) = Ref ID# N-33; P(CCC)55R(CGC) = Ref ID# N-08; T(ACC)53I(ATC) = Ref ID# N-30; P(CCC)55L(CTC) = Ref ID# N-21. M. leprae DDS-susceptible strains: Wild-type = Thai-53 (control); Ref ID# B-01; Ref ID# B-08; Ref ID# B-11; Ref ID# B-23; Ref ID# B-32; and Ref ID# B-55.
Fig 4
Fig 4. Determination of RMP-resistant mutant allele detection limit in a mixture of wild-type/RMP-resistant mutant DRDRs using M. leprae qPCR-HRM DST.
A) Difference plot graphic displays of the post-PCR HRM melting curve analysis of the rpoB drug resistance determining region (DRDR) of wild-type (Thai-53) and RMP-R strains (Ze-4 and Ze-9) and mixtures of Thai-53 and these RMP-R mutants; B) PCR-DNA sequencing of the rpoB DRDRs of 3:2 ratio of Ze-4; and C) PCR-direct DNA sequencing of the rpoB DRDRs of 1:1 ratio of Ze-9. Yellow highlights identify codon where mutations were observed.

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