qPCR detection of Mycobacterium leprae in biopsies and slit skin smear of different leprosy clinical forms

Abstract Leprosy, whose etiological agent is Mycobacterium leprae, is a chronic infectious disease that mainly affects the skin and peripheral nervous system. The diagnosis of leprosy is based on clinical evaluation, whereas histopathological analysis and bacilloscopy are complementary diagnostic tools. Quantitative PCR (qPCR), a current useful tool for diagnosis of infectious diseases, has been used to detect several pathogens including Mycobacterium leprae. The validation of this technique in a robust set of samples comprising the different clinical forms of leprosy is still necessary. Thus, in this study samples from 126 skin biopsies (collected from patients on all clinical forms and reactional states of leprosy) and 25 slit skin smear of leprosy patients were comparatively analyzed by qPCR (performed with primers for the RLEP region of M. leprae DNA) and routine bacilloscopy performed in histological sections or in slit skin smear. Considering clinical diagnostic as the gold standard, 84.9% of the leprosy patients were qPCR positive in skin biopsies, resulting in 84.92% sensitivity, with 84.92 and 61.22% positive (PPV) and negative (NPV) predictive values, respectively. Concerning bacilloscopy of histological sections (BI/H), the sensitivity was 80.15% and the PPV and NPV were 80.15 and 44.44%, respectively. The concordance between qPCR and BI/H was 87.30%. Regarding the slit skin smear, 84% of the samples tested positive in the qPCR. Additionally, qPCR showed 100% specificity, since all samples from different mycobacteria, from healthy individuals, and from other granulomatous diseases presented negative results. In conclusion, the qPCR technique for detection of M. leprae using RLEP primers proved to be specific and sensitive, and qPCR can be used as a complementary test to diagnose leprosy irrespective of the clinical form of disease.

qPCR detection of Mycobacterium leprae in biopsies and slit skin smear of different leprosy clinical forms.

Leprosy, whose etiological agent is Mycobacterium leprae, is a chronic infectious disease that mainly affects the skin and peripheral nervous system. The diagnosis of leprosy is based on clinical evaluation, whereas histopathological analysis and bacilloscopy are complementary diagnostic tools. Quantitative PCR (qPCR), a current useful tool for diagnosis of infectious diseases, has been used to detect several pathogens including Mycobacterium leprae. The validation of this technique in a robust set of samples comprising the different clinical forms of leprosy is still necessary. Thus, in this study samples from 126 skin biopsies (collected from patients on all clinical forms and reactional states of leprosy) and 25 slit skin smear of leprosy patients were comparatively analyzed by qPCR (performed with primers for the RLEP region of M. leprae DNA) and routine bacilloscopy performed in histological sections or in slit skin smear. Considering clinical diagnostic as the gold standard, 84.9% of the leprosy patients were qPCR positive in skin biopsies, resulting in 84.92% sensitivity, with 84.92 and 61.22% positive (PPV) and negative (NPV) predictive values, respectively. Concerning bacilloscopy of histological sections (BI/H), the sensitivity was 80.15% and the PPV and NPV were 80.15 and 44.44%, respectively. The concordance between qPCR and BI/H was 87.30%. Regarding the slit skin smear, 84% of the samples tested positive in the qPCR. Additionally, qPCR showed 100% specificity, since all samples from different mycobacteria, from healthy individuals, and from other granulomatous diseases presented negative results. In conclusion, the qPCR technique for detection of M. leprae using RLEP primers proved to be specific and sensitive, and qPCR can be used as a complementary test to diagnose leprosy irrespective of the clinical form of disease.

qPCR detection of Mycobacterium leprae in biopsies and slit skin smear of different leprosy clinical forms

Leprosy, whose etiological agent is Mycobacterium leprae, is a chronic infectious disease that mainly affects the skin and peripheral nervous system. The diagnosis of leprosy is based on clinical evaluation, whereas histopathological analysis and bacilloscopy are complementary diagnostic tools. Quantitative PCR (qPCR), a current useful tool for diagnosis of infectious diseases, has been used to detect several pathogens including Mycobacterium leprae. The validation of this technique in a robust set of samples comprising the different clinical forms of leprosy is still necessary. Thus, in this study samples from 126 skin biopsies (collected from patients on all clinical forms and reactional states of leprosy) and 25 slit skin smear of leprosy patients were comparatively analyzed by qPCR (performed with primers for the RLEP region of M. leprae DNA) and routine bacilloscopy performed in histological sections or in slit skin smear. Considering clinical diagnostic as the gold standard, 84.9% of the leprosy patients were qPCR positive in skin biopsies, resulting in 84.92% sensitivity, with 84.92 and 61.22% positive (PPV) and negative (NPV) predictive values, respectively. Concerning bacilloscopy of histological sections (BI/H), the sensitivity was 80.15% and the PPV and NPV were 80.15 and 44.44%, respectively. The concordance between qPCR and BI/H was 87.30%. Regarding the slit skin smear, 84% of the samples tested positive in the qPCR. Additionally, qPCR showed 100% specificity, since all samples from different mycobacteria, from healthy individuals, and from other granulomatous diseases presented negative results. In conclusion, the qPCR technique for detection of M. leprae using RLEP primers proved to be specific and sensitive, and qPCR can be used as a complementary test to diagnose leprosy irrespective of the clinical form of disease.

Extração de DNA do Mycobacterium leprae a partir de esfregaços de lâminas de baciloscopia coradas por Ziehl-Neelsen para amplificação dos genes associados à resistência a drogas em hanseníase

A emergência de resistência a drogas pode influenciar no controle/eliminação dahanseníase, no entanto, os dados sobre resistência são escassos, nos países endêmicos. Relatosde resistência do M. leprae a drogas da poliquimioterapia se tornaram mais freqüentes após apadronização dos métodos de sequenciamento gênico. A biópsia de lesão é amplamenteutilizada para esse fim, porém pode causar desconforto ao paciente. Como alternativa, o DNAdo bacilo obtido a partir de esfregaços de raspado intradérmico (baciloscopia) corados porZiehl-Neelsen (ZN), tem sido utilizado com sucesso em alguns protocolos de análisemolecular. O objetivo deste estudo foi verificar a viabilidade da utilização de lâminas deraspado intradérmico para detecção de resistência a drogas por métodos moleculares, a partirda extração do DNA do bacilo utilizando-se diferentes protocolos. Para padronização foramutilizadas inicialmente lâminas coradas por ZN a frio, contendo suspensões de bacilos emdiluições seriadas, obtidas de camundongos nude previamente inoculados. Foramselecionadas lâminas coradas contendo esfregaços intradérmicos de pacientes multibacilares,diagnosticados entre 2009/2012, com índice baciloscópicos (IB) de 0 a 5+. Do total de 36pacientes, 29 possuíam duas lâminas, 6 uma lâmina e um três lâminas (n=67). A extração doDNA foi feita com Chelex100(CH) 5% (n=17), 10% (n=17) e com o kit DNaesyQiagen®(DN) (n=33). O excesso de óleo de imersão foi removido com papel absorvente,adicionados 90μl de água ultrapura, os esfregaços removidos com lâmina de bisturi epipetados em microtubo. Em 17 amostras foram adicionados 100μl de CH 5%, incubadas a97°C/10min e então centrifugadas a 13000rpm/2min. Em outras 17 foram adicionados 100μlde CH 10%, incubadas a 97°C/30 min e então centrifugadas a 13000rpm/10min. Apósincubação overnigth/4°C, as amostra foram centrifugadas e o sobrenadante (DNA) transferidopara outro microtubo. A extração pelo DN (n=33) foi realizada de acordo...