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2025-01-01
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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
Biópsia
Pacientes
Hanseníase
Avaliação em tempo real
Segmentação por PCR convencional
Conventional PCR Targeting
Evaluation of Real-Time
Skin Biopsy
Leprosy
Patients
Author
Affilliation
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Micobacterioses. Laboratório de Hanseníase. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Bioquímica e Biologia Molecular. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Micobacterioses. Laboratório de Hanseníase. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Micobacterioses. Laboratório de Hanseníase. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Micobacterioses. Laboratório de Hanseníase. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Micobacterioses. Laboratório de Hanseníase. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Micobacterioses. Laboratório de Hanseníase. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Bioquímica e Biologia Molecular. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Micobacterioses. Laboratório de Hanseníase. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Micobacterioses. Laboratório de Hanseníase. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Micobacterioses. Laboratório de Hanseníase. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Micobacterioses. Laboratório de Hanseníase. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Micobacterioses. Laboratório de Hanseníase. Rio de Janeiro, RJ, Brasil.
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 85Bcoding
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.
Keywords in Portuguese
DNA de Mycobacterium lepraeBiópsia
Pacientes
Hanseníase
Avaliação em tempo real
Segmentação por PCR convencional
Keywords
Mycobacterium leprae DNAConventional PCR Targeting
Evaluation of Real-Time
Skin Biopsy
Leprosy
Patients
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