RESUMO
Leprosy urgently needs a precise and early diagnostic tool. The sensitivity of the direct (bacilli staining, Mycobacterium leprae DNA) and indirect (antibody levels, T cell assays) diagnostics methods vary based on the clinical form. Recently, PCR-based M. leprae DNA detection has been shown to differentially diagnose leprosy from other dermatological conditions. However, accuracy can still be improved, especially for use with less invasive clinical samples. We tested different commercial DNA extraction kits: DNeasy Blood & Tissue, QIAamp DNA Microbiome, Maxwell 16 DNA Purification, PowerSoil DNA Isolation; as well as in-house phenol-chloroform and Trizol/FastPrep methods. Extraction was performed on M. leprae-infected mouse footpads and different clinical samples of leprosy patients (skin biopsies and scrapings, lesion, oral and nasal swabs, body hair, blood on FTA cards, peripheral whole blood). We observed that the Microbiome kit was able to enrich for mycobacterial DNA, most likely due the enzymatic digestion cocktail along with mechanical disruption involved in this method. Consequently, we had a significant increase in sensitivity in skin biopsies from paucibacillary leprosy patients using a duplex qPCR targeting 16S rRNA (M. leprae) and 18S rRNA (mammal) in the StepOnePlus system. Our data showed that the presence of M. leprae DNA was best detected in skin biopsies and skin scrapings, independent of the extraction method or the clinical form. For multibacillary patients, detection of M. leprae DNA in nasal swabs indicates the possibility of having a much less invasive sample that can be used for the purposes of DNA sequencing for relapse analysis and drug resistance monitoring. Overall, DNA extracted with the Microbiome kit presented the best bacilli detection rate for paucibacillary cases, indicating that investments in extraction methods with mechanical and DNA digestion should be made.
Assuntos
DNA Bacteriano/isolamento & purificação , Mycobacterium leprae/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Animais , DNA Bacteriano/genética , Humanos , Camundongos , Mycobacterium leprae/genética , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Sensibilidade e EspecificidadeRESUMO
Leprosy is difficult to diagnose since it is caused by a bacterium that does not grow in vitro. Bacilli direct detection or the presence of specific antibodies can vary greatly depending on the clinical form. M. leprae direct DNA detection can aid clinical diagnosis, although invasive skin biopsies are still necessary to detect the pathogen or histological features consistent with leprosy. Here we show that a kit combining mechanical and chemical lysis efficiently removes host DNA and enriches for M. leprae DNA, allowing better detection of paucibacillary cases. We believe our findings can contribute to improving disease diagnosis, as well as early detection and that could help monitoring strategies(AU).
