สถาบันวิจัยวิทยาศาสตร์สาธารณสุข

National Institute of Health of Thailand

A simplified reverse transcription polymerase chain reaction for rapid detection of Mycobacterium leprae in skin specimens

Authors : Benjawan Phetsuksiri*, Janisara Rudeeaneksin*, Prapaporn Supapkul*, Suksawat Wachapong**, Krisada Mahotarn**, and Patrick J. Brennan ***

 

Affiliations:    *Sasakawa Research Building, Thai National Institute of Health, Department of Medical Sciences, Ministry of Public Health, Nonthaburi, Thailand      
** Raj-Prach-Samasai Institute, Department of Disease Control, Ministry of Public Health, Nonthaburi, Thailand
*** Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado USA 80523
 
Source:           International Journal of Leprosy and other Mycobacterial Diseases 2002;
70(4): 56A                       
 
Language:         English
 
Abstract:
 
Diagnosis of leprosy based on detection of Mycobacterium leprae RNA is difficult. To simplify detection, a one-step reverse transcription polymerase chain reaction (RT-PCR) was established and evaluated for its potential in the rapid and sensitive detection of leprosy. RNA and DNA could be simultaneously isolated by the commercially available ready-to-use solution, Tri-reagent. Based on M. leprae-specific primers targeting a 171-bp fragment of the M. leprae 16s RNA gene, RT-PCR designed for convenience and reproducibility, resulted in detection of M. leprae in both slit skin smears and skin biopsies. The assay was specific for M. leprae in comparison to other mycobacteria, and the specificity to leprosy was absolute when compared to skin specimens from healthy volunteers and patients with other skin diseases. The use of a digoxigenin-labelled DNA probe enhanced the positivity signal of the amplified RT-PCR product such that the method could detect as low as one bacterium. When used directly on skin specimens collected from leprosy patients, 16s rRNA of M. leprae was detected in 34 of 36 multibacillary (MB) and 13 of 24 paucibacillary (PB) cases. Skin slit smears from cases suspected of leprosy but negative for acid-fast bacilli, were positive by RT-PCR. The method was also effective in monitoring bacterial clearance in leprosy patients during chemotherapy; after treatment with the standard regimen of multidrug therapy for 6 months resulting in bacterial clearance, 16 of 36MB and 3 of 24 PB patients tested were still positive for 16s rRNA of M. leprae, suggesting the advisability of a more prolonged treatment course. This form of RT-PCR is of value in terms of simplicity and sensitivity to identify M. leprae in routine skin specimens, especially when acid-fast bacilli are not discernable.