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.

BackgroundSince leprosy is both treated and controlled by multidrug therapy (MDT) it is important to monitor recurrent cases for drug resistance and to distinguish between relapse and reinfection as a means of assessing therapeutic efficacy. All three objectives can be reached with single nucleotide resolution using next generation sequencing and bioinformatics analysis of Mycobacterium leprae DNA present in human skin.MethodologyDNA was isolated by means of optimized extraction and enrichment methods from samples from three recurrent cases in leprosy patients participating in an open-label, randomized, controlled clinical trial of uniform MDT in Brazil (U-MDT/CT-BR). Genome-wide sequencing of M. leprae was performed and the resultant sequence assemblies analyzed in silico.Principal findingsIn all three cases, no mutations responsible for resistance to rifampicin, dapsone and ofloxacin were found, thus eliminating drug resistance as a possible cause of disease recurrence. However, sequence differences were detected between the strains from the first and second disease episodes in all three patients. In one case, clear evidence was obtained for reinfection with an unrelated strain whereas in the other two cases, relapse appeared more probable.Conclusions/SignificanceThis is the first report of using M. leprae whole genome sequencing to reveal that treated and cured leprosy patients who remain in endemic areas can be reinfected by another strain. Next generation sequencing can be applied reliably to M. leprae DNA extracted from biopsies to discriminate between cases of relapse and reinfection, thereby providing a powerful tool for evaluating different outcomes of therapeutic regimens and for following disease transmission.

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Background Leprosy has been treated with multidrug therapy, which has been distributed for free across the globe and regarded as highly efficient. However, the impossibility of growing Mycobacterium leprae in axenic media has historically impaired assessments of M. leprae resistance, a parameter only recently detectable through molecular methods. Methods A systematic, population-based search for M. leprae resistance in suspected leprosy relapse cases and contacts was performed in Prata Village, an isolated, hyperendemic, former leprosy colony located in the Brazilian Amazon. Results led to an extended active search involving the entire Prata population. Confirmed leprosy cases were investigated for bacterial resistance using a combination of in vivo testing and direct sequencing of resistance genes folP1, rpoB, and gyrA. A molecular epidemiology analysis was performed using data from 17 variable number tandem repeats (VNTR). Results Mycobacterium leprae was obtained from biopsies of 37 leprosy cases (18 relapses and 19 new cases): 16 (43.24%) displayed drug-resistance variants. Multidrug resistance to rifampicin and dapsone was observed in 8 relapses and 4 new cases. Single resistance to rifampicin was detected in 1 new case. Resistance to dapsone was present in 2 relapses and 1 new case. Combined molecular resistance and VNTR data revealed evidence of intra-familial primary transmission of resistant M. leprae. Conclusions A comprehensive, population-based systematic approach to investigate M. leprae resistance in a unique population revealed an alarming scenario of the emergence and transmission of resistant strains. These findings may be used for the development of new strategies for surveillance of drug resistance in other populations.

Leprosy, caused by Mycobacterium leprae, is an important infectious disease that is still endemic in many countries around the world, including Brazil. There are currently no known methods for growing M. leprae in vitro, presenting a major obstacle in the study of this pathogen in the laboratory. Therefore, the maintenance and growth of M. leprae strains are preferably performed in athymic nude mice (NU-Foxn1 nu ). The laboratory conditions for using mice are readily available, easy to perform, and allow standardization and development of protocols for achieving reproducible results. In the present report, we describe a simple protocol for purification of bacilli from nude mouse footpads using trypsin, which yields a suspension with minimum cell debris and with high bacterial viability index, as determined by fluorescent microscopy. A modification to the standard method for bacillary counting by Ziehl-Neelsen staining and light microscopy is also demonstrated. Additionally, we describe a protocol for freezing and thawing bacillary stocks as an alternative protocol for maintenance and storage of M. leprae strains.

Leprosy remains an endemic disease in Brazil, with almost 40,000 new cases diagnosed each year. As it is difficult to perform laboratory procedures in the field, operational classification is determined by counting lesions, which can cause underdiagnosis of multibacillary cases and failures in treatment.To evaluate a new tool to diagnose MB cases, the ML Flow test, 21/77 (27.3%) patients with untreated borderline leprosy (6 BL and 15 BT) with 1 to 5 cutaneous lesions were evaluated according to the R&J Classification. The ML Flow test was positive in 14/21 (66.6%) patients; 7/21 (33.3%) cases, 5 BT and 2 BL, showed negative results. Classification of leprosy based only on the number of lesions can fail to diagnose MB leprosy. The ML Flow test is a useful tool to diagnose borderline leprosy in patients with 1 to 5 cutaneous lesions. Key-words:Borderline leprosy. Classification. Serology. Pathology. RESUMOA hanseníase ainda é doença endêmica no Brasil, com cerca de 40.000 novos casos por ano. Devido à dificuldade na realização de exames laboratoriais em campo, classifica-se a forma clínica contando-se lesões, o que pode causar subdiagnóstico de casos multibacilares e falha terapêutica. Para avaliar uma nova ferramenta para diagnóstico de hanseníase multibacilar, o teste ML Flow, foi realizado em 21/77 (27,3%) pacientes com hanseníase dimorfa (6 DV e 15 DT) não tratados, com até cinco lesões de pele, avaliados de acordo com a classificação de Ridley & Jopling (R&J). O teste ML Flow foi positivo em 14/21 (66,6%) pacientes (4 DV e 10 DT); em 7/21 (33,3%) pacientes (5 DT e 2 DV) o resultado foi negativo. A classificação da hanseníase baseada somente na contagem de lesões pode falhar em diagnosticar casos MB. O ML Flow é ferramenta útil no diagnóstico de hanseníase dimorfa com até cinco lesões cutâneas. Palavras-chaves:Hanseníase dimorfa. Classificação. Sorologia. Patologia.

Comunicação Breve/Short Communication Resumo Objetivo: As infecções de trato urinário (ITU) estão entre as doenças infecciosas mais comuns na prática clínica. O objetivo do presente estudo foi investigar a prevalência de microrganismos patogênicos, analisando a faixa etária e o gênero mais acometido bem como o perfil de resistência aos antimicrobianos. Métodos: O estudo caracterizou-se por uma pesquisa histórica documental, na qual se analisaram registros de 605 uroculturas realizadas pelo Laboratório de Microbiologia do Instituto Lauro de Souza Lima, na cidade de Bauru, SP, no período de outubro de 2010 a outubro de 2015. Foram incluídos pacientes de ambos os gêneros e de todas as idades. Os dados foram coletados a partir do livro de registro de exames do laboratório e do sistema de informatização SIGH e transcritos para uma planilha eletrônica utilizando o programa Microsoft Office Excel. Resultados e Discussão: Das 605 uroculturas analisadas, 24,2% apresentaram resultados positivos para ITU. Dentre as positivas, 75,3% foram de pacientes do gênero feminino. Analisando a incidência dos microrganismos, a bactéria Escherichia coli foi a mais isolada, apresentando maior resistência à penicilina G, quinolonas e trimetoprim-sulfamatoxazol. Conclusão: Pode-se concluir que o correto diagnóstico é imprescindível na escolha e na instituição da antibioticoterapia mais adequada, evitando o uso indiscriminado de antimicrobianos Palavras-chave Infecções urológicas; Escherichia coli; Farmacorresistência bacteriana

Background: Mycobacterium peregrinum is a rapidly growing mycobacterium (RGM) that rarely causes skin infections. The correct identification of the specific RGM infecting the skin will enhance therapeutic success. Objective: To highlight the importance of rapid and precise identification of the Mycobacterium involved in skin infections in order to enhance therapeutic success. Methods: We describe an RGM skin infection in an immunocompetent patient. Results: Classic methods (biochemical tests and culture) of RGM identification are time-consuming, and the histopathological features are not specific. Some molecular methods are reliable but expensive. The PRAhsp-65 is a simple procedure that is helpful in identifying the specific agent of an RGM. Conclusion: Although skin infections caused by M peregrinum are rare, they represent a substantial clinical challenge. Specific and more effective treatment options depend on the development of precise and rapid methods for identifying mycobacterial species.