Detection of mycobacteria in coalho cheese sold in Northeastern Brazil / Detecção de micobactérias em queijo de coalho comercializado no Nordeste do Brasil

The aim of the present study was to detect and identify Mycobaterium spp. in 50 samples of coalho cheese sold in the Northeast region of Brazil. Of the 50 analyzed samples, 35 were produced by the artisanal process, using raw milk, and 15 originated from industrialized establishments that pasteurize milk. Conventional and real-time nested PCR for the rv2807 gene of the M. tuberculosis complex was performed directly from the 50 analyzed samples. Samples of coalho cheese were grown simultaneously in Stonebrink medium, and conventional PCR was performed from the bacterial isolates with primers that flank differentiation region 4 (DR4), specific to M. bovis, mb400F. Bacterial isolates negative by PCR for RD4 were subjected to PCR for hsp65 of Mycobacterium spp., with subsequent DNA sequencing. The cultures were negative for the M. tuberculosis complex, but two samples (4%) from the artisanal process (with raw milk) exhibited identity with hsp65 of Mycobacterium lehmanii (Sequence ID: KY933786.1, identities: 312/363 [86%]); and Mycobacterium rutilum (sequence ID: LT629971.1, identities: 331/371 [89%]), showing to be indicative environmental contamination. Non-tuberculous mycobacteria are emergent and ubiquitous microorganisms; therefore, they deserve greater attention in the cheese production chain, both in terms of Good Agricultural Practices (GAP)

O presente estudo teve como objetivo detectar e identificar Mycobaterium spp. em 50 amostras de queijo de coalho comercializados na região Nordeste do Brasil. Das 50 amostras analisadas, 35 foram produzidas pelo processo artesanal, com leite cru e 15 provenientes de estabelecimentos industrializados que realizam a pasteurização do leite. Nested-PCR convencional e em tempo real para o gene rv2807 do Complexo M. tuberculosis foi realizada diretamente das cinquenta amostras analisadas. Paralelamente, as amostras de queijo de coalho foram cultivadas em meio de Stonebrink e dos isolados bacterianos foi realizada PCR convencional com iniciadores que flanqueiam a região de diferenciação 4 (RD4), específica de M. bovis, mb400F. Os isolados bacterianos negativos na PCR para RD4 foram submetidos à PCR para hsp65 de Mycobacterium spp., com posterior sequenciamento do DNA. As culturas mostraram-se negativas para o Complexo M. tuberculosis, porém duas amostras oriundas do processo artesanal (com leite cru) (4%) apresentaram identidade com hsp65 de Mycobacterium lehmanii (ID da sequência: KY933786.1, identidades: 312/363 [86%]); e Mycobacterium rutilum (ID da sequência: LT629971.1, identidades: 331/371 [89%]), apontando-se como indicativos de contaminação ambiental. As micobactérias-não-tuberculosas (MNT) são microrganismos emergentes e de natureza ubíqua. Devido a essas características merecem maior atenção na cadeia produtiva de queijos, tanto nas boas práticas agropecuárias (BPAs) quanto nas boas práticas de fabricação de alimentos (BPFs).

Detection of mycobacteria in coalho cheese sold in Northeastern Brazil / Detecção de micobactérias em queijo de coalho comercializado no Nordeste do Brasil

The aim of the present study was to detect and identify Mycobaterium spp. in 50 samples of coalho cheese sold in the Northeast region of Brazil. Of the 50 analyzed samples, 35 were produced by the artisanal process, using raw milk, and 15 originated from industrialized establishments that pasteurize milk. Conventional and real-time nested PCR for the rv2807 gene of the M. tuberculosis complex was performed directly from the 50 analyzed samples. Samples of coalho cheese were grown simultaneously in Stonebrink medium, and conventional PCR was performed from the bacterial isolates with primers that flank differentiation region 4 (DR4), specific to M. bovis, mb400F. Bacterial isolates negative by PCR for RD4 were subjected to PCR for hsp65 of Mycobacterium spp., with subsequent DNA sequencing. The cultures were negative for the M. tuberculosis complex, but two samples (4%) from the artisanal process (with raw milk) exhibited identity with hsp65 of Mycobacterium lehmanii (Sequence ID: KY933786.1, identities: 312/363 [86%]); and Mycobacterium rutilum (sequence ID: LT629971.1, identities: 331/371 [89%]), showing to be indicative environmental contamination. Non-tuberculous mycobacteria are emergent and ubiquitous microorganisms; therefore, they deserve greater attention in the cheese production chain, both in terms of Good Agricultural Practices (GAP)(AU)

O presente estudo teve como objetivo detectar e identificar Mycobaterium spp. em 50 amostras de queijo de coalho comercializados na região Nordeste do Brasil. Das 50 amostras analisadas, 35 foram produzidas pelo processo artesanal, com leite cru e 15 provenientes de estabelecimentos industrializados que realizam a pasteurização do leite. Nested-PCR convencional e em tempo real para o gene rv2807 do Complexo M. tuberculosis foi realizada diretamente das cinquenta amostras analisadas. Paralelamente, as amostras de queijo de coalho foram cultivadas em meio de Stonebrink e dos isolados bacterianos foi realizada PCR convencional com iniciadores que flanqueiam a região de diferenciação 4 (RD4), específica de M. bovis, mb400F. Os isolados bacterianos negativos na PCR para RD4 foram submetidos à PCR para hsp65 de Mycobacterium spp., com posterior sequenciamento do DNA. As culturas mostraram-se negativas para o Complexo M. tuberculosis, porém duas amostras oriundas do processo artesanal (com leite cru) (4%) apresentaram identidade com hsp65 de Mycobacterium lehmanii (ID da sequência: KY933786.1, identidades: 312/363 [86%]); e Mycobacterium rutilum (ID da sequência: LT629971.1, identidades: 331/371 [89%]), apontando-se como indicativos de contaminação ambiental. As micobactérias-não-tuberculosas (MNT) são microrganismos emergentes e de natureza ubíqua. Devido a essas características merecem maior atenção na cadeia produtiva de queijos, tanto nas boas práticas agropecuárias (BPAs) quanto nas boas práticas de fabricação de alimentos (BPFs).(AU)

Mycobacteria in Minas cheese commercialized in open fairs in São Paulo, Brazil / Mycobacteria em queijos tipo Minas comercializados em feiras-livres de São Paulo, Brasil

Mycobacterium bovis is the causative agent of bovine tuberculosis, a disease that affects dairy herds throughout the Brazilian territory, constituting a neglected zoonosis transmitted by raw milk and its derivatives. In this study, we evaluated the presence of M. bovis and other mycobacteria in Minas cheese obtained from open fairs in the city of São Paulo between 2012 and 2013. Samples (n = 133) were decontaminated using hexa-cetylpyridinium chloride and seeded on StonebrinkLeslie medium. The isolates were submitted to molecular identification by TB Multiplex PCR targeting the 16S rRNA gene and amplicon nucleotide sequencing. From 16 cheese samples (12%), we obtained 26 putative colonies of Mycobacterium spp., none of which belonged to any of the Mycobacterium tuberculosis, Mycobacterium avium, or Mycobacterium intracellulare complexes. Phylogenetic analysis showed that sample sequences were grouped in a clade that includes only non-tuberculous mycobacteria with proximity to sequences obtained from Mycobacterium novocastrense (3 sequences), Mycobacterium holsaticum (1 sequence), andMycobacterium elephantis (2 sequences). Although no epidemiological evidence was found regarding the importance of oral transmission of mycobacteria in healthy people, their importance in the immunosuppressed population remains uncertain.

Mycobacterium bovis é o agente da tuberculose bovina, doença que acomete o rebanho em todo território brasileiro e é uma negligenciada zoonose transmitida pelo leite e seus derivados. Este trabalho avaliou a presença de M. bovis e outras micobactérias, em queijo minas meia-cura, obtidos em feiras-livres na cidade de São Paulo, entre os anos de 2012 e 2013. As amostras (n = 133) foram descontaminadas pelo método HPC (hexa-cetyl-pyridinium chloride) e semeadas em meio Stonebrink Leslie. Os isolados foram submetidos à identificação molecular por PCR TB multiplex, pesquisando-se o gene 16S rRNA, e ao sequenciamento nucleotídico. Dezesseis amostras (12%) possuiam 26 colônias sugestivas de Mycobacterium spp., mas nenhuma delas pertencia aos complexos Mycobacterium tuberculosis, Mycobacterium avium e Mycobacterium intracellulare. A análise filogenética mostrou que todas as amostras estavam agrupadas em clados que incluem apenas micobactérias não tuberculosas (MNT), sendo que algumas possuiam proximidade com sequências obtidas de Mycobacterium novocastrense (3 sequências), Mycobacterium hosaticum (1 sequência) e Mycobacterium elephantis (2 sequências). Embora no momento não haja evidência epidemiológica da importância da transmissão oral das micobactérias pra indivíduos saudáveis, sua importância na população imunossuprimida ainda é incerta.

Mycobacteria in Minas cheese commercialized in open fairs in São Paulo, Brazil / Mycobacteria em queijos tipo Minas comercializados em feiras-livres de São Paulo, Brasil

Mycobacterium bovis is the causative agent of bovine tuberculosis, a disease that affects dairy herds throughout the Brazilian territory, constituting a neglected zoonosis transmitted by raw milk and its derivatives. In this study, we evaluated the presence of M. bovis and other mycobacteria in Minas cheese obtained from open fairs in the city of São Paulo between 2012 and 2013. Samples (n = 133) were decontaminated using hexa-cetylpyridinium chloride and seeded on Stonebrink­Leslie medium. The isolates were submitted to molecular identification by TB Multiplex PCR targeting the 16S rRNA gene and amplicon nucleotide sequencing. From 16 cheese samples (12%), we obtained 26 putative colonies of Mycobacterium spp, none of which belonged to any of the Mycobacterium tuberculosis, Mycobacterium avium, or Mycobacterium intracellulare complexes. Phylogenetic analysis showed that sample sequences were grouped in a clade that includes only non-tuberculous mycobacteria with proximity to sequences obtained from Mycobacterium novocastrense (3 sequences), Mycobacterium holsaticum (1 sequence), and Mycobacterium elephantis (2 sequences). Although no epidemiological evidence was found regarding the importance of oral transmission of mycobacteria in healthy people, their importance in the immunosuppressed population remains uncertain.(AU)

Mycobacterium bovis é o agente da tuberculose bovina, doença que acomete o rebanho em todo território brasileiro e é uma negligenciada zoonose transmitida pelo leite e seus derivados. Este trabalho avaliou a presença de M. bovise outras micobactérias, em queijo minas meia-cura, obtidos em feiras-livres na cidade de São Paulo, entre os anos de 2012 e 2013. As amostras (n = 133) foram descontaminadas pelo método HPC (hexa-cetyl-pyridinium chloride) e semeadas em meio Stonebrink Leslie. Os isolados foram submetidos à identificação molecular por PCR TB multiplex, pesquisando-se o gene 16S rRNA, e ao sequenciamento nucleotídico. Dezesseis amostras (12%) possuiam 26 colônias sugestivas de Mycobacterium spp, mas nenhuma delas pertencia aos complexos Mycobacterium tuberculosis, Mycobacterium avium e Mycobacterium intracellulare. A análise filogenética mostrou que todas as amostras estavam agrupadas em clados que incluem apenas micobactérias não tuberculosas (MNT), sendo que algumas possuiam proximidade com sequências obtidas de Mycobacterium novocastrense (3 sequências), Mycobacterium hosaticum(1 sequência) e Mycobacterium elephantis (2 sequências). Embora no momento não haja evidência epidemiológica da importância da transmissão oral das micobactérias pra indivíduos saudáveis, sua importância na população imunossuprimida ainda é incerta.(AU)

Mycobacteria in Minas cheese commercialized in open fairs in São Paulo, Brazil / Mycobacteria em queijos tipo Minas comercializados em feiras-livres de São Paulo, Brasil

Mycobacterium bovis is the causative agent of bovine tuberculosis, a disease that affects dairy herds throughout the Brazilian territory, constituting a neglected zoonosis transmitted by raw milk and its derivatives. In this study, we evaluated the presence of M. bovis and other mycobacteria in Minas cheese obtained from open fairs in the city of São Paulo between 2012 and 2013. Samples (n = 133) were decontaminated using hexa-cetylpyridinium chloride and seeded on StonebrinkLeslie medium. The isolates were submitted to molecular identification by TB Multiplex PCR targeting the 16S rRNA gene and amplicon nucleotide sequencing. From 16 cheese samples (12%), we obtained 26 putative colonies of Mycobacterium spp., none of which belonged to any of the Mycobacterium tuberculosis, Mycobacterium avium, or Mycobacterium intracellulare complexes. Phylogenetic analysis showed that sample sequences were grouped in a clade that includes only non-tuberculous mycobacteria with proximity to sequences obtained from Mycobacterium novocastrense (3 sequences), Mycobacterium holsaticum (1 sequence), andMycobacterium elephantis (2 sequences). Although no epidemiological evidence was found regarding the importance of oral transmission of mycobacteria in healthy people, their importance in the immunosuppressed population remains uncertain.

Mycobacterium bovis é o agente da tuberculose bovina, doença que acomete o rebanho em todo território brasileiro e é uma negligenciada zoonose transmitida pelo leite e seus derivados. Este trabalho avaliou a presença de M. bovis e outras micobactérias, em queijo minas meia-cura, obtidos em feiras-livres na cidade de São Paulo, entre os anos de 2012 e 2013. As amostras (n = 133) foram descontaminadas pelo método HPC (hexa-cetyl-pyridinium chloride) e semeadas em meio Stonebrink Leslie. Os isolados foram submetidos à identificação molecular por PCR TB multiplex, pesquisando-se o gene 16S rRNA, e ao sequenciamento nucleotídico. Dezesseis amostras (12%) possuiam 26 colônias sugestivas de Mycobacterium spp., mas nenhuma delas pertencia aos complexos Mycobacterium tuberculosis, Mycobacterium avium e Mycobacterium intracellulare. A análise filogenética mostrou que todas as amostras estavam agrupadas em clados que incluem apenas micobactérias não tuberculosas (MNT), sendo que algumas possuiam proximidade com sequências obtidas de Mycobacterium novocastrense (3 sequências), Mycobacterium hosaticum (1 sequência) e Mycobacterium elephantis (2 sequências). Embora no momento não haja evidência epidemiológica da importância da transmissão oral das micobactérias pra indivíduos saudáveis, sua importância na população imunossuprimida ainda é incerta.

Mycobacteria in Minas cheese commercialized in open fairs in São Paulo, Brazil / Mycobacteria em queijos tipo Minas comercializados em feiras-livres de São Paulo, Brasil

Mycobacterium bovis is the causative agent of bovine tuberculosis, a disease that affects dairy herds throughout the Brazilian territory, constituting a neglected zoonosis transmitted by raw milk and its derivatives. In this study, we evaluated the presence of M. bovis and other mycobacteria in Minas cheese obtained from open fairs in the city of São Paulo between 2012 and 2013. Samples (n = 133) were decontaminated using hexa-cetylpyridinium chloride and seeded on StonebrinkLeslie medium. The isolates were submitted to molecular identification by TB Multiplex PCR targeting the 16S rRNA gene and amplicon nucleotide sequencing. From 16 cheese samples (12%), we obtained 26 putative colonies of Mycobacterium spp., none of which belonged to any of the Mycobacterium tuberculosis, Mycobacterium avium, or Mycobacterium intracellulare complexes. Phylogenetic analysis showed that sample sequences were grouped in a clade that includes only non-tuberculous mycobacteria with proximity to sequences obtained from Mycobacterium novocastrense (3 sequences), Mycobacterium holsaticum (1 sequence), andMycobacterium elephantis (2 sequences). Although no epidemiological evidence was found regarding the importance of oral transmission of mycobacteria in healthy people, their importance in the immunosuppressed population remains uncertain.(AU)

Mycobacterium bovis é o agente da tuberculose bovina, doença que acomete o rebanho em todo território brasileiro e é uma negligenciada zoonose transmitida pelo leite e seus derivados. Este trabalho avaliou a presença de M. bovis e outras micobactérias, em queijo minas meia-cura, obtidos em feiras-livres na cidade de São Paulo, entre os anos de 2012 e 2013. As amostras (n = 133) foram descontaminadas pelo método HPC (hexa-cetyl-pyridinium chloride) e semeadas em meio Stonebrink Leslie. Os isolados foram submetidos à identificação molecular por PCR TB multiplex, pesquisando-se o gene 16S rRNA, e ao sequenciamento nucleotídico. Dezesseis amostras (12%) possuiam 26 colônias sugestivas de Mycobacterium spp., mas nenhuma delas pertencia aos complexos Mycobacterium tuberculosis, Mycobacterium avium e Mycobacterium intracellulare. A análise filogenética mostrou que todas as amostras estavam agrupadas em clados que incluem apenas micobactérias não tuberculosas (MNT), sendo que algumas possuiam proximidade com sequências obtidas de Mycobacterium novocastrense (3 sequências), Mycobacterium hosaticum (1 sequência) e Mycobacterium elephantis (2 sequências). Embora no momento não haja evidência epidemiológica da importância da transmissão oral das micobactérias pra indivíduos saudáveis, sua importância na população imunossuprimida ainda é incerta.(AU)

Diagnostics of tuberculosis and differentiation of nonspecific tuberculin reactions in animals

Tuberculosis is a serious disease of humans and animals, caused by bacteria of the Mycobacterium genus. This leads to complications in the life of the sick person, and subsequently to death. The cattle, who have been diagnosed with this bacterium, are usually sent to the slaughter, with the result that their livestock is reduced. Mycobacteriosis is also a disease, after determining which cattle are most often sent to slaughter. Such a reduction in livestock numbers has a negative effect on the economy. Of the 300 samples from the animals, 25 cultures of atypical bacteria responding to tuberculin were isolated. A series of tests - intravenous tuberculin test, ophthalmic test, palpebral test, "ZhAT" test, showed that most of the tuberculosis changes in cattle were found in regional lymph nodes more often than in internal organs. In healthy for tuberculosis cows, at the age of 4-9 years, seasonal nonspecific sensitivity to tuberculin is observed. Implementation of the developed express method of glutaraldehyde test on farms in healthy tuberculosis will speed up the diagnosis of tuberculosis and mycobacteriosis in animals that reacted to tuberculin and will exclude short-term nonspecific sensitization of their organism to tuberculin. The introduction of this methodology can be used to diagnose and clearly differentiate the diagnoses of "tuberculosis" and "mycobacteriosis" in cattle. This will cure part of the livestock and reduce the amount of slaughter.(AU)

Use of green fluorescent protein labeled non-tuberculous mycobacteria to evaluate the activity quaternary ammonium compound disinfectants and antibiotics

Although infections with NonTuberculous Mycobacteria have become less common in AIDS patients, they are important opportunistic infections after surgical procedures, likely because they are ubiquitous and not efficiently killed by many commonly used disinfectants. In Venezuela there have recently been many non-tuberculous mycobacteria soft tissue infections after minor surgical procedures, some apparently related to the use of a commercial disinfectant based on a Quaternary Ammonium Compound. We studied the activity of this and other quaternary ammonium compounds on different non-tuberculous mycobacteria by transforming the mycobacteria with a dnaA-gfp fusion and then monitoring fluorescence to gauge the capacity of different quaternary ammonium compounds to inhibit bacterial growth. The minimum inhibitory concentration varied for the different quaternary ammonium compounds, but M. chelonae and M. abscessus were consistently more resistant than M. smegmatis, and M. terrae more resistant than M. bovis BCG.(AU)

Desempenho da cultura líquida MGIT após implementação em uma rede de laboratórios públicos do estado de São Paulo / Performance of liquid culture MGIT after implementation in a network of public laboratories of Sao Paulo state

A OMS, em 2007, recomendou a implementação da cultura líquida para o diagnóstico da tuberculose (TB) e teste de sensibilidade para países de baixa e média renda. Neste estudo foi avaliado o desempenho da cultura líquida MGIT em condição de rotina após dois anosde implantação em uma rede de laboratórios públicos. Foi efetuada análise retrospectiva de dados da cultura líquida, realizadas em dez laboratórios regionais do Instituto Adolfo Lutz, de janeiro a março de 2010. Foram incluídas amostras submetidas a baciloscopia, cultura líquida MGIT automatizada ou manual e identificação presuntiva do complexo Mycobacterium tuberculosis (CMTB). Foram detectadas 1.159 culturas positivas. Destas, 113 (9,7%) contaminaram, e 1.046 foram analisadas, sendo 850 (81,3%) CMTB, 116 (11,1%) micobactérias não tuberculosas e 6 (0,6%) Nocardia sp A taxa de contaminação foi de 2,2% e o acréscimo da cultura para o diagnóstico da TB foi de 29,9%. A média do tempo de detecção da cultura foi de 14,7 dias (DP+/- 11,7 dias). A acurácia da identificação presuntiva foi de 91,3%. A cultura líquida MGIT demonstrou ser excelente alternativa para efetuar diagnóstico da TB e das micobacterioses, em razão da rapidez possibilitando uma intervenção rápida e eficaz no tratamento.

In 2007, WHO recommended the implementation of liquid culture for tuberculosis (TB) diagnosis and drug-susceptibility test in low and middle-income countries. This study evaluated the performance of MGIT culture in routine condition after two years of its implementation in a public laboratories network.This is a retrospective study, which analyzed the data on the liquid culture performed in ten regional laboratories of the Institute Adolfo Lutz, from January to March 2010. The data included clinical samples submitted to microscopy, automated or manual MGIT culture and presumptive M. tuberculosis complex (MTBC) identification by analyzing the cord formation. Culture was positive in 1,159 samples. Of these, 113 (9.7%) contaminated, and 1,046 were analyzed, of which 850 (81.3%) were identified as MTBC, 116 (11.1%) as non-tuberculous mycobacteria and 6 (0.6%) as Nocardia sp. Contamination rate was 2.2% and the contribution of culture to the TB diagnosis was 29.9%. The detection mean time was 14.7 days (SD+/-11.7 days). The accuracy of the presumptive identification of MTBC was 91.3%. MGIT liquid culture demonstrated to be an excellent alternative for diagnosing TB and mycobacterioses, because of the rapidity of diagnosis, thus allowing an immediate and effective treatment.

Desempenho da cultura líquida MGIT após implementação em uma rede de laboratórios públicos do estado de São Paulo / Performance of liquid culture MGIT after implementation in a network of public laboratories of Sao Paulo state

A OMS, em 2007, recomendou a implementação da cultura líquida para o diagnóstico da tuberculose (TB) e teste de sensibilidade para países de baixa e média renda. Neste estudo foi avaliado o desempenho da cultura líquida MGIT em condição de rotina após dois anosde implantação em uma rede de laboratórios públicos. Foi efetuada análise retrospectiva de dados da cultura líquida, realizadas em dez laboratórios regionais do Instituto Adolfo Lutz, de janeiro a março de 2010. Foram incluídas amostras submetidas a baciloscopia, cultura líquida MGIT automatizada ou manual e identificação presuntiva do complexo Mycobacterium tuberculosis (CMTB). Foram detectadas 1.159 culturas positivas. Destas, 113 (9,7%) contaminaram, e 1.046 foram analisadas, sendo 850 (81,3%) CMTB, 116 (11,1%) micobactérias não tuberculosas e 6 (0,6%) Nocardia sp A taxa de contaminação foi de 2,2% e o acréscimo da cultura para o diagnóstico da TB foi de 29,9%. A média do tempo de detecção da cultura foi de 14,7 dias (DP+/- 11,7 dias). A acurácia da identificação presuntiva foi de 91,3%. A cultura líquida MGIT demonstrou ser excelente alternativa para efetuar diagnóstico da TB e das micobacterioses, em razão da rapidez possibilitando uma intervenção rápida e eficaz no tratamento.(AU)

In 2007, WHO recommended the implementation of liquid culture for tuberculosis (TB) diagnosis and drug-susceptibility test in low and middle-income countries. This study evaluated the performance of MGIT culture in routine condition after two years of its implementation in a public laboratories network.This is a retrospective study, which analyzed the data on the liquid culture performed in ten regional laboratories of the Institute Adolfo Lutz, from January to March 2010. The data included clinical samples submitted to microscopy, automated or manual MGIT culture and presumptive M. tuberculosis complex (MTBC) identification by analyzing the cord formation. Culture was positive in 1,159 samples. Of these, 113 (9.7%) contaminated, and 1,046 were analyzed, of which 850 (81.3%) were identified as MTBC, 116 (11.1%) as non-tuberculous mycobacteria and 6 (0.6%) as Nocardia sp. Contamination rate was 2.2% and the contribution of culture to the TB diagnosis was 29.9%. The detection mean time was 14.7 days (SD+/-11.7 days). The accuracy of the presumptive identification of MTBC was 91.3%. MGIT liquid culture demonstrated to be an excellent alternative for diagnosing TB and mycobacterioses, because of the rapidity of diagnosis, thus allowing an immediate and effective treatment.(AU)

Occurrence of Mycobacterium bovis and non-tuberculous mycobacteria (NTM) in raw and pasteurized milk in the northwestern region of Paraná, Brazil

Milk is widely consumed in Brazil and can be the vehicle of agent transmission. In this study, was evaluated the occurrence of Mycobacterium bovis and non-tuberculous mycobacteria (NTM) in raw and pasteurized milk consumed in the northwestern region of Paraná, Brazil. Fifty-two milk samples (20 pasteurized and 32 raw) from dairy farms near the municipality of Maringa, Parana State, Brazil were collected. Milk samples were decontaminated using 5% oxalic acid method and cultured on Lowenstein-Jensen and Stonebrink media at 35 °C and 30 °C, with and without 5-10% CO2. Mycobacteria isolates were identified by morphological features, PCR-Restriction Fragment Length Polymorphism Analysis (PCR-PRA) and Mycolic acids analysis. Thirteen (25%) raw and 2 (4%) pasteurized milk samples were positive for acid fast bacilli growth. Nine different species of NTM were isolated (M. nonchromogenicum, M. peregrinum, M. smegmatis, M. neoaurum, M. fortuitum, M. chelonae, M. flavescens, M. kansasii and M. scrofulaceum). M. bovis was not detected. Raw and pasteurized milk may be considered one source for NTM human infection. The paper reinforces the need for intensification of measures in order to avoid the milk contamination and consequently prevent diseases in the south of Brazil.

Comportamento da tuberculose humana no Rio Grande do Norte e pesquisa de Mycobacterium spp. em queijos de coalho comercializados no município de Caicó-RN, Brasil

A tuberculose (Tb) é mundialmente uma das 10 principais causas de morte humana e é tida como a principal causadora de óbitos por um único agente infeccioso. As Américas representam 3% da prevalência mundial da doença e se atribui que 33% dos casos notificados neste continente são no Brasil. Esse, é um dos 22 países priorizados pela OMS, que juntos representam 80% da ocorrência mundial desta bacteriose. Uma das formas da doença é a Tuberculose zoonótica, cujo agente causador é o M. bovis, cuja principal via de transmissão para a população humana é por meio do consumo de leite e derivados (queijos e iogurtes) elaborados sem a adoção de Boas Práticas Agropecuárias (BPAs) e de Fabricação (BPFs). O presente trabalho disserta sobre dois assuntos: No Capítulo primeiro, aborda-se o comportamento da tuberculose humana no estado do Rio Grande do Norte, Brasil, no período de 2008 a 2017, por meio de estudos descritivo e retrospectivo, avaliando-se a incidência anual da tuberculose no Estado e ocorrência de suas formas clínicas. Utilizamos dados primários obtidos junto ao Programa de Tuberculose no Rio Grande do Norte SUVIGE/SESAP-RN (fazer a mesma descrição que vc fez acima com BPAs); e de dados secundários, colhidos junto ao Sinan/Ministério da Saúde. Como resultado, observou-se haver divergência entre os coeficientes de incidência calculados com os dados primários daqueles que estão publicados em Boletins Epidemiológicos: 37,1 a 27,0, por grupo de 100 mil hab; além disso, a doença apresentou tendência linear de crescimento no Rio Grande do Norte. Recomendado-se medidas amplas de proteção à população e ao meio ambiente, associadas ao fortalecimento dos serviços de saúde para que se consiga atingir as metas propostas no programa End TB, sejam de controle e/ou de erradicação. O Capítulo seguinte, avaliou-se s qualidade microbiológica do queijo de coalho comercializado no município de Caicó-RN, pesquisando-se a presença de Mycobaterium spp. Por meio de cultivo bacteriano e diagnóstico molecular. Foram analisadas 50 amostras de queijo de coalho obtidas aleatoriamente no comércio local, sendo: 35 elaboradas a partir de leite cru (artesanal) e 15 com leite pasteurizado (industrializado), das quais se extraiu materiais que foram submetidos a processos: para obtenção de DNA; cultivo (Stonebrink); PCR convencional para pesquisa de M. bovis; e, finalmente, nested-PCR para Mycobacterium spp. Na pesquisa laboratorial, não se conseguiu isolar micobactérias tuberculosas nos queijos estudados. Porém, detectou-se que duas amostras (4%) apresentaram crescimento de micobactérias não tuberculosas (MNT) em meio de cultura, que através de diagnóstico molecular apresentaram identidade com hsp65 de: Mycobacterium lehmanii (ID da sequência: KY933786.1, valor e: 2e-133, identidades: 312/363 [86%]); e Mycobacterium rutilum (ID da sequência: LT629971.1, valor e: 3e-108, identidades: 331/371 [89%]), apontando-se como indicativos de contaminação ambiental. Há na literatura resultados publicados sobre micobacterioses em humanos causadas por agentes com alta similaridade genética aos encontrados neste estudo, bem como infecções em tecidos animais. Conhecendo-se as características das MNT, considerando-as como microrganismos emergentes e de natureza ubíqua, sugere-se maior atenção na cadeia produtiva de queijos, tanto nas boas práticas agropecuárias (BPAs) quanto nas boas práticas de fabricação de alimentos (BPFs).

Tuberculosis (TB) is one of the leading causes of human death and is one of the main death´s culprit by a single infectious agent. The region of the Americas represents 3% of the world's disease, of which 33% are attributed to Brazil, what is one of the 22 countries prioritized by WHO, who together represent 80% of the world's occurrence by this microrganism. One of the forms of the disease on humans is zoonotic tuberculosis, which is caused by Mycobacterium bovis, and its main route of transmission to population is through the consumption of milk and dairy products (cheeses and yoghurts) prepared without the adoption of Good Agricultural Practices (GAPs) and Manufacturing (GMPs). In the first chapter, the behavior of human tuberculosis in the state of Rio Grande do Norte, Brazil, from 2008 to 2017, is analyzed through descriptive and retrospective studies, evaluating the annual incidence of tuberculosis in the State and occurrence of their clinical forms. The primary data obtained from the Tuberculosis Program in Rio Grande do Norte - SUVIGE / SESAP-RN were used; and secondary data collected from Sinan / Ministry of Health. As a result, there was a divergence between the incidence coefficients calculated with the primary data from those published in Epidemiological Bulletins: 37.1 to 27.0, per group of 100 thousand inhabitants; in addition, the disease showed a linear growth trend in Rio Grande do Norte. It is recommended that comprehensive measures be taken to protect the population and the environment associated with strengthening health services so that the goals proposed in the End TB program can be achieved, be they control and / or eradication. The following chapter evaluated the microbiological quality of the Coalho cheese marketed in the municipality of Caicó-RN, by investigating the presence of Mycobaterium spp. through bacterial culture and molecular diagnosis. Fifty samples of Coalho cheese randomly from local commerce were analyzed: 35 were made from raw milk (artisanal) and 15 with pasteurized (industrialized) milk, from which materials were extracted and submitted to processes: to obtain DNA; cultivation (Stonebrink); conventional PCR for M. bovis; and finally, nested-PCR for Mycobacterium spp. In the laboratory research, it was not possible to isolate tuberculous mycobacteria from cheeses studied. However, the growth of non-tuberculous mycobacteria (NTM) in culture medium was detected in two samples (4%), which showed hsp65 identity of: Mycobacterium lehmanii (sequence ID: KY933786.1, value-e: 2e-133, identities: 312/363 [86%]); and Mycobacterium rutilum (sequence ID: LT629971.1, value-e: 3e-108, identities: 331/371 [89%]), considerated as indicative of environmental contamination. Knowing more about the characteristics of the MNT and considering them as emerging microorganisms and ubiquitous, it is suggested that more attention be given to the cheese production chain, both in good agricultural practices (GAPs) and in good food manufacturing practices (GMPs).

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.

Detection of mycobacteria in ornamental fish in Iran by culture and Ziehl-Neelsen staining methods

Background: Mycobacterial infections in fish is largely chronic to subacute in nature and affects fishes in fresh water, brackish water and salt water. In addition to their known infectivity to fishes, aquatic mycobacteria pose significant zoonotic concerns. Due to the zoonotic character of the disease, increasing importance of aquariology, and lack of any clinical signs in early steps of mycobacteriosis, present study was undertaken to analyze the distribution of mycobacteria in diseased and apparently healthy ornamental fish from some local aquarium fish shops in four different cities in Iran by culture and Ziehl-Neelsen staining. Materials, Methods & Results: One hundred and one fresh water aquarium fish of 22 species from some local shops in four cities in Iran were examined. Before decontamination, smears of homogenized samples stained with Ziehl-Neelsen. For bacterial culture, samples were inoculated on Lowenstein-Jensen medium. Culture plates were examined daily for four weeks. The rate of growth at different temperature, colony morphology and pigmentation were evaluated for species identification. Among 79 moribund fish examined, 16 individuals were positive for acid fast rods at microscopic examination. Seven fish out of 22 apparently healthy individuals also gave positive microscopic results. Using the culture method, 29 and 10 Mycobacterium isolates were obtained from moribund and healthy fish. The following Mycobacterium species were isolated from unhealthy fish: Mycobacterium fortuitum, M. marinum, and M. smegmatis. The number of different species of Mycobacterium from apparently healthy fish was: M. fortuitum (one fish), M. marinum (one fish), M. terrae (one fish) and M. flavescens (one fish). Discussion: Based on the results in both moribund and apparently healthy fish examined, culture examination showed more mycobacteria than Ziehl-Neelsen staining detection. Lower proportion of Ziehl-Neelsen positive results compared with culture method was reported in aquarium fish in Slovania. Using positive microscopic results, 13 isolates were obtained where as 29 samples gave positive culture results for mycobacteria. Similar result was also observed in clinically healthy ornamental fish and their aquarium environment. The identification of mycobacteria by Ziehl-Neelsen staining is a traditional method. However, acid-fast bacilli may not always be found through direct microscopy because the destruction of mycobacteria or their low number may sometimes happen. Culture examination is a more sensitive method than direct microscopy. However, killing of mycobacteria caused by host defense mechanisms, a low number of viable mycobacteria in the tissue, or by destruction of the mycobacteria during the preparation of the sample could result in negative cultivation results. Species of Mycobacterium identified in unhealthy fish were M. fortuitum, M. marinum and M. smegmatis. High frequency of identifying M. fortuitum and M. marinum (6 out of 7) in the samples provide more evidence that these species are common Mycobacterium species to be found in diseased aquarium fish. Numerous studies showed the common isolation of these two species from aquarium fish. M. marinum infection may be an occupational hazard for certain professionals such as pet shop workers. Many infections may also occur in fish fanciers who keep an aquarium at home. Less common than M. marinum, M. fortuitum is also capable of infecting human. In this study, the occurrence of M. marinum and M. fortuitum in both unhealthy and apparently healthy aquarium fish shows the importance of recognizing fish mycobacteriosis in order to prevent their transmission to human.

Isolamento de micobactérias provenientes de amostras clínicas da região de Rio Claro: análise da frequência / Mycobacterial isolates from patients clinical samples from Rio Claro region: frequency analysis

Os agentes etiológicos da tuberculose pertencem ao Complexo Mycobacterium tuberculosis (MT), porém outras espécies do gênero e de importância médica, denominadas micobactérias não causadoras de tuberculose (MNT), são frequentemente isoladas. A identificação correta e rápida das MNT é obrigatória para o diagnóstico, auxiliando o clínico na conduta terapêutica. A frequência da MT e MNT foi avaliada no período de 2003 a 2009, analisando-se 6.460 amostras clínicas, sendo 5.696 (88,2 por cento) culturas negativas para micobactéria, 715 (11,1 por cento) culturas positivas e 49 (0,7 por cento) não avaliadas. Os isolados mais frequentes foram MT 78,6 por cento (562). Dentre as MNT, a espécie mais isolada foi M. gordona e em 3,09 por cento(22) das amostras, seguida por M. fortuitum em 2,39 por cento (17), Complexo M. avium (MAC) em 2,25 por cento (16),M. kansasii em 1,97 por cento (14) e M. abscessus em 1,83 por cento (13). Quando relacionado ao número de pacientes,o complexo MAC foi o mais isolado (19,4 por cento). Foi observado um declínio na frequência de isolamento de MT no período estudado, enquanto a frequência de MNT teve um aumento de 22,8 por cento. A alta frequênciade MT nas amostras é uma constante preocupação para a saúde pública, mas o aumento de doentes com MNT salienta a importância da notificação e do monitoramento epidemiológico desses pacientes. (AU)

Estudo de micobactérias em animais silvestres mantidos em cativeiro

O gênero Mycobacterium compreende inúmeras espécies estudadas por sua capacidade de causar doenças. Surtos de tuberculose e micobacteriose em zoológicos, aquários, centros de pesquisa e criadouros evidenciaram a importância desses organismos em relação à saúde de animais silvestres mantidos em cativeiro. Duzentas e onze amostras, de 101 animais entre mamíferos, aves, répteis, anfíbios e peixes, foram submetidas à cultura. Foram isolados M. tuberculosis, M. bovis e 13 espécies de MNT associadas à doença. A natureza crônica dessas doenças, seu diagnóstico ante mortem limitado, juntamente com as características de interação social de cada espécie e o confinamento, favorecem a transmissão das micobactérias pelo contato com humanos ou outros animais infectados. Apesar de a presença de M. tuberculosis e M. bovis em instituições brasileiras já ter sido constatada, o presente estudo descreveu infecções em espécies animais utilizadas para consumo humano, e em espécies conhecidas pela suscetibilidade, mas que ainda não foram documentadas no Brasil. Doença causada por MNT foi relatada, mas sua importância deve ser considerada de acordo com a espécie animal acometida.

Mycobacterium genus comprises several species, which have been studied for their capacity to cause diseases. Outbreaks of tuberculosis and mycobacteriosis in zoos, aquariums, research and breeding facilities have shown the importance of these organisms to health of wildlife animal maintained in captivity. Two hundred and eleven samples, from 101 animals among mammal, birds, reptiles, amphibians and fishes, were cultured. Mycobacterium tuberculosis, M. bovis and 13 species of nontuberculous mycobacteria associated to diseases were isolated. The chronical nature of tuberculosis and mycobacteriosis, their limited ante mortem diagnosis, along with social behavior of each species and confinement, favor mycobacterial transmission through contact with infected humans and other animals. Despite the presence of M. tuberculosis and M. bovis has already been observed in Brazilian institutions, the present study described infections in animal species destined to human consumption, and species known for their susceptiblility, yet not documented in Brazil. Disease caused by non-tuberculous mycobacteria was reported, though its importance must be considered according to the animal species affected.

Mycobacterial isolation from Felis concolor in captivity / Isolamento de micobactérias em Felis concolor em cativeiro

This study was made in a wildlife preserve from Argentina where a previous tuberculosis report in Felis concolor has been done. The aim was to identify mycobacterial species isolated from the orpharynx of five American lions using bacteriological and molecular biology techniques on cases with nonspecific clinical signals. Samples were collected after sedation. They were treated in order to isolate Mycobacterium. Bacteriological differentiation was made using biochemical tests. Polymerase chain reaction has been performed to detect hsp65, IS6110 and IS1081. Acid fast bacilli were present infour specimens and from them were isolated slowly growing mycobacteria. The strains were differentiated as M. gordonae in two cases and M. simiae, M. scrofulaceum and M. avium/intracellulare in one case each other. The strains were identified as M. gordonae in three cases and M. avium III or M. simiae in two by PRA. The role of feral cats in the epidemiology of nontuberculous mycobacterial diseases remains to be further investigated.(AU)

Este trabalho foi realizado em uma reserva natural da Argentina com antecedentes de tuberculose em uma suçuarana adulta. O objetivo foi identificar por meio de técnicas bacteriológicas e de biologia molecular as espécies isoladas da orofaringe de cinco suçuaranas que apresentavam sinais clínicos inespecíficos. As amostras foram colhidas das suçuaranas após sedação. Posteriormente foram processadas para obtenção do isolamento e identificação por meio de provas bioquímicas do gênero Mycobacterium pela técnica de PCR. Investigou-se a presença das seqüências de inserção IS6110 e IS1081 e hsp65. Obtiveram-se resultados positivos à coloração de Ziehl-Neelsen de quatro amostras, isolando cinco cepas de crescimento lento. As cepas foram classificadas como M. gordonae em dois casos e M. simiae, Mscrofulaceum e M. avium/intracellulare em um. Por PRA, identificou-se o padrão de M. gordonae em três cepas e M. avium III ou M.simiae em dois.(AU)

Identification of non-tuberculous mycobacteria from the Central Public Health Laboratory from Mato Grosso do Sul and analysis of clinical relevance

Non-tuberculous mycobacteria isolated at the Central Public Health Laboratory from Mato Grosso do Sul in 2003 and 2004 were identified by conventional phenotypic methods (TI) and by PCR-Restriction Enzyme Analysis (PRA) using the hsp65 gene as target (PRA-hsp65). With 15 of the 32 analysed isolates, results of both methods were concordant, being 8 Mycobacterium avium, 3 M. fortutium, 1 M. kansasii, 1 M. flavescens, 1 M. peregrinum and 1 Nocardia brasiliensis. TI of 12 isolates was inconclusive. Novel PRA-hsp65 patterns were observed with 11 isolates. Medical data were evaluated for inference of clinical relevance of these isolates.

Micobactérias não-tuberculosas isoladas no Laboratório Central de Saúde Pública de Mato Grosso do Sul em 2003 e 2004 foram identificadas usando métodos fenotípicos convencionais (TI) e PCR-Restriction Enzyme Analysis (PRA) tendo o gene hsp65 como alvo (PRA-hsp65). Em 15 dos 32 isolados analisados os resultados obtidos com ambos métodos foram concordantes, sendo 8 Mycobacterium avium, 3 M. fortutium, 1 M. kansasii, 1 M. flavescens, 1 M. peregrinum e 1 Nocardia brasiliensis. TI de 12 isolados não foi conclusiva. Perfis não descritos de PRA-hsp65 foram observados com 11 isolados. Dados dos prontuários médicos foram avaliados para inferir a relevância clínica dos isolados.

Síndrome do granuloma lepróide canino: relato de caso / Canine leproid Granuloma Syndrome: a case report

A Síndrome Granuloma Lepróide Canino (SGLC) é uma infecção causada por micobactérias atípicas, provenientes do gênero Mycobacterium sp. Possui curso crônico e recidivante, caracterizada por lesões paniculares e granulomatosas, nodulares e firmes, de abrangência epidermodermal e subcutânea com tendência a ulceração, normalmente envolvendo face e pescoço, mas com evidente tendência ao acometimento da região dorsal dos pavilhões auriculares. Uma maior ocorrência da SGLC é observada e, cães de grande porte e pelagem curta, sendo a maioria dos casos representados pela raça Boxer e mestiços. Achados clínicos, citológicos e dermatohistopatológicos subsidiaram o diagnóstico(AU)

Canine Leproid Granuloma Syndrome (CLGS) is an ui infection caused by atypical mycobacteria, genus Mycobacterium sp. It has a chronic and relapsing progress, characterized by granulomatous lesions, nodular and firm, with epidermodermal and subcutaneous coverage, tending to ulceration. Usually involves the face and neck, but it tends to affect the dorsal region of the ears. A higher occurrence of CLGS is large dog, and the most of the cases are represented by Boxer and mixed breeds. Clinical, cytological and histopathological supported the presented diagnosis(AU)

Síndrome do granuloma lepróide canino: relato de caso / Canine leproid Granuloma Syndrome: a case report

A Síndrome Granuloma Lepróide Canino (SGLC) é uma infecção causada por micobactérias atípicas, provenientes do gênero Mycobacterium sp. Possui curso crônico e recidivante, caracterizada por lesões paniculares e granulomatosas, nodulares e firmes, de abrangência epidermodermal e subcutânea com tendência a ulceração, normalmente envolvendo face e pescoço, mas com evidente tendência ao acometimento da região dorsal dos pavilhões auriculares. Uma maior ocorrência da SGLC é observada e, cães de grande porte e pelagem curta, sendo a maioria dos casos representados pela raça Boxer e mestiços. Achados clínicos, citológicos e dermatohistopatológicos subsidiaram o diagnóstico

Canine Leproid Granuloma Syndrome (CLGS) is an ui infection caused by atypical mycobacteria, genus Mycobacterium sp. It has a chronic and relapsing progress, characterized by granulomatous lesions, nodular and firm, with epidermodermal and subcutaneous coverage, tending to ulceration. Usually involves the face and neck, but it tends to affect the dorsal region of the ears. A higher occurrence of CLGS is large dog, and the most of the cases are represented by Boxer and mixed breeds. Clinical, cytological and histopathological supported the presented diagnosis