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Journal of Clinical Microbiology, September 2006, p. 3154-3159, Vol. 44, No. 9
0095-1137/06/$08.00+0     doi:10.1128/JCM.02250-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Evaluation of Real-Time and Conventional PCR Targeting Complex 85 Genes for Detection of Mycobacterium leprae DNA in Skin Biopsy Samples from Patients Diagnosed with Leprosy

Alejandra N. Martinez,1 Constança F. P. C. Britto,2 José A. C. Nery,1 Elizabeth P. Sampaio,1 Márcia R. Jardim,1 Euzenir N. Sarno,1 and Milton O. Moraes1*

Leprosy Laboratory, Department of Mycobacterioses, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro, RJ, Brazil,1 Department of Biochemistry and Molecular Biology, Oswaldo Cruz Institute, Fiocruz, Av. Brasil 4365, Manguinhos, 21045-900, Rio de Janeiro, Brazil2

Received 26 October 2005/ Returned for modification 23 December 2005/ Accepted 11 June 2006

In spite of the decrease in the number of registered leprosy patients, the number of new cases diagnosed each year (400,000) has remained essentially unchanged. Leprosy diagnosis is difficult due to the low sensitivity of current methodologies to identify new cases. In this study, conventional and TaqMan real-time PCR assays for detection of Mycobacterium leprae DNA were compared to current classification based on clinical, bacteriological, and histological evaluation. M. leprae DNA was extracted from frozen skin biopsy specimens from 69 leprosy patients enrolled in the study and was amplified using specific primers for either the antigen 85B-coding gene or the 85A-C intergenic region by using conventional and real-time PCR. The detection rate was 100% among multibacillary (MB) patients and ranged from 62.5% to 79.2% among paucibacillary (PB) patients according to the assay used. The TaqMan system for 85B gene amplification showed the highest sensitivity, although conventional PCR using the 85A-C gene as a target was also efficient. The cycle threshold (CT) values obtained using the TaqMan system were able to statistically (P < 0.0001) differentiate MB (mean CT, 28.06; standard deviation [SD], 4.51) from PB (mean CT, 33.06; SD, 2.24) patients. Also, there was a correlation between CT values and the bacteriological index for MB patients (Pearson's r, –0.444; P = 0.008). Within the PB patients' group, we tested normal skin from six patients exhibiting the pure neuritic form of leprosy (PNL). Five out of six PNL patients were positive for the presence of M. leprae DNA, even in the absence of skin lesions. In conclusion, the TaqMan real-time PCR developed here seems to be a useful tool for rapidly detecting and quantifying M. leprae DNA in clinical specimens in which bacilli were undetectable by conventional histological staining.

* Corresponding author. Mailing address: Leprosy Laboratory, Mycobacterioses Department, Fiocruz, Av. Brasil 4365, Manguinhos, Rio de Janeiro, 21040-360, Brazil. Phone: (21) 25984467. Fax: (21) 22709997. E-mail: mmoraes{at}fiocruz.br.

Journal of Clinical Microbiology, September 2006, p. 3154-3159, Vol. 44, No. 9
0095-1137/06/$08.00+0     doi:10.1128/JCM.02250-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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  • Lini, N., Shankernarayan, N. P., Dharmalingam, K. (2009). Quantitative real-time PCR analysis of Mycobacterium leprae DNA and mRNA in human biopsy material from leprosy and reactional cases. J Med Microbiol 58: 753-759 [Abstract] [Full Text]  
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