Genetic diversity of Mycobacterium leprae in the state of São Paulo, an area of low-leprosy incidence in Brazil

ABSTRACT Background: Brazil has the second largest number of leprosy cases worldwide, and the state of São Paulo has been considered non-endemic since 2006. Methods: We analyzed 16 variable number tandem repeats loci and three single nucleotide polymorphisms loci of Mycobacterium leprae (M. leprae) in 125 clinical isolates from patients in different municipalities in the state. Results: The clustering pattern of M. leprae indicated that the transmission of leprosy persisted in the state and included scenarios of intra-extra-familial transmission in areas with low endemicity. Conclusions: A significantly active circulation of M. leprae was observed. Therefore, surveillance and control measures must be implemented.

Tuberculosis drug resistance profiling based on machine learning: A literature review

Abstract Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is one of the top 10 causes of death worldwide. Drug-resistant tuberculosis (DR-TB) poses a major threat to the World Health Organization's "End TB" strategy which has defined its target as the year 2035. In 2019, there were close to 0.5 million cases of DRTB, of which 78% were resistant to multiple TB drugs. The traditional culture-based drug susceptibility test (DST - the current gold standard) often takes multiple weeks and the necessary laboratory facilities are not readily available in low-income countries. Whole genome sequencing (WGS) technology is rapidly becoming an important tool in clinical and research applications including transmission detection or prediction of DR-TB. For the latter, many tools have recently been developed using curated database(s) of known resistance conferring mutations. However, documenting all the mutations and their effect is a time-taking and a continuous process and therefore Machine Learning (ML) techniques can be useful for predicting the presence of DR-TB based on WGS data. This can pave the way to an earlier detection of drug resistance and consequently more efficient treatment when compared to the traditional DST.

Heterogeneous persistence of Mycobacterium leprae in oral and nasal mucosa of multibacillary patients during multidrug therapy

BACKGROUND Leprosy is curable by multidrug therapy (MDT) treatment regimen ranging from six to 12 months. The variable levels of tolerance and adherence among patients can, however, result in treatment failure and the emergence of drug-resistant strains. OBJECTIVES Describe the impact of MDT over Mycobacterium leprae viability in patient's oral and nasal mucosa along treatment. METHODS Mycobacterium leprae viability was monitored by quantitative polymerase chain reaction (qPCR) quantification of 16S rRNA in lateral and contralateral scrapings of oral and nasal mucosa of 10 multibacillary patients along the initial five months of treatment. FINDINGS The results demonstrated high heterogenicity of M. leprae viability among patients and between nasal and oral samples. Of six patients who presented good adherence and tolerance to the treatment, only four displayed absence of M. leprae viability in both samples three months after the first MDT dose, while for the other two, the absence of M. leprae viability in the oral and nasal cavities was only detected five months after the first dose. MAIN CONCLUSIONS We concluded that qPCR of 16S rRNA for the determination of M. leprae viability in nasal and oral scraping samples could represent an interesting approach to monitor treatment efficacy.

Spoligotyping, genotyping, and spatial distribution of Mycobacterium bovis in cattle in the state of Bahia, Brazil / Genotipagem por spoligotyping e distribuição espacial de Mycobacterium bovis em bovinos no estado da Bahia, Brasil

Mycobacterium bovis is responsible for bovine and buffalo tuberculosis, an important zoonotic disease with global distribution. The knowledge of the distribution and the precise identification of this disease, including advanced diagnoses such as spoligotyping, allows choosing the best strategies to fight the disease's progress. The present work aimed to investigate mycobacteria's presence, genotype their strains, and evaluate tuberculosis cases' spatial distribution from suggestive lesions in carcasses of bovine and buffalo inspected in slaughterhouses under an official inspection regime in the state of Bahia, Brazil. The study investigated 453,417 animals. Among these, 31 (0.007%) from 17 municipalities were suspected of tuberculosis. Among the culture medium growth, 95% of these were categorized as alcohol-acid resistant bacilli (BAAR). All isolates were subjected to spoligotyping and 95% were confirmed as M. bovis (SB0120, SB0121, SB0852, SB0828, SB0295, SB0881, SB1648, SB6119, SB0140, SB1055). The strain SB0120 was the most prevalent, and this profile has been described in cases of human tuberculosis by M. bovis, highlighting the zoonotic potential of this profile. This study also identified strains never reported in Bahia, highlighting a distinctive pattern from other parts of Brazil, besides mixed infections. Besides, to identify strains never before described in the state, highlighting a distinctive pattern in Brazil (SB6119 and SB0852, respectively). An unpublished profile was identified and inserted in the international database (Mbovis.org), named SB2715.(AU)

O Mycobacterium bovis é o responsável pela tuberculose bovina e bubalina, doença zoonótica importante e com distribuição global. O conhecimento da distribuição e a identificação precisa dessa enfermidade, incluindo diagnósticos mais avançados como o spoligotyping, permite escolher as melhores estratégias de combate ao avanço da doença. O presente trabalho objetivou investigar a presença de micobactérias, genotipar suas estirpes e avaliar a distribuição espacial dos casos de tuberculose a partir de lesões sugestivas nas carcaças de bovinos e bubalinos inspecionadas em frigoríficos sob regime de inspeção oficial no estado da Bahia. Foram investigados 453.417 animais dentre os quais 31 (0,007%) foram suspeitos de doença e provenientes de 17 municípios. Após o crescimento em meio de cultura, 95% foram categorizados como bacilos álcool-ácido resistentes (BAAR). Todos os isolados foram submetidos à spoligotyping e 95% foram confirmados M. bovis (SB0120, SB0121, SB0852, SB0828, SB0295, SB0881, SB1648, SB6119, SB0140, SB1055). A cepa SB0120 foi a mais prevalente e este perfil vem sendo descrito na literatura com casos de tuberculose humana por M. bovis ressaltando o potencial zoonótico deste perfil. Este estudo também identificou cepas nunca relatadas no estado da Bahia, destacando um padrão distinto de outras partes do Brasil, além da existência de infecções mistas. Permitiu ainda relatar linhagens nunca antes descritas no estado com destaque para um padrão novo no Brasil (SB6119 e SB0852 respectivamente). Um perfil inédito identificado foi identificado e inserido no banco de dados internacional (Mbovis.org), nomeado SB2715.(AU)

Spoligotyping, genotyping, and spatial distribution of Mycobacterium bovis in cattle in the state of Bahia, Brazil

ABSTRACT: Mycobacterium bovis is responsible for bovine and buffalo tuberculosis, an important zoonotic disease with global distribution. The knowledge of the distribution and the precise identification of this disease, including advanced diagnoses such as spoligotyping, allows choosing the best strategies to fight the diseases progress. The present work aimed to investigate mycobacterias presence, genotype their strains, and evaluate tuberculosis cases spatial distribution from suggestive lesions in carcasses of bovine and buffalo inspected in slaughterhouses under an official inspection regime in the state of Bahia, Brazil. The study investigated 453,417 animals. Among these, 31 (0.007%) from 17 municipalities were suspected of tuberculosis. Among the culture medium growth, 95% of these were categorized as alcohol-acid resistant bacilli (BAAR). All isolates were subjected to spoligotyping and 95% were confirmed as M. bovis (SB0120, SB0121, SB0852, SB0828, SB0295, SB0881, SB1648, SB6119, SB0140, SB1055). The strain SB0120 was the most prevalent, and this profile has been described in cases of human tuberculosis by M. bovis, highlighting the zoonotic potential of this profile. This study also identified strains never reported in Bahia, highlighting a distinctive pattern from other parts of Brazil, besides mixed infections. Besides, to identify strains never before described in the state, highlighting a distinctive pattern in Brazil (SB6119 and SB0852, respectively). An unpublished profile was identified and inserted in the international database (Mbovis.org), named SB2715.

RESUMO: O Mycobacterium bovis é o responsável pela tuberculose bovina e bubalina, doença zoonótica importante e com distribuição global. O conhecimento da distribuição e a identificação precisa dessa enfermidade, incluindo diagnósticos mais avançados como o spoligotyping, permite escolher as melhores estratégias de combate ao avanço da doença. O presente trabalho objetivou investigar a presença de micobactérias, genotipar suas estirpes e avaliar a distribuição espacial dos casos de tuberculose a partir de lesões sugestivas nas carcaças de bovinos e bubalinos inspecionadas em frigoríficos sob regime de inspeção oficial no estado da Bahia. Foram investigados 453.417 animais dentre os quais 31 (0,007%) foram suspeitos de doença e provenientes de 17 municípios. Após o crescimento em meio de cultura, 95% foram categorizados como bacilos álcool-ácido resistentes (BAAR). Todos os isolados foram submetidos à spoligotyping e 95% foram confirmados M. bovis (SB0120, SB0121, SB0852, SB0828, SB0295, SB0881, SB1648, SB6119, SB0140, SB1055). A cepa SB0120 foi a mais prevalente e este perfil vem sendo descrito na literatura com casos de tuberculose humana por M. bovis ressaltando o potencial zoonótico deste perfil. Este estudo também identificou cepas nunca relatadas no estado da Bahia, destacando um padrão distinto de outras partes do Brasil, além da existência de infecções mistas. Permitiu ainda relatar linhagens nunca antes descritas no estado com destaque para um padrão novo no Brasil (SB6119 e SB0852 respectivamente). Um perfil inédito identificado foi identificado e inserido no banco de dados internacional (Mbovis.org), nomeado SB2715.

Comparative study of RFLP-IS6110 and MIRU-VNTR from Mycobacterium tuberculosis isolated in the state of Minas Gerais, Brazil

Abstract DNA genotyping of Mycobacterium tuberculosis has been widely applied in the understanding of disease transmission in many countries. The purpose of this study was to genotype the strains of M. tuberculosis isolated in patients with new tuberculosis (TB) cases in Minas Gerais, as well as to compare the similarity, discriminatory power, and agreement of the clusters between the IS6110 Restriction Fragment Length Polymorfism (RFLP) and 12 loci Variable Number Tandem Repeat - Mycobacterial Interspersed Repetitive Units (MIRU-VNTR) techniques. It was observed that 32% (66/204) of the isolated strains in the RFLP-IS6110 and 50.9% (104/204) of the isolated strains in the MIRU-VNTR presented a similarity of equal to or above 85%. The RFLP-IS6110 and MIRU-VNTR proved to contain a high discriminatory power. The similarity index resulting from the RFLP showed no recent transmission. Good agreement was observed between the techniques when clusters were detected; however, the best epidemiological relationship was found when using the RFLP-IS6110.

Mixed infection and two different resistance profiles of Treponema pallidum to macrolides identified in some clinical specimens of the same patient / Infecção mista e resistência diferente a macrolídeos em Treponema pallidum identificadas por variadas espécimes clínicos em um mesmo paciente

Syphilis represents a global public health problem. The resistance of Treponema pallidum to macrolides is related to the mutation in the 23S rRNA gene (A2058G). We reported a case of secondary syphilis in a 52-year-old man presenting two profiles: the first one of susceptibility, and the other one of resistance, when we analyzed the 23S rRNA gene sequence from two different clinical specimens of the same infectious episode. DNA from T. pallidum from skin biopsy presented resistance profile, whereas T. pallidum DNA from blood presented a profile of susceptibility to macrolides. These results suggest it was mixed infection or reinfection.

A sífilis representa um problema de saúde pública mundial. A resistência de Treponema pallidum aos macrolídeos está relacionada à mutação no gene 23S rRNA (A2058G). Relatamos um caso de sífilis secundária, em um homem de 52 anos, com um perfil de suscetibilidade e outro de resistência, ao analisarmos a sequência do gene 23S rRNA de dois espécimes clínicos diferentes, do mesmo episódio infeccioso. A amostra de DNA de T. pallidum proveniente de raspado dérmico da lesão apresentou um perfil de resistência, enquanto aquele que derivou de sangue apresentou perfil de suscetibilidade aos macrolídeos. Esses resultados sugerem tratar-se de infecção mista ou de reinfecção.

Risk factors for human Mycobacterium bovis infections in an urban area of Brazil

BACKGROUND The World Health Organization (WHO) has classified human zoonotic tuberculosis (TB) due to Mycobacterium bovis as a neglected issue in the developing world. In a recent cross-sectional study in Brazil, three of 189 TB patients presented with a coinfection of M. bovis and M. tuberculosis and were selected as cases for this study. OBJECTIVE The aim was to evaluate risk factors (RF) for zoonotic TB in an urban area of Brazil in order to guide preventive programmes. METHODS A matched case-control study was carried out nested within a cross-sectional study. For each of the three cases, 14 age- and sex-matched controls (TB due to M. tuberculosis) were selected. FINDINGS Zoonotic potential exposures (ZE) and extrapulmonary TB (EPTB) were independently associated with zoonotic TB in multivariate analyses. CONCLUSIONS ZE by occupation and consumption of raw milk and derivative products that place individuals in direct and indirect contact with animals and their excretions/secretions increase the risk for zoonotic TB in Brazil, especially among those with EPTB. Therefore, measures such as efficient control of bovine TB, distribution of pasteurised milk and its derivative products, and the diagnosis and monitoring of zoonotic TB in humans are essential steps, especially in developing countries where bovine TB is enzootic, and further studies are necessary.

Whole genome sequence of Mycobacterium kansasii isolates of the genotype 1 from Brazilian patients with pulmonary disease demonstrates considerable heterogeneity

Mycobacterium kansasii is an opportunistic pathogen and one of the most commonly encountered species in individuals with lung disease. We here report the complete genome sequence of 12 clinical isolates of M. kansasii from patients with pulmonary disease in Brazil.

Tuberculosis associated factors caused by Mycobacterium tuberculosis of the RDRio genotype

BACKGROUND Tuberculosis (TB) continues to be a disease that affects many countries around the world, including Brazil. Recently, a subtype of Latin American-Mediterranean family strain was identified and characterised by RDRio. The strain has been associated with different characteristics of the disease. OBJECTIVES In the present study we investigated the association of epidemiological, clinical, radiological and bacteriological variables with pulmonary tuberculosis caused by RDRioMycobacterium tuberculosis strain in large regions of São Paulo. METHODS We conducted a cross-sectional study in 530 patients with pulmonary tuberculosis, diagnosed using sputum culture, from two regions of the São Paulo state in Brazil. The samples were brought to São Paulo reference laboratories for epidemiological, clinical, radiological and bacteriological analyses, and the data were obtained from a TB notification system. RDRio genotyping and Spoligotyping of the samples were performed. For the analysis of the categorical variables we used the chi-square test or the Fisher’s exact test, and for the continuous variables, the Mann-Whitney test. In addition, a logistic regression was used for multivariate analysis. Differences with p < 0.05 were considered significant. FINDINGS The RDRio deletion was identified in 152 (28.7%) samples. In the univariate analysis, both the age groups above 25 years and alcohol consumption were associated with the RDRio deletion. The multivariate analysis confirmed the association of the RDRio deletion with the age groups: 25-35 years old [OR: 2.28 (1.02-5.07; p = 0.04)] and 36-60 years old (OR: 2.36 (1.11-5.05); p = 0.03], and also with alcohol consumption [OR: 1.63 (1.05-2.54); p = 0,03]. MAIN CONCLUSIONS In this study, we identified new factors associated with the M. tuberculosis of the RDRio deletion strains infection.

Clinical and epidemiological profiles of individuals with drug-resistant tuberculosis

Drug-resistant tuberculosis (TB) is a growing global threat. Approximately 450,000 people developed multidrug-resistant TB worldwide in 2012 and an estimated 170,000 people died from the disease. This paper describes the sociodemographic, clinical-epidemiological and bacteriological aspects of TB and correlates these features with the distribution of anti-TB drug resistance. Mycobacterium tuberculosis (MT) cultures and drug susceptibility testing were performed according to the BACTEC MGIT 960 method. The results demonstrated that MT strains from individuals who received treatment for TB and people who were infected with human immunodeficiency virus were more resistant to TB drugs compared to other individuals (p < 0.05). Approximately half of the individuals received supervised treatment, but most drug-resistant cases were positive for pulmonary TB and exhibited positive acid-fast bacilli smears, which are complicating factors for TB control programs. Primary healthcare is the ideal level for early disease detection, but tertiary healthcare is the most common entry point for patients into the system. These factors require special attention from healthcare managers and professionals to effectively control and monitor the spread of TB drug-resistant cases.

In house reverse membrane hybridisation assay versus GenoType MTBDRplus and their performance to detect mutations in the genes rpoB, katG and inhA

Drug-resistant tuberculosis (TB) threatens global TB control and is a major public health concern in several countries. We therefore developed a multiplex assay (LINE-TB/MDR) that is able to identify the most frequent mutations related to rifampicin (RMP) and isoniazid (INH) resistance. The assay is based on multiplex polymerase chain reaction, membrane hybridisation and colorimetric detection targeting of rpoB and katG genes, as well as the inhA promoter, which are all known to carry specific mutations associated with multidrug-resistant TB (MDR-TB). The assay was validated on a reference panel of 108 M. tuberculosis isolates that were characterised by the proportion method and by DNA sequencing of the targets. When comparing the performance of LINE-TB/MDR with DNA sequencing, the sensitivity, specificity and agreement were 100%, 100% and 100%, respectively, for RMP and 77.6%, 90.6% and 88.9%, respectively, for INH. Using drug sensibility testing as a reference standard, the performance of LINE-TB/MDR regarding sensitivity, specificity and agreement was 100%, 100% and 100% (95%), respectively, for RMP and 77%, 100% and 88.7% (82.2-95.1), respectively, for INH. LINE-TB/MDR was compared with GenoType MTBDRplus for 65 isolates, resulting in an agreement of 93.6% (86.7-97.5) for RIF and 87.4% (84.3-96.2) for INH. LINE-TB/MDR warrants further clinical validation and may be an affordable alternative for MDR-TB diagnosis.

Direct detection of Mycobacterium tuberculosis complex in bovine and bubaline tissues through nested-PCR

Post-mortem bacterial culture and specific biochemical tests are currently performed to characterize the etiologic agent of bovine tuberculosis. Cultures take up to 90 days to develop. A diagnosis by molecular tests such as PCR can provide fast and reliable results while significantly decreasing the time of confirmation. In the present study, a nested-PCR system, targeting rv2807, with conventional PCR followed by real-time PCR, was developed to detect Mycobacterium tuberculosis complex (MTC) organisms directly from bovine and bubaline tissue homogenates. The sensitivity and specificity of the reactions were assessed with DNA samples extracted from tuberculous and non-tuberculous mycobacteria, as well as other Actinomycetales species and DNA samples extracted directly from bovine and bubaline tissue homogenates. Regarding the analytical sensitivity, DNA of the M. bovis AN5 strain was detected up to 1.5 pg by nested-PCR, whereas DNA of M. tuberculosis H37Rv strain was detected up to 6.1 pg. The nested-PCR system showed 100% analytical specificity for MTC when tested with DNA of reference strains of non-tuberculous mycobacteria and closely-related Actinomycetales. A clinical sensitivity level of 76.7% was detected with tissues samples positive for MTC by means of the culture and conventional PCR. A clinical specificity of 100% was detected with DNA from tissue samples of cattle with negative results in the comparative intradermal tuberculin test. These cattle exhibited no visible lesions and were negative in the culture for MTC. The use of the nested-PCR assay to detect M. tuberculosis complex in tissue homogenates provided a rapid diagnosis of bovine and bubaline tuberculosis.

Múltiplas estirpes de isolados de Mycobacteriumbovis identificados por tipagem molecular em bovinos abatidos em matadouros-frigoríficos / Multiple strains of Mycobacterium bovis isolates identified by molecular typing of bovine animals slaughtered in slaughterhouse

O objetivo do presente trabalho foi utilizar métodos bacteriológicos e moleculares para a identificação do Mycobacteriumbovis em lesões observadas em carcaças de bovinos durante a inspeção postmortem de rotina em matadouros-frigoríficos com serviço de inspeção oficial. Foi acompanhado o abate e a inspeção de 825.394 bovinos, sadios ao exame ante mortem pelo serviço de inspeção oficial em dez matadouros-frigoríficos do estado da Bahia. Carcaça de 180 bovinos apresentaram lesões sugestivas de tuberculose e por outras linfadenites. No isolamento bacteriano, 25 amostras apresentaram crescimento disgônico de colônias de coloração creme-amareladas em meio de cultura Stonebrink-Leslie. Desses isolados, 14 foram identificados como M. bovis PCR multiplex e pela técnica do spoligotyping foram discriminados oito diferentes espoligotipos do M. bovis, sendo sete descritos na literatura e um novo spoligotipo sem descrição anterior. O espoligotipo majoritário foi o SB0121, com cinco amostras, sendo descrito no Brasil e em outros países, seguidos por dois clusters, SB295 e SB1055, com dois isolados cada. O espoligotipo SB1145 e SB1648 foram referidos apenas no Brasil e Dinamarca, respectivamente. O espoligotipo SB140 já foi encontrado no Brasil, Argentina, Uruguai e Paraguai. Estes resultados demonstram que os espoligotipos obtidos são compartilhados, até o momento, entre estados brasileiros e entre países da América Latina e Europa. Sendo assim, a discriminação molecular de isolados de M. bovis através do Spoligotyping constitui-se numa ferramenta para estudos epidemiológicos da tuberculose bovina no Estado da Bahia.

The aim of this study was to use bacteriological and molecular methods to identify Mycobacteriumbovis in lesions observed in cattle carcasses during routine post-mortem inspection in slaughterhouses with official inspection service. It was accompanied the slaughter and inspection of 825,394 cattle, healthy ante mortem examination by the official inspection service in ten slaughterhouses in the state of Bahia. Carcasses of 180 cattle presented lesions suggestive of tuberculosis and other lymphadenitis. In bacterial isolation, 25 samples showed dysgonic growth of colonies of creamy-yellow in medium-Stonebrink Leslie. From these isolates, 14 were identified as M. bovis and the multiplex PCR technique spoligotyping was discriminated against eight different spoligotypes of M. bovis, seven previously described in the literature and a new spoligotypes without former description. The major spoligotypes was SB0121, with five samples which has been described in Brazil and other countries, followed by two clusters, SB295 and SB1055, with two isolates each. The SB1145 and SB1648 spoligotypes were reported only in Brazil and Denmark, respectively. The spoligotypes SB140 has been found in Brazil, Argentina, Uruguay and Paraguay. These results demonstrate that the spoligotypes obtained are shared, so far, among Brazilian states and among Latin America and Europe. Thus, molecular discrimination of isolates of M. bovis by Spoligotyping constitutes a tool for epidemiological studies of bovine tuberculosis in the state of Bahia.

Nontuberculous mycobacteria in respiratory samples from patients with pulmonary tuberculosis in the state of Rondonia, Brazil

The main cause of pulmonary tuberculosis (TB) is infection with Mycobacterium tuberculosis (MTB). We aimed to evaluate the contribution of nontuberculous mycobacteria (NTM) to pulmonary disease in patients from the state of Rondônia using respiratory samples and epidemiological data from TB cases. Mycobacterium isolates were identified using a combination of conventional tests, polymerase chain reaction-based restriction enzyme analysis of hsp65 gene and hsp65 gene sequencing. Among the 1,812 cases suspected of having pulmonary TB, 444 yielded bacterial cultures, including 369 cases positive for MTB and 75 cases positive for NTM. Within the latter group, 14 species were identified as Mycobacterium abscessus, Mycobacterium avium, Mycobacterium fortuitum, Mycobacterium intracellulare, Mycobacterium gilvum, Mycobacterium gordonae, Mycobacterium asiaticum, Mycobacterium tusciae, Mycobacterium porcinum, Mycobacterium novocastrense, Mycobacterium simiae, Mycobacterium szulgai, Mycobacterium phlei and Mycobacterium holsaticum and 13 isolates could not be identified at the species level. The majority of NTM cases were observed in Porto Velho and the relative frequency of NTM compared with MTB was highest in Ji-Paraná. In approximately half of the TB subjects with NTM, a second sample containing NTM was obtained, confirming this as the disease-causing agent. The most frequently observed NTM species were M. abscessus and M. avium and because the former species is resistant to many antibiotics and displays unsatisfactory cure rates, the implementation of rapid identification of mycobacterium species is of considerable importance.

Tuberculosis patients co-infected with Mycobacterium bovis and Mycobacterium tuberculosis in an urban area of Brazil

In this cross-sectional study, mycobacteria specimens from 189 tuberculosis (TB) patients living in an urban area in Brazil were characterised from 2008-2010 using phenotypic and molecular speciation methods (pncA gene and oxyR pseudogene analysis). Of these samples, 174 isolates simultaneously grew on Löwenstein-Jensen (LJ) and Stonebrink (SB)-containing media and presented phenotypic and molecular profiles of Mycobacterium tuberculosis, whereas 12 had molecular profiles of M. tuberculosis based on the DNA analysis of formalin-fixed paraffin wax-embedded tissue samples (paraffin blocks). One patient produced two sputum isolates, the first of which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, and the second of which only grew on SB media and presented phenotypic profiles of Mycobacterium bovis. One patient provided a bronchial lavage isolate, which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, but had molecular profiles of M. bovis from paraffin block DNA analysis, and one sample had molecular profiles of M. tuberculosis and M. bovis identified from two distinct paraffin blocks. Moreover, we found a low prevalence (1.6%) of M. bovis among these isolates, which suggests that local health service procedures likely underestimate its real frequency and that it deserves more attention from public health officials.

Mycobacterium leprae in six-banded (Euphractus sexcinctus) and nine-banded armadillos (Dasypus novemcinctus) in Northeast Brazil

Human beings are the main reservoir of the causative agent of leprosy, Mycobacterium leprae. In the Americas, nine-banded armadillos (Dasypus novemcinctus) also act as a reservoir for the bacillus. In the state of Ceará (CE), which is located in Northeast Brazil and is an endemic area of leprosy, there are several species of armadillos, including D. novemcinctus and Euphractus sexcinctus (six-banded armadillo). Contact between humans and armadillos occur mainly through hunting, cleaning, preparing, cooking and eating. This study identified M. leprae DNA in the two main species of armadillos found in Northeast Brazil. A total of 29 wild armadillos (27 D. novemcinctus and 2 E. sexcinctus) were captured in different environments of CE countryside. Samples from the ear, nose, liver and spleen from each of these animals were tested by a nested M. leprae-specific repetitive element polymerase chain reaction assay. The samples that tested positive were confirmed by DNA sequencing. M. leprae was detected in 21% (6/29) of the animals, including five D. novemcinctus and one E. sexcinctus. This is the first Brazilian study to identify the presence of a biomarker of M. leprae in wild armadillos (D. novemcinctus and E. sexcinctus) in a leprosy hyperendemic area where there is continuous contact between humans and armadillos.

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.

Spoligotyping and variable number tandem repeat analysis of Mycobacterium bovis isolates from cattle in Brazil

We performed spoligotyping and 12-mycobacterial interspersed repetitive unit-variable number tandem repeats (MIRU-VNTRs) typing to characterise Mycobacterium bovis isolates collected from tissue samples of bovines with lesions suggestive for tuberculosis during slaughter inspection procedures in abattoirs in Brazil. High-quality genotypes were obtained with both procedures for 61 isolates that were obtained from 185 bovine tissue samples and all of these isolates were identified as M. bovis by conventional identification procedures. On the basis of the spoligotyping, 53 isolates were grouped into nine clusters and the remaining eight isolates were unique types, resulting in 17 spoligotypes. The majority of the Brazilian M. bovis isolates displayed spoligotype patterns that have been previously observed in strains isolated from cattle in other countries. MIRU-VNTR typing produced 16 distinct genotypes, with 53 isolates forming eight of the groups, and individual isolates with unique VNTR profiles forming the remaining eight groups. The allelic diversity of each VNTR locus was calculated and only two of the 12-MIRU-VNTR loci presented scores with either a moderate (0.4, MIRU16) or high (0.6, MIRU26) discriminatory index (h). Both typing methods produced similar discriminatory indexes (spoligotyping h = 0.85; MIRU-VNTR h = 0.86) and the combination of the two methods increased the h value to 0.94, resulting in 29 distinct patterns. These results confirm that spoligotyping and VNTR analysis are valuable tools for studying the molecular epidemiology of M. bovis infections in Brazil.

Genetic polymorphisms of NAT2, CYP2E1 and GST enzymes and the occurrence of antituberculosis drug-induced hepatitis in Brazilian TB patients

Isoniazid (INH), one of the most important drugs used in antituberculosis (anti-TB) treatment, is also the major drug involved in hepatotoxicity. Differences in INH-induced toxicity have been attributed to genetic variability at several loci, such as NAT2, CYP2E1, GSTM1 and GSTT1, that code for drug-metabolising enzymes. Our goal was to examine the polymorphisms in these enzymes as susceptibility factors to anti-TB drug-induced hepatitis in Brazilian individuals. In a case-control design, 167 unrelated active tuberculosis patients from the University Hospital of the Federal University of Rio de Janeiro, Brazil, were enrolled in this study. Patients with a history of anti-TB drug-induced acute hepatitis (cases with an increase to 3 times the upper limit of normal serum transaminases and symptoms of hepatitis) and patients with no evidence of anti-TB hepatic side effects (controls) were genotyped for NAT2, CYP2E1, GSTM1 and GSTT1 polymorphisms. Slow acetylators had a higher incidence of hepatitis than intermediate/rapid acetylators [22 percent (18/82) vs. 9.8 percent (6/61), odds ratio (OR), 2.86, 95 percent confidence interval (CI), 1.06-7.68, p = 0.04). Logistic regression showed that slow acetylation status was the only independent risk factor (OR 3.59, 95 percent CI, 2.53-4.64, p = 0.02) for the occurrence of anti-TB drug-induced hepatitis during anti-TB treatment with INH-containing schemes in Brazilian individuals.