Affiliation:
1. Leprosy Laboratory, Department of Mycobacterioses, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro, RJ, Brazil
2. Department of Biochemistry and Molecular Biology, Oswaldo Cruz Institute, Fiocruz, Av. Brasil 4365, Manguinhos, 21045-900, Rio de Janeiro, Brazil
Abstract
ABSTRACT
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 (
C
T
) values obtained using the TaqMan system were able to statistically (
P
< 0.0001) differentiate MB (mean
C
T
, 28.06; standard deviation [SD], 4.51) from PB (mean
C
T
, 33.06; SD, 2.24) patients. Also, there was a correlation between
C
T
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.
Publisher
American Society for Microbiology
Reference26 articles.
1. Almeida, E. C., A. N. Martinez, V. C. Maniero, A. M. Sales, N. C. Duppre, E. N. Sarno, A. R. Santos, and M. O. Moraes. 2004. Detection of Mycobacterium leprae DNA by polymerase chain reaction in the blood and nasal secretion of Brazilian household contacts. Mem. Inst. Oswaldo Cruz99:509-511.
2. Beyene, D., A. Aseffa, M. Harboe, D. Kidane, M. Macdonald, P. R. Klatser, G. A. Bjune, and W. C. Smith. 2003. Nasal carriage of Mycobacterium leprae DNA in healthy individuals in Lega Robi village, Ethiopia. Epidemiol. Infect.131:841-848.
3. Simple and Fast Lateral Flow Test for Classification of Leprosy Patients and Identification of Contacts with High Risk of Developing Leprosy
4. Chae, G. T., M. J. Kim, T. J. Kang, S. B. Lee, H. K. Shin, J. P. Kim, Y. H. Ko, S. H. Kim, and N. H. Kim. 2002. DNA-PCR and RT-PCR for the 18-kDa gene of Mycobacterium leprae to assess the efficacy of multi-drug therapy for leprosy. J. Med. Microbiol.51:417-422.
5. Feitosa, M. F., I. Borecki, H. Krieger, B. Beiguelman, and D. C. Rao. 1995. The genetic epidemiology of leprosy in a Brazilian population. Am. J. Hum. Genet.56:1179-1185.
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