Genotyping of Mycobacterium leprae present on Ziehl-Neelsen-stained microscopic slides and in skin biopsy samples from leprosy patients in different geographic regions of Brazil

We analysed 16 variable number tandem repeats (VNTR) and three single-nucleotide polymorphisms (SNP) in Mycobacterium leprae present on 115 Ziehl-Neelsen (Z-N)-stained slides and in 51 skin biopsy samples derived from leprosy patients from Ceará (n = 23), Pernambuco (n = 41), Rio de Janeiro (n = 22) and Rondônia (RO) (n = 78). All skin biopsies yielded SNP-based genotypes, while 48 of the samples (94.1%) yielded complete VNTR genotypes. We evaluated two procedures for extracting M. leprae DNA from Z-N-stained slides: the first including Chelex and the other combining proteinase and sodium dodecyl sulfate. Of the 76 samples processed using the first procedure, 30.2% were positive for 16 or 15 VNTRs, whereas of the 39 samples processed using the second procedure, 28.2% yielded genotypes defined by at least 10 VNTRs. Combined VNTR and SNP analysis revealed large variability in genotypes, but a high prevalence of SNP genotype 4 in the Northeast Region of Brazil. Our observation of two samples from RO with an identical genotype and seven groups with similar genotypes, including four derived from residents of the same state or region, suggest a tendency to form groups according to the origin of the isolates. This study demonstrates the existence of geographically related M. leprae genotypes and that Z-N-stained slides are an alternative source for M. leprae genotyping.

Concordance between expected and observed bacilloscopy results of clinical forms of leprosy: a 6-year retrospective study in Recife, State of Pernambuco, Brazil.

INTRODUCTION: Operational classification of leprosy based on the number of skin lesions was conceived to screen patients presenting severe forms of the disease to enable their reception of a more intense multidrug regimen without having to undergo lymph smear testing. We evaluated the concordance between operational classification and bacilloscopy to define multibacillary and paucibacillary leprosy. METHODS: We selected 1,213 records of individuals with leprosy, who were untreated (new cases) and admitted to a dermatology clinic in Recife, Brazil, from 2000 to 2005, and who underwent bacteriological examination at diagnosis for ratification of the operational classification. RESULTS: Compared to bacilloscopy, operational classification demonstrated 88.6% sensitivity, 76.9% specificity, a positive predictive value of 61.8%, and a negative predictive value of 94.1%, with 80% accuracy and a moderate kappa index. Among the bacilloscopy-negative cases, 23% had more than 5 skin lesions. Additionally, 11% of the bacilloscopy-positive cases had up to 5 lesions, which would have led to multibacillary cases being treated as paucibacillary leprosy if the operational classification had not been confirmed by bacilloscopy. CONCLUSIONS: Operational classification has limitations that are more obvious in borderline cases, suggesting that in these cases, lymph smear testing is advisable to enable the selection of true multibacillary cases for more intense treatment, thereby contributing to minimization of resistant strain selection and possible relapse.

Concordance between expected and observed bacilloscopy results of clinical forms of leprosy: a 6-year retrospective study in Recife, State of Pernambuco, Brazil / Concordância entre os resultados observados e esperados da forma clinica da hanseníase de acordo com a baciloscopia: um estudo retrospectivo de seis anos em Recife, Estado de Pernambuco, Brasil

INTRODUCTION: Operational classification of leprosy based on the number of skin lesions was conceived to screen patients presenting severe forms of the disease to enable their reception of a more intense multidrug regimen without having to undergo lymph smear testing. We evaluated the concordance between operational classification and bacilloscopy to define multibacillary and paucibacillary leprosy. METHODS: We selected 1,213 records of individuals with leprosy, who were untreated (new cases) and admitted to a dermatology clinic in Recife, Brazil, from 2000 to 2005, and who underwent bacteriological examination at diagnosis for ratification of the operational classification. RESULTS: Compared to bacilloscopy, operational classification demonstrated 88.6% sensitivity, 76.9% specificity, a positive predictive value of 61.8%, and a negative predictive value of 94.1%, with 80% accuracy and a moderate kappa index. Among the bacilloscopy-negative cases, 23% had more than 5 skin lesions. Additionally, 11% of the bacilloscopy-positive cases had up to 5 lesions, which would have led to multibacillary cases being treated as paucibacillary leprosy if the operational classification had not been confirmed by bacilloscopy. CONCLUSIONS: Operational classification has limitations that are more obvious in borderline cases, suggesting that in these cases, lymph smear testing is advisable to enable the selection of true multibacillary cases for more intense treatment, thereby contributing to minimization of resistant strain selection and possible relapse.

INTRODUÇÃO: A classificação operacional da hanseníase baseada no número de lesões de pele foi concebida para selecionar pacientes que apresentam formas graves da doença para receber regime terapêutico mais intenso com múltiplas drogas sem o exame de baciloscopia da linfa. Nós avaliamos a concordância entre a classificação operacional e a baciloscopia para a definição de hanseníase multibacilar e paucibacilar. MÉTODOS: Nós selecionamos 1.213 registros de indivíduos com hanseníase não tratada (casos novos), atendidos em um Ambulatório de Dermatologia, em Recife, Brasil, no período de 2000 a 2005, que foram submetidos a exame bacteriológico ao diagnóstico para a ratificação da classificação operacional. RESULTADOS: Comparando com a baciloscopia, a classificação operacional baseada no número de lesões cutâneas mostrou sensibilidade de 88,6%, especificidade 76,9%, valor preditivo positivo de 61,8% e valor preditivo negativo de 94,1%, com uma precisão de 80% e um moderado índice kappa. Entre os casos com baciloscopia negativa, 23% tinham mais de cinco lesões de pele, recebendo um tratamento mais intensivo. Além disso, 11% dos casos baciloscopia positiva tinham até cinco lesões, o que induziriam casos multibacilares de serem tratados com hanseníase paucibacilar se a classificação operacional não tivesse sido confirmada pela baciloscopia. CONCLUSÕES: Concluímos que a classificação operacional tem limitações mais visíveis nos casos borderline, sugerindo que, nestes casos, o esfregaço seria aconselhável por permitir que os verdadeiros casos multibacilares fossem selecionados para um tratamento mais intenso, contribuindo para minimizar a seleção de cepas resistentes e uma possível recidiva.

Genotyping of Mycobacterium leprae present on Ziehl-Neelsen-stained microscopic slides and in skin biopsy samples from leprosy patients in different geographic regions of Brazil.

We analysed 16 variable number tandem repeats (VNTR) and three single-nucleotide polymorphisms (SNP) in Mycobacterium leprae present on 115 Ziehl-Neelsen (Z-N)-stained slides and in 51 skin biopsy samples derived from leprosy patients from Ceará (n = 23), Pernambuco (n = 41), Rio de Janeiro (n = 22) and Rondônia (RO) (n = 78). All skin biopsies yielded SNP-based genotypes, while 48 of the samples (94.1%) yielded complete VNTR genotypes. We evaluated two procedures for extracting M. leprae DNA from Z-N-stained slides: the first including Chelex and the other combining proteinase and sodium dodecyl sulfate. Of the 76 samples processed using the first procedure, 30.2% were positive for 16 or 15 VNTRs, whereas of the 39 samples processed using the second procedure, 28.2% yielded genotypes defined by at least 10 VNTRs. Combined VNTR and SNP analysis revealed large variability in genotypes, but a high prevalence of SNP genotype 4 in the Northeast Region of Brazil. Our observation of two samples from RO with an identical genotype and seven groups with similar genotypes, including four derived from residents of the same state or region, suggest a tendency to form groups according to the origin of the isolates. This study demonstrates the existence of geographically related M. leprae genotypes and that Z-N-stained slides are an alternative source for M. leprae genotyping.

Mannose-binding lectin serum levels in patients with leprosy are influenced by age and MBL2 genotypes.

BACKGROUND: Mannose-binding lectin (MBL) activates the complement system promoting opsonophagocytosis, which could represent an advantage for Mycobacterium leprae, an intracellular pathogen. Therefore, a single nucleotide polymorphism (SNP) in the MBL2 gene associated with low levels of MBL could confer protection against the development of leprosy disease. METHODS: In this study, we investigated SNPs of the MBL2 gene and MBL levels in 228 Brazilian leprosy patients and 232 controls. RESULTS: There were no differences in the frequencies of variant genotypes and haplotypes of MBL2 between patients and controls, or between the different clinical forms of leprosy. In the group of patients with a genotype for high expression of MBL2, those aged>40 years had decreased MBL levels compared to patients aged ≤ 40 years (p = 0.037). CONCLUSION: Our results demonstrate that age could influence the phenotype of MBL2, but no evidence was found for an association of MBL2 polymorphism with susceptibility to leprosy or its clinical forms.