title: LightCycler real-time PCR for rapid detection and quantitation of Mycobacterium leprae in skin specimens.
abstract: Diagnosis of leprosy is usually based on clinical features and skin smear results including the number of skin lesions. Mycobacterium leprae is not cultivable and bacterial enumeration by microscopic examination is required for leprosy classification, choice in choosing and monitoring chemotherapy regimens, and diagnosis of relapse. However, detection and quantification using standard microscopy yields results of limited specificity and sensitivity. We describe an extremely sensitive and specific assay for the detection and quantification of M. leprae in skin biopsy specimens. Primers that amplified a specific 171-bp fragment of M. leprae 16S rRNA gene were chosen and specificity was verified by amplicon melting temperature. The method is sensitive enough to detect as low as 20 fg of M. leprae DNA, equivalent to four bacilli. The assay showed 100% concordance with clinical diagnosis in cases of multibacillary patients, and 50% of paucibacillary leprosy. The entire procedure of DNA extraction and PCR could be performed in c. 3 h. According to normalized quantitative real-time PCR, the patients in this study had bacilli numbers in the range of 1.07 x 10(2) -1.65 x 10(8) per 6-mm3 skin biopsy specimen. This simple real-time PCR assay is a facile tool with possible applications for rapid detection and simultaneous quantification of leprosy bacilli in clinical samples.
authors.affiliation: Mycobacteria Laboratory, Sasakawa Research Building, National Institute of Health, Department of Medical Sciences, Ministry of Public Health, Nonthaburi, Thailand.
authors.forename: Janisara
authors.initials: J
authors.lastname: Rudeeaneksin
authors.forename: Sopa
authors.initials: S
authors.lastname: Srisungngam
authors.forename: Pathom
authors.initials: P
authors.lastname: Sawanpanyalert
authors.forename: Thaverit
authors.initials: T
authors.lastname: Sittiwakin
authors.forename: Sirirat
authors.initials: S
authors.lastname: Likanonsakul
authors.forename: Supannee
authors.initials: S
authors.lastname: Pasadorn
authors.forename: Prasit
authors.initials: P
authors.lastname: Palittapongarnpim
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Brennan
authors.forename: Benjawan
authors.initials: B
authors.lastname: Phetsuksiri
mesh_terms: D006801:Humans; D007918:Leprosy / Q000175:diagnosis*; D009166:Mycobacterium leprae / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012680:Sensitivity and Specificity; D012867:Skin / Q000382:microbiology* / Q000473:pathology
chemical_list: D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S