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

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 (T = 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 × 10-8.02 × 10, and in SB samples from MB patients were 1.87 × 10-1.50 × 10. 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.

Leprosy: A Review of Epidemiology, Clinical Diagnosis, and Management

“…Over the past 30 years, PCR methods have been developed to amplify various gene targets in M. leprae . PCR techniques have been used to detect possible environmental sources for the dissemination of M. leprae as well as the aerosol route of infection by means of nasal carriage [ 74 – 77 ]. The summary sensitivity of the PCR test was 75.3% (95% CI 67.9–81.5), and the specificity was 94.5% (91.4–96.5) [ 70 ].…”

Utilidad de las técnicas de biología molecular en el diagnóstico de las infecciones cutáneas

“…En general, la PCR tiene una elevada sensibilidad en los estadios tempranos de las infecciones por Borrelia. En la enfermedad de Lyme (Borrelia burgdorferi) y, en especial, los casos de eritema migrans que clı ´nica e histolo ´gicamente no son claros, la PCR estarı ´a indicada con un buen rendimiento (67-71%), a pesar de no existir un me ´todo definitivo de referencia [15][16][17] . Por ello, los resultados moleculares deberı ´an correlacionarse con los hallazgos clı ´nicos y serolo ´gicos 3 .…”

Perfil epidemiológico e tendência da hanseníase em menores de 15 anos

“…This bacillus is transmitted mainly via the upper respiratory tract and its primordial attraction of invasion are Schwann cells, located in the Peripheral Nervous System (PNS). This invasion may lead to diverse forms of clinical presentation, which are classified according to the immune response of each host (4) .…”