Evaluation of 16S rRNA qPCR for detection of Mycobacterium leprae DNA in nasal secretion and skin biopsy samples from multibacillary and paucibacillary leprosy cases

Pathog Glob Health. 2018 Mar;112(2):72-78. doi: 10.1080/20477724.2017.1415736. Epub 2017 Dec 26.

Abstract

Mycobacterium leprae bacilli are mainly transmitted by the dissemination of nasal aerosols from multibacillary (MB) patients to susceptible individuals through inhalation. The upper respiratory tract represents the main entry and exit routes of M. leprae. Therefore, this study aimed to evaluate the sensitivity and specificity of real-time quantitative polymerase chain reaction (qPCR) in detecting M. leprae in nasal secretion (NS) and skin biopsy (SB) samples from MB and paucibacillary (PB) cases. Fifty-four NS samples were obtained from leprosy patients at the Dona Libânia National Reference Centre for Sanitary Dermatology in Ceará, Brazil. Among them, 19 MB cases provided both NS and SB samples. Bacilloscopy index assays were conducted and qPCR amplification was performed using specific primers for M. leprae 16S rRNA gene, generating a 124-bp fragment. Primer specificity was verified by determining the amplicon melting temperature (Tm = 79.5 °C) and detection limit of qPCR was 20 fg of M. leprae DNA. Results were positive for 89.7 and 73.3% of NS samples from MB and PB cases, respectively. SB samples from MB patients were 100% positive. The number of bacilli detected in NS samples were 1.39 × 103-8.02 × 105, and in SB samples from MB patients were 1.87 × 103-1.50 × 106. Therefore, qPCR assays using SYBR Green targeting M. leprae 16S rRNA region can be employed in detecting M. leprae in nasal swabs from leprosy patients, validating this method for epidemiological studies aiming to identify healthy carriers among household contacts or within populations of an endemic area.

Keywords: Mycobacterium leprae; biopsy; nasal cavity; paucibacillary leprosy; quantitative real-time PCR.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biopsy*
  • Body Fluids / microbiology*
  • Brazil
  • DNA, Bacterial / genetics
  • DNA, Ribosomal / genetics
  • Humans
  • Leprosy / diagnosis*
  • Leprosy / microbiology
  • Molecular Diagnostic Techniques / methods
  • Mycobacterium leprae / isolation & purification*
  • Nasal Cavity / microbiology*
  • RNA, Ribosomal, 16S / genetics
  • Real-Time Polymerase Chain Reaction / methods*
  • Sensitivity and Specificity
  • Skin / microbiology*

Substances

  • DNA, Bacterial
  • DNA, Ribosomal
  • RNA, Ribosomal, 16S