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

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

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.

Publication types

  • Evaluation Study

MeSH terms

  • Bacterial Proteins / genetics
  • DNA, Bacterial / genetics
  • Dapsone / pharmacology
  • Drug Resistance, Bacterial / genetics*
  • Humans
  • Leprostatic Agents / pharmacology
  • Leprosy / drug therapy*
  • Leprosy / microbiology
  • Microbial Sensitivity Tests / methods*
  • Mycobacterium leprae / drug effects*
  • Mycobacterium leprae / isolation & purification
  • Ofloxacin / pharmacology
  • Real-Time Polymerase Chain Reaction / methods*
  • Reproducibility of Results
  • Rifampin / pharmacology
  • Sensitivity and Specificity
  • Sequence Analysis, DNA
  • Skin / microbiology
  • Skin / pathology

Substances

  • Bacterial Proteins
  • DNA, Bacterial
  • Leprostatic Agents
  • Dapsone
  • Ofloxacin
  • Rifampin

Grants and funding

This study was financially supported in Brazil with grants from: Foundation for Research Support of the State of Minas Gerais (Fundação de Amparo à Pesquisa do Estado de Minas Gerais - FAPEMIG), Brazilian National Council for Scientific and Technological Development (Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq), Brazilian Coordinating Office for the Improvement of Higher Education Personnel (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES), and by the National Fund for Health/Brazilian Ministry of Health and the Damien Foundation. In the US the study was supported by the U.S. Department of Health and Human Services (DHHS), Health Resources and Services Administration HRSA), Healthcare Systems Bureau (HSB), National Hansen’s Disease Programs. SA received a PDSE fellowship from CAPES during the conduction of the experiments in the NHDP facilities, National Institutes of Health/ NIAID YI-AA-2646. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.