An RNA-based assay is an additional molecular tool for leprosy diagnosis and determination of the viability of leprosy bacilli. To simplify RNA detection, a one-step reverse transcriptase PCR (RT-PCR) was established and evaluated. RNA and DNA could be isolated simultaneously. With the use of Mycobacterium leprae-specific primers targeting a 171-bp fragment of the M. leprae 16S RNA gene, RT-PCR resulted in detection of M. leprae in both slit skin smears and skin biopsy specimens. To enhance the positive signal, a digoxigenin-labeled DNA was developed, and successfully detected the amplified RT-PCR product. The method is sensitive, as it could detect one leprosy bacillus. When it was used directly on skin specimens collected from leprosy patients, 34 of 36 multibacillary (MB) and 13 of 24 paucibacillary (PB) cases showed positive results. The assay was also effective in monitoring bacterial clearance in leprosy patients during chemotherapy; after treatment with the multidrug therapy for 6 months, resulting in bacterial clearance, 16 of 36 MB patients and three of 24 PB patients tested were still positive for the 16S rRNA gene 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 in identifying M. leprae in routine skin specimens, especially when acid-fast bacilli are not discernable.

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.

SUMMARY:A cross-sectional study was conducted on the performance of the tuberculin skin test (TST) and QuantiFERON -TB Gold In-Tube test (QFT-IT) for detecting latent tuberculosis infection among Thai healthcare workers (HCWs). Each HCW underwent both the TST and QFT-IT during the annual health screening. Among the 260 HCWs enrolled, the median age was 30 years (range 19-60 years), 92z were women, 64z were nurses and nurse assistants, 78z were Bacillus Calmette Gu áerin vaccinated, and 37z had previously taken the TST. Correlation between TST reaction size and the interferon-g level was weak (r ＝ 0.29; P ＜ 0.001). Of the HCWs, 38z and 20z had a reactive TST and a positive QFT-IT, respectively. Using QFT-IT positivity as a standard for latent tuberculosis diagnosis, the cut-off for TST reactivity with the best performance was 13 mm with a sensitivity, specificity, false positivity, and false negativity of 71z, 70z, 30z, and 29z, respectively (area under the curve 0.73; P ＜ 0.001). The independent factor associated with a false reactive TST was a previous TST (adjusted odds ratio 1.83; P ＝ 0.04). Our findings suggest that the QFT-IT may be the preferred test among HCWs with previous TST. In settings where the QFT-IT is not available, appropriate cut-offs for TST reactivity should be evaluated for use among HCWs.