Nontuberculous mycobacteria in patients with suspected tuberculosis and the genetic diversity of Mycobacterium avium in the extreme south of Brazil / Micobactérias não tuberculosas em pacientes com suspeita de tuberculose e a diversidade genética de Mycobacterium avium no extremo sul do Brasil

ABSTRACT Objective: Nontuberculous mycobacteria (NTM) are a heterogeneous group of bacteria that are widely distributed in nature and associated with opportunistic infections in humans. The aims of this study were to identify NTM in patients with suspected tuberculosis who presented positive cultures and to evaluate the genetic diversity of strains identified as Mycobacterium avium. Methods: We studied pulmonary and extrapulmonary samples obtained from 1,248 patients. The samples that tested positive on culture and negative for the M. tuberculosis complex by molecular identification techniques were evaluated by detection of the hsp65 and rpoB genes and sequencing of conserved fragments of these genes. All strains identified as M. avium were genotyped using the eight-locus mycobacterial interspersed repetitive unit-variable-number tandem-repeat method. Results: We found that NTM accounted for 25 (7.5%) of the 332 mycobacteria isolated. Of those 25, 18 (72%) were M. avium, 5 (20%) were M. abscessus, 1 (4%) was M. gastri, and 1 (4%) was M. kansasii. The 18 M. avium strains showed high diversity, only two strains being genetically related. Conclusions: These results highlight the need to consider the investigation of NTM in patients with suspected active tuberculosis who present with positive cultures, as well as to evaluate the genetic diversity of M. avium strains.

RESUMO Objetivo: As micobactérias não tuberculosas (MNT) são um grupo heterogêneo de bactérias amplamente distribuídas na natureza e relacionadas com infecções oportunistas em seres humanos. Os objetivos deste estudo foram identificar MNT em pacientes com suspeita de tuberculose e culturas positivas e avaliar a diversidade genética de cepas identificadas como Mycobacterium avium. Métodos: Foram estudadas amostras pulmonares e extrapulmonares provenientes de 1.248 pacientes. As amostras que apresentaram resultado positivo em cultura e negativo para o complexo M. tuberculosis na identificação molecular foram avaliadas por meio da detecção dos genes hsp65 e rpoB e de sequenciamento de fragmentos conservados desses genes. Todas as cepas identificadas como M. avium foram genotipadas pelo método mycobacterial interspersed repetitive unit-variable-number tandem-repeat com oito loci. Resultados: Das 332 micobactérias isoladas, 25 (7,5%) eram MNT. Dessas 25, 18 (72%) eram M. avium, 5 (20%) eram M. abscessus, 1 (4%) era M. gastri e 1 (4%) era M. kansasii. As 18 cepas de M. avium apresentaram alta diversidade, e apenas duas eram geneticamente relacionadas. Conclusões: Esses resultados mostram a necessidade de considerar a investigação de MNT em pacientes com suspeita de tuberculose ativa e culturas positivas e de avaliar a diversidade genética de cepas de M. avium.

Paratuberculosis: new histopathological findings in red deer (Cervus elaphus) and fallow deer (Dama dama) in Chile / Paratuberculosis: novos achados histopatológicos em Veado Vermelho (Cervus elaphus) e Gamo (Dama dama) no Chile

Paratuberculosis is a disease caused by Mycobacterium avium subsp. paratuberculosis (MAP) that affects domestic and wild ruminants. The most common gross lesions are emaciation and corrugation and thickening of the mucosa of the small intestine. Mesenteric lymph nodes might be enlarged. For the present study, 14 red deer and 9 fallow deer from game reserves or venison farms were analyzed. The lesions found correspond to those found by other authors in other geographic locations, except for some differences in histopathological examinations. Among these differences, stands out that intestinal lesions were concentrated mostly in the ileum and granulomas were shown to be more frequent in this section of the intestine than in the corresponding lymph node. Furthermore, in multibacillary lesions the inflammatory infiltrate in the lymph nodes was mainly composed of macrophages. These differences may be due to individual variations of the animals, the stage of disease or a different strain of the pathogen. This study allowed to obtain basic information about the disease and to describe patterns of lesions found in red deer and fallow deer with prediagnosis of clinical paratuberculosis which were not described in the literature before.(AU)

Paratuberculosis é uma doença causada por Mycobacterium avium subsp. paratuberculosis (MAP) que afecta ruminantes selvagens e domésticos. As lesões macroscópicas mais comuns são ondulação e espessamento da mucosa do intestino delgado. Os linfonodos mesentéricos podem aparecer com volume aumentado. Para este estudo, foram analisados 14 veados vermelhos e 9 veados gamo de reservas de caça e fazendas de carne. As lesões encontradas correspondem à encontrada por outros autores em outras localizações geográficas, com exceção de algumas diferenças no exame histopatológico. Entre essas diferenças, sobressai que as lesões intestinais se concentraram principalmente no íleo, os granulomas ocorreram com maior frequência nesta seção do intestino que no seu correspondente linfonodo. Além disso, nas lesões bacterianas, o infiltrado inflamatório linfonodos linfáticos era composta principalmente por macrófagos. Estas diferenças podem ser devidas a variações individuais dos animais, o estádio da doença ou de uma estirpe diferente do agente patogénico. Este estudo permitiu obter informação básica sobre a doença e descrever padrões de lesões encontradas em veados e em gamos com pré-diagnóstico, de paratuberculosis clínica nunca antes descritas na literatura.(AU)

Broncho-Pleural Fistula with Hydropneumothorax at CT: Diagnostic Implications in Mycobacterium avium Complex Lung Disease with Pleural Involvement

OBJECTIVE: To determine the patho-mechanism of pleural effusion or hydropneumothorax in Mycobacterium avium complex (MAC) lung disease through the computed tomographic (CT) findings. MATERIALS AND METHODS: We retrospectively collected data from 5 patients who had pleural fluid samples that were culture-positive for MAC between January 2001 and December 2013. The clinical findings were investigated and the radiological findings on chest CT were reviewed by 2 radiologists. RESULTS: The 5 patients were all male with a median age of 77 and all had underlying comorbid conditions. Pleural fluid analysis revealed a wide range of white blood cell counts (410-100690/microL). The causative microorganisms were determined as Mycobacterium avium and Mycobacterium intracellulare in 1 and 4 patients, respectively. Radiologically, the peripheral portion of the involved lung demonstrated fibro-bullous changes or cavitary lesions causing lung destruction, reflecting the chronic, insidious nature of MAC lung disease. All patients had broncho-pleural fistulas (BPFs) and pneumothorax was accompanied with pleural effusion. CONCLUSION: In patients with underlying MAC lung disease who present with pleural effusion, the presence of BPFs and pleural air on CT imaging are indicative that spread of MAC infection is the cause of the effusion.

Caracterização histológica e imuno-histoquímica das lesões de tuberculose em bovinos e de linfadenite granulomatosa em suínos / Histological and immunohistochemical characterization of tuberculosis lesions in cattle and of granulomatous lymphadenitis in swine

Mycobacterium sp. induz inflamação granuloma-tosa em diferentes espécies animais. Mycobacterium bovis e o complexo Mycobacterium avium são importantes patógenos de bovinos e suínos e podem causar infecção em humanos, principalmente imunossuprimidos. Perdas na produção, barreiras comerciais e prejuízos por condenação de carcaças em abatedouro/frigorífico estão atrelados à ocorrência dessas infecções, com prejuízos econômicos significativos. Foi realizado um estudo de casos diagnosticados como tuberculose em bovinos e linfadenite granulomatosa em suínos no Setor de Patologia Veterinária da Universidade Federal do Rio Grande do Sul (SPV-UFRGS) no período de janeiro de 2007 a dezembro de 2011. Dados referentes à raça, ao sexo, à idade e ao histórico clínico foram compilados dos livros de registro e analisados. As características histológicas das lesões em linfonodos e pulmões foram avaliadas em Hematoxilina-Eosina, com predomínio de células gigantes nas lesões de tuberculose bovina e de macrófagos epitelioides em suínos. As técnicas histoquímicas de Ziehl-Neelsen e Tricrômico de Masson foram utilizadas para evidenciar, respectivamente, bacilos álcool-ácido resistentes e tecido conjuntivo fibroso nas lesões. A técnica de imuno-histoquímica foi utilizada em aproximadamente 30% dos casos estudados de cada espécie, selecionados aleatoriamente, para a caracterização do infiltrado linfocítico. Foram utilizados os anticorpos anti-CD3 para a marcação de linfócitos T e anti-CD79cy para a marcação de linfócitos B. Linfócitos T predominaram nas lesões em ambas as espécies, com diferença estatisticamente significativa entre as médias dos linfócitos T e linfócitos B. Foi usado o teste t pareado, com t=5,501 (p<0,001) nas lesões dos bovinos e t=5,826 (p<0,001) para as lesões de linfadenite dos suínos...

Mycobacterium sp. induces granulomatous inflammation in different animal species. Mycobacterium bovis and the Mycobacterium avium complex are important cattle and swine pathogens that can also infect humans, especially those immunosuppressed. Losses in production, commercial barriers and carcasses condemnations in slaughtering are related to this infection, which implies in large economic losses. It was carried out a study on bovine tuberculosis lesions and granulomatous lymphadenitis in pigs, diagnosed by the Setor de Patologia Veterinária from the Universidade Federal do Rio Grande do Sul (SPV-UFRGS), Brazil, between January 2007 and December 2011. Data concerning breed, sex, age and clinical history were retrieved from the files and analyzed. Histological features of the lesions in lymph nodes and lungs were evaluated in Hematoxylin-Eosin staining. Multinucleated giant cells were predominant in bovine tuberculosis lesions and epithelioid macrophages were abundant in swine. Ziehl-Neelsen and Masson's trichrome techniques were used respectively to demonstrate the alcohol-acid resistant bacillus and fibrous connective tissue in the lesions. The immunohistochemistry technique was performed to characterize the lymphocytic infiltrate. Anti-CD3 antibodies were utilized to immunolabeling lymphocytes T, and anti-CD79cy to lymphocytes B. Lymphocytes T were predominant in both species lesions, confirmed statistically by paired t test, which showed significantly differ means of T and B lymphocytes, with t=5,501 (p<0.001) for the bovines tuberculosis lesions, and t=5.826 (p<0.001), for the cases of pigs lymphadenitis...

Evaluation of a MPB70-ELISA to differentiate Mycobacterium bovis from M. avium-sensitized swine / Avaliação de um MPB70-ELISA para diferenciar entre suínos sensibilizados com Mycobacterium bovis ou M. avium

Swine are susceptible to different mycobacteria species, being Mycobacterium bovis an agent of tuberculosis, with most significant zoonotic risks, while M. avium determines a granulomatous lymphadenitis with low zoonotic risk. Currently performed intradermal tests present some important limitations, such as the lack of ability to detect anergic animals or to differentiate among mycobacterial species. In order to improve the TB diagnosis, serological assays have been developed, with encouraging results. The purpose of this study was to evaluate the performance of a MPB70-ELISA in 82 piglets divided into four groups: sensitized by inactivated M. bovis, M. avium, inoculated with oil adjuvant, or with saline solution. The test was able to discriminate between an animal sensitized by M. bovis and animals of the three other groups, including M. avium-sensitized animals; for this reason, we suggest that MPB70-ELISA could be used as a complementary tool for discriminating the agent of the mycobacteriosis, and therefore to diagnose tuberculosis in a swine herd.(AU)

Suínos são suscetíveis a diferentes espécies de micobactérias, sendo Mycobacterium bovis agente de tuberculose (TB), com claro risco zoonótico, enquanto M. avium determina uma linfadenite granulomatosa (LG) de baixo risco zoonótico. Os testes intradérmicos atualmente realizados apresentam algumas limitações importantes, como a falta de habilidade em detectar animais anérgicos ou de diferenciar entre as espécies micobacterianas. Com o intuito de melhorar o diagnóstico de TB, testes sorológicos têm sido desenvolvidos, com resultados encorajadores. O objetivo do presente estudo foi avaliar um MPB70-ELISA em 82 leitões divididos em quatro grupos: sensibilizados por M. bovis, por M. avium, inoculados com óleo adjuvante ou com solução salina. O teste foi capaz de discriminar entre os animais sensibilizados com M. bovis dos demais três grupos, incluindo aqueles que foram sensibilizados com M. avium; desta forma, sugere-se que o MPB70-ELISA poderia ser utilizado como ferramenta complementar para discriminar o agente da micobacteriose, e portanto diagnosticar TB em um plantel de suínos.(AU)

Padronização do teste imunoalérgico aplicado ao diagnóstico da tuberculose e micobacterioses em suínos (Sus scrofa) experimentalmente sensibilizados com suspensões oleosas de Mycobacterium bovis ou M. avium inativados / Standardization of the immunoallergic skin test applied for the diagnosis of tuberculosis and mycobacterioses in swine (Sus scrofa) experimentally sensitized with oil suspensions of inactivated Mycobacterium bovis or M. avium

Foi investigado o valor diagnóstico da resposta alérgica cutânea em leitões experimentalmente sensibilizados, pela via intramuscular, com suspensões oleosas de Mycobacterium bovis ou M. avium inativados pelo calor.Foram utilizados 91 animais, divididos em quatro grupos: grupos A e B, cada um com 25 indivíduos, grupos C e D com 21 e 20 indivíduos respectivamente, balanceando-se as características de raça, linhagem, faixa etária e sexo. Aos 30 dias de idade, todos os animais foram submetidos a uma triagem com a aplicação de tuberculina PPD bovina, pela via intradérmica na base da orelha e não houve qualquer tipo de reação. Decorridos 60 dias do teste tuberculínico de triagem, o grupo A recebeu injeção intramuscular de 0,5 mL de uma suspensão oleosa de M. avium estirpe D4; o grupo B recebeu 0,5 mL de uma suspensão oleosa de M. bovis estirpe AN5; o grupo C (controle I), recebeu 0,5 mL do adjuvante oleoso; e o grupo D (controle II), recebeu 0,5 mL de solução fisiológica. Após 30 dias da sensibilização foi realizada a prova de tuberculinização comparativa com reação medida pela variação da espessura da pele com cutímetro de mola às 0h, 24h, 48h e 72h, após a aplicação das tuberculinas. No teste comparativo, lido às 48 ou 72 horas, a reação foi considerada negativa quando a diferença das reações entre o PPD bovino e o PPD aviário foi menor que 6,7 mm; suspeito ou inconclusivo quando a diferença se situou na faixa de 6,7 a 7,5 mm; e positiva de acordo com o tipo de PPD, considerando-se tuberculose para PPD M. bovis e micobacteriose para PPD M. avium, quando a diferença da reação foi superior a 7,5 mm.

The diagnostic value of the cutaneous allergic response to tuberculin in piglets experimentally sensitized intramuscularly with the oily suspensions of heat inactivated M. bovis or M. avium was investigated. Ninety-one animals were used and divided into four groups: groups A and B were formed each with 25 individuals, and groups C and D, with 21 and 20 individuals, respectively, balancing the characteristics of race, ancestry, age and sex. At the age of 30 days, all the animals were submitted to the screening test with the use of M. bovis PPD, by the intradermal route at the base of the ear and no reaction was detected. Sixty days after the screening tuberculin test, animals of the group A were injected intramuscularly with 0.5 mL of oily suspension of M. avium D4 strain; animals of the group B received 0.5 mL of an oily suspension of M. bovis, AN5 strain; group C (control I) received 0.5 mL of an oily adjuvant; and the individuals of the group D (control II) received 0.5 mL of saline solution. Following 30 days of sensitization, comparative skin reactions were measured by the variation in skin thickness with a caliper at 0h, 24h, 48h an 72h after applications of tuberculins. In the comparative test measured at 48 or 72h, the reaction was considered negative when the difference of the reactions between bovine PPD and avian PPD was less than 6.7 mm; suspected or inconclusive, when the difference stood in the range of 6.7 to 7.5 mm; and positive according to the type of PPD, considering tuberculosis the M. bovis PPD and mycobacteriosis the M. avium PPD, when the difference of the reaction was greater than 7.5 mm.

Occurrence of Non-Tuberculous Mycobacterium in clinical samples - a potential pathogen.

Background: Silent presence of non-tuberculous mycobacterium (NTM) has been observed since the last 100 years, but now the increasing incidence of NTM is of great concern for clinical microbiologists as well as clinicians. Although many advanced efforts are being made for identification and control of Mycobacterium tuberculosis, still the silently growing menace of non-tuberculous mycobacteria is receiving negligible attention. Objectives: This study was aimed to find NTMs in positive cultures and identify them up to species level. Material & Methods: During the study period, i.e. from January 2009 to June 2011, a total of 4104 positive cultures were subjected to species identification by different morphological and biochemical tests. All the tests for identification were performed as per standard procedure along with the standard strains of NTM provided by JALMA, Agra. Results: The identification of positive cultures showed 4044/15581 (25.95%) Mycobacterium tuberculosis complex and 60/15581(0.38%) NTM. The mycobacterium species identification results showed that out of total 60 NTM, 21 different species of NTM were found and they belonged to all the four groups of runyon. The most common species identified in this study was M.simiae (07) followed by M.avium(06), M.gordonae(05), M.kansasii(05), M.fortuitum(05), M.chelonae(05), M.pheli(05), M.terrae(04), M.szulgai(02), M.vaccae(02), M.flavescens(02), M. trivale(02), M.malmoense(01), M.scrofulaceum(01), M.intracellulare(01), M.xenopi(01), M.ulcerans(01), M.tusciae(01), M.triplex(01), M.septicum(01), M.mucogenicum(01). Conclusion: The isolation of NTMs from different clinical samples indicated that they may be the causative agents for pulmonary and extra-pulmonary non-tuberculous diseases. Elaborate and focused studies are needed to differentiate NTMs amongst commensal/colonizer, pathogen and laboratory contaminants.

Relationship between Paratuberculosis and the microelements Copper, Zinc, Iron, Selenium and Molybdenum in Beef Cattle

To study the deficiency of minerals and its relationship with Paratuberculosis, blood, serum, and fecal samples were obtained from 75 adult bovines without clinical symptoms of the disease and from two bovines with clinical symptoms of the disease, from two beef herds with a previous history of Paratuberculosis in the Province of Buenos Aires, Argentina. Serum samples were processed by ELISA and feces were cultured in Herrolds medium. Copper, zinc and iron in serum were quantified by spectrophotometry and selenium was measured by the activity of glutathione peroxidase. We also determined copper, zinc, iron and molybdenum concentrations in pastures and the concentration of sulfate in water. Mycobacterium avium subsp paratuberculosis (Map) was isolated from 17.3% of fecal samples of asymptomatic animals and from the fecal samples from the two animals with clinical symptoms. All the Map-positive animals were also ELISA-positive or suspect, and among them, 84.6% presented low or marginal values of selenium and 69.2% presented low or marginal values of copper. The two animals with clinical symptoms, and isolation of Map from feces and organs were selenium-deficient and had the lowest activity of glutathione peroxidase of all the animals from both herds. All the animals negative to Map in feces and negative to ELISA had normal values of Se, while 13.8% of animals with positive ELISA or suspect and culture negative presented low levels of Se. Half of the animals that were negative both for ELISA and culture in feces were deficient in copper but none of them presented low values of selenium. The content of molybdenum and iron in pasture was high, 2.5 ppm and 1.13 ppm in one herd and 2.5 ppm and 2.02 ppm in the other, respectively, whereas the copper:molybdenum ratio was 1.5 and 5.2, respectively. These results do not confirm an interaction between imbalances of the micronutrients and clinical Paratuberculosis, but show evidence of the relationship between selenium...

Diagnosis of paratuberculosis in cattle: microbiological culture, serology and PCR

The aim of this study was to confirm clinical diagnosis of paratuberculosis in two cows showing suggestive clinical signs of the disease. Based on clinical signs, in culture and in IS900 PCR results from the individual milk samples it was possible to diagnose paratuberculosis in the cows studied.

Parameter estimation and use of gamma interferon assay for the diagnosis of bovine tuberculosis in Brazil / Estimação dos parâmetros e uso do teste do interferon gama para o diagnóstico da tuberculose bovina no Brasil

This study aimed to evaluate the interference of tuberculin test on the gamma-interferon (INFg) assay, to estimate the sensitivity and specificity of the INFg assay in Brazilian conditions, and to simulate multiple testing using the comparative tuberculin test and the INFg assay. Three hundred-fifty cattle from two TB-free and two TB-infected herds were submitted to the comparative tuberculin test and the INFg assay. The comparative tuberculin test was performed using avian and bovine PPD. The INFg assay was performed by the BovigamTM kit (CSL Veterinary, Australia), according to the manufacturer's specifications. Sensitivity and specificity of the INFg assay were assessed by a Bayesian latent class model. These diagnostic parameters were also estimate for multiple testing. The results of INFg assay on D0 and D3 after the comparative tuberculin test were compared by the McNemar's test and kappa statistics. Results of mean optical density from INFg assay on both days were similar. Sensitivity and specificity of the INFg assay showed results varying (95% confidence intervals) from 72 to 100% and 74 to 100% respectively. Sensitivity of parallel testing was over 97.5%, while specificity of serial testing was over 99.7%. The INFg assay proved to be a very useful diagnostic method.(AU)

O presente estudo avalia a interferência do teste de tuberculinização no teste do interferon gama (INFg), estima a sensibilidade e a especificidade do INFg em condições brasileiras e simula a utilização dos testes múltiplos usando a tuberculinização comparada e o teste do INFg. Trezentos e cinquenta animais oriundos de dois rebanhos livres e dois rebanhos positivos foram submetidos à tuberculinização comparada e ao teste de INFg. A tuberculinização comparada foi realizada utilizando PPD aviária e bovina. O teste de INFg foi realizado utilizando o kit Bovigam® (CSL Veterinary, Austrália) de acordo com as especificações do fabricante. A sensibilidade e especificidade do teste de INFg foram calculadas pelo Modelo Bayesiano de Classe Latente. Esses parâmetros foram também estimados para os testes múltiplos. Os resultados do teste de INFg no D0 e D3 após o teste de tuberculinização foram comparados pelos testes estatísticos de McNemar e kappa. Os resultados das médias de densidade ótica do teste de INFg em ambos os dias foram similares. A sensibilidade e a especificidade do teste de INFg apresentaram resultados variando (95% intervalo de confiança) de 72 a 100% e 74 a 100%, respectivamente. A sensibilidade do teste em paralelo foi acima de 97,5% enquanto a especificidade do teste em série foi acima de 99,7%. O teste do INFg provou ser um método de diagnóstico muito útil.(AU)

Comentarios clínicos acerca de las micobacteriosis pulmonares / Pulmonary mycobacterioses: clinical commentaries

Las micobacteriosis pulmonares son afecciones provocadas por micobacterias ambientales, de evolución crónica y clínicamente similares a la tuberculosis. Se analiza una serie de 26 casos asistidos en el Hospital Muñiz de la Ciudad de Buenos Aires, con una edad promedio de 59.2 años, 73.1% de sexo femenino y 80.1% de los casos de nivel socioeconómico mediano a alto. Se hallaron enfermedades predisponentes en el 88.5% de los casos (tuberculosis previa, EPOC, silicosis, reflujo gastroesofágico). Desde el punto de vista radiológico se observaron nódulos, bronquiectasias y cavidades pequeñas en 14 casos y lesiones bilaterales cavitarias extensas en 12. Se destacó la mayor frecuencia de Mycobacterium avium complex como patógeno, la mejor evolución de los pacientes con menor compromiso pulmonar y la de los afectados por M. avium en relación con otras micobacterias. Los fármacos más utilizados en el tratamiento fueron claritromicina y azitromicina, asociados a etambutol. La proporción global de curaciones fue baja (57.7%) pero superior en los casos provocados por M. avium (86.7%).

Detection of Mycobacterium avium in pet birds / Detecção de Mycobacterium avium em aves de companhia

The present study is a report on the presence of Mycobacterium avium in four birds of the psittaciform order kept as pets. Anatomopathological diagnosis showed lesions suggestive of the agent and presence of alcohol-acid resistant bacilli (AARB) shown by the Ziehl-Neelsen staining. The identification of Mycobacterium avium was performed by means of PRA (PCR Restriction Analysis). DNA was directly extracted from tissue of the lesions and blocked in paraffin. The role of this agent in pet bird infection is discussed, as well as its zoonotic potential.

Este estudo relata a presença de Mycobacterium avium em quatro aves da ordem Psitaciformes, mantidos como animais de estimação. O exame anatomopatológico revelou a presença de bacilos álcool ácido resistentes na coloração de Ziehl-Neelsen, e o diagnostico definitivo foi feito pelo método PRA (PCR Restriction Analysis) a partir de tecidos emblocados em parafina. Este estudo visa alertar o possível potencial zoonótico deste agente em aves mantidas domiciliadas.

Fiebre de origen desconocido en pacientes con sida en el Hospital "José María Vargas" Caracas-Venezuela: ¿Mycobacterium avium complex (MAC) o tuberculosis diseminada / Fever of unknown origin in AIDS patients in the Hospital José María Vargas Caracas-Venezuela: ¿mycobacterium avium complex (MAC) tuberculosis disseminated

La micobacteriosis diseminada, es la infección con especies de micobacterias, diferentes a las pertenecientes al complejo de Mycobacterium tuberculosis. Es una enfermedad oportunista frecuente en pacientes con SIDA, particularmente las ocasionadas por Mycobacterium avium complex (MAC) la información de la prevalencia en personas VIH positivas en Latinoamerica son escasos. Entre junio 2005 y febrero 2006 se efectuó un estudio prospectivo para determinar dicha prevalencia en el Hospital "José María Vargas". Un total de 75 pacientes con SIDA C, en los cuales se sospechaba una infección diseminada por micobacterias, basados en la presencia de fiebre de origen desconocido por más de 2 semanas y pérdida de peso del más del 10 por ciento de la masa corporal durante un período de tres meses, ingresaron al estudio. De cada paciente se recolectaron dos muestras de sangre y una muestra de médula ósea que se procesaron para el aislamiento de microbacterias. El crecimiento de Mycobacterium tuberculosis se reportó en el 9,3 por ciento (7/75) pacientes, con un paciente co-infectado con M.kansasii. La letalidad fue de 100 por ciento en estos pacientes. La muestra de sangre diagnósticó el 100 por ciento de los casos, mientras que la médula ósea sólo 50 por ciento. Se concluye que existe un sub-diagnóstico de tuberculosis diseminada y alta mortalidad por la misma en pacientes con SIDA y fiebre de origen desconocido. Micobacteriosis parece ser infrecuente en el hospital.

Mycobacteriosis is defined as an infection with mycobacteria others than those belonging to the Mycobacterium tuberculosis complex. It has been reported as avery common opportunistic infection in AIDS patients, particulary disseminated mycobacteriosis caused by the Mycobacterium avium complex (MAC). Data on the prevalence of mycobacteriosis in HIV patients in Venezuela are scarce. We conducted between June 2005 and February 2006 a prospective study to determine the prevalence of MAC infection in the Hospital "José María Vargas" in Caracas, Venezuela. A total of 75 AIDS-C patients with fever of unknown origin for more then 2 weeks and weinght loss of more than 10 % of their body mass over a period of 3 months, were suspected having a disseminated mycobacterial infection and entered in this study. From each patients, two whole blood samples and one bone marrow sample were collected and processed for isolation of mycobacteria. The growth of Mycobacterium tuberculosis was reported for 9,3 % (7/75) of the patients, with one patient co-infected with M.kansasii. The mortality was 100 % un these patients. The diagnostic yield of the blood samples was 100 % whereas the bone marrow sample detected only 50 % of the cases. We conclude that in our hospital there is a sub-diagnosis of disseminated tuberculosis with a high mortality in patients with AIDS and fever of unknown origin. MAC infectious was not detected and is probably rare in our setting.

Identification of non-tuberculous mycobacteria from the Central Public Health Laboratory from Mato Grosso do Sul and analysis of clinical relevance / Identificação de micobactérias não-tuberculosas do Laboratório Central de Saúde Pública de Mato Grosso de Sul e análise de dados clínicos dos pacientes

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.

Similaridades clinicopatológicas entre paratuberculosis y enfermedad de crohn: ¿posible vínculo zoonótico? / Clinical and pathological similarities between paratuberculosis and crohn’s disease: ¿a possible zoonotic link?

EL artículo presenta a la luz de la literatura científica actual la asociación del Mycobacterium avium subespecie paratuberculosis con la enfermedad de Crohn, las principales similaridades clinicopatológicas. La revisión se limitó a las publicaciones contenidas en el programa HINARI de la WHO, en especial de Elservier Science, Bioline Internacional, Blackwell Publishing, BMJ Publishing, Lippincott Williams & Wilkins, Nature Publishing, PubMed, Springer Science y publicaciones de la Asociación Internacional para estudio de Paratuberculosis. Evidencias significativas apoyan un posible vinculo zoonótico entre las enfermedades. El aislamiento del Mycobacterium avium subespecie paratuberculosis en pacientes con enfermedad de Crohn desde muestras de intestino, leche, sangre periférica y nódulos linfáticos; de igual manera la respuesta inmune especifica a algunos antígenos de Mycobacterium en pacientes con la enfermedad; aunque estos hallazgos tienen cada uno sus propias controversias. La evidencia actualmente disponible no es suficiente para validar o negar al Mycobacterium avium subespecie paratuberculosis como agente causal de por lo menos algunos casos de enfermedad de Crohn. Los estudios son inconclusos en aceptar un vinculo zoonótico dada la naturaleza multifactorial de esta enfermedad. Se requieren estudios para determinar sí Mycobacterium es un agente espectador ó patogénico; paralelamente realizar estudios epidemiológicos a gran escala que permitan analizar la distribución geográfica y temporal de ambas enfermedades.

Linfadenite granulomatosa em suínos: linfonodos afetados e diagnóstico patológico da infecção causada por agentes do Complexo Mycobacterium avium / Granulomatous lymphadenitis in swine: lymph nodes affected and pathologic diagnosis of the infection caused by Mycobacterium avium Complex agents

Realizou-se um estudo com o objetivo de avaliar a distribuição de lesões granulomatosas nos linfonodos das carcaças de suínos abatidos e de comparar os métodos de isolamento do Complexo Mycobacterium avium (MAC), coloração de Ziehl-Neelsen (ZN), exames histopatológico e de imunohisto-química (IHQ) para o diagnóstico da linfadenite granulomatosa causada por micobactérias do MAC. Foram utilizadas 431 amostras de linfonodos colhidos de 394 carcaças de suínos abatidos em 12 frigoríficos da Região Sul do Brasil, com o Serviço de Inspeção Federal (SIF). Os linfonodos que apresentavam lesões granulomatosas foram submetidos aos exames histológicos, ZN e IHQ com anticorpo monoclonal produzido com extrato celular de M. avium. A concordância entre os exames foi medida pelo teste Kappa, com nível de confiança de 95 por cento. O exame macroscópico realizado pelo SIF identifica corretamente 90,3 por cento das lesões granulomatosas, quando comparado com o exame histológico e a maioria das carcaças (92,5) apresentam lesões apenas nos linfonodos cadeia alimentar. O exame histológico confirmou a presença de lesões granulo-matosas em 90,3 por cento dos linfonodos. As concordâncias entre os exames histopatológico e coloração de ZN (Kappa: 0,342) e de IHQ e o isolamento do MAC (Kappa: 0,102) foram baixas, porém alta entre os exames de IHQ e histológico com a presença de granulomas típicos nos linfonodos (Kappa: 0,973). O exame de IHQ associado ao exame histopatológico mostrou-se eficiente na identificação das lesões de linfadenite granulomatosa causadas pelo MAC.

A study to evaluate the distribution of granulomatous lesions in the lymph nodes of swine carcasses was accomplished. The main objective was to compare the methods of isolation of mycobacteria of the Mycobacterium avium Complex (MAC), Ziehl-Neelsen (ZN) staining, histopathological examination and immunohistochemistry (IHC) for the diagnosis of granulomatous lymphadenitis caused by MAC. A total of 431 samples of lymph nodes from 394 swine carcasses kept in 12 cold storage rooms of southern Brazil, which had the Service of Federal Inspection (SFI), were analyzed. Lymph nodes with granulomatous lesions were submitted to histological examination, ZN staining and IHC with monoclonal antibody produced with cellular extract of M. avium. The agreement between the exams was measured by the Kappa test at a 95 percent confidence level. The macrocospic examination done by SFI correctly identified 90.3 percent of granulomatous lesions, when compared with histological examination, and the majority of carcasses (92.5 percent) showed the lesions only in lymph nodes of the alimentary chain. Histological examination confirmed the presence of granulomatous lesions in 90.3 percent of the lymph nodes. The agreements between histological examination, ZN staining (Kappa: 0.342), IHC and the isolation of MAC (Kappa: 0.102) were low, however high between IHC and histology with typical presence of granulomas in the lymph nodes (Kappa: 0.973). IHC together with histological examination revealed to be efficient for identification of the granulomatous lymphadenitis caused by MAC.

Resposta imune específica de bovinos experimentalmente sensibilizados com inóculos inativados de Mycobacterium bovis e Mycobacterium avium / Specific immune response of cattle to experimental sensibilization by inactivated Mycobacterium bovis and Mycobacterium avium

O diagnóstico presuntivo da tuberculose bovina é baseado na análise da resposta imune celular a antígenos micobacterianos. Procedeu-se à simulação experimental de sensibilização por Mycobacterium bovis e Mycobacterium avium inativados em bovinos a fim de acompanhar a resposta imune a partir do teste cervical comparativo e da evolução da produção específica de interferon-gama, além de identificar a interferência de reações inespecíficas por M. avium nos resultados dos testes. Verificou-se que os animais desencadearam resposta de hipersensibilidade tardia contra os bacilos inativados, e que ambos os testes diagnósticos da tuberculose bovina foram eficientes na identificação dos animais sensibilizados com M. bovis e na discriminação das reações geradas pela inoculação dos bovinos com M. avium.

The presumptive diagnosis of bovine tuberculosis is based on analysis of the immune response to micobacterial antigens. This experimental simulation of sensibilization by Mycobacterium bovis and Mycobacterium avium in cattle aimed to verify the immune response by both the cervical comparative test and the evolution of the specific production of gamma-interferon, and also to identify interference of unspecified reactions by M. avium on the test results. The results support that the experimental animals started a response of delayed hypersensitivity to the inactivated bacilli, and that both diagnostic tests for bovine tuberculosis were efficient for the identification of animals sensitized with M. bovis and for discrimination of reactions generated by inoculation of cattle with M. avium.

Comparação entre métodos de tuberculinização no diagnóstico da infecção por agentes do complexo Mycobacterium avium ou M. bovis em suínos / Comparative study of methods of tuberculin skin test with the avium and bovine tuberculin in the diagnosis of the infection by agents of the Mycobacterium avium-complex or M. bovis in swine

Avaliou-se a eficiência do teste de tuberculina intradérmico comparado, para identificar rebanhos suínos infectados com o complexo Mycobacterium avium (MAC), seu poder de discriminação entre rebanhos infectados com MAC e Mycobacterium bovis, e para estudar diferentes critérios de leitura e interpretação. Foram realizados três experimentos com rebanhos infectados naturalmente com MAC, com suínos inoculados com MAC e com suínos inoculados com M. bovis. Os testes foram comparados com a presença ou não de lesões macro e/ou microscópicas nos linfonodos de suínos em idade de abate. Verificaram-se reações cruzadas entre os testes intradérmicos, tanto com animais infectados com o MAC como com M. bovis. Reações acima de 0,5cm de diâmetro ou acima de 0,20cm no aumento da espessura da dobra da pele, entre a aplicação e a leitura, foram consideradas positivas no cálculo da média para rebanhos. Rebanhos infectados com MAC podem ser identificados por meio do teste intradérmico com PPD aviário e bovino. Tanto a medida do diâmetro quanto o aumento da espessura da dobra da pele das reações foram eficientes no diagnóstico da infecção por MAC. Recomenda-se a utilização do método da medição do diâmetro.

The efficiency of the tuberculin delayed-type skin hypersensitivity test was evaluated in swine. A comparison of the tuberculin skin tests was performed with avian and bovine protein derivatives (PPD), in the detection of Mycobacterium avium-Complex or M. bovis infected swine. The methods were evaluated as to define criteria for readings and results interpretation, to determine their capacity in detecting Mycobacterium avium complex (MAC) infected swine and their performance in differentiating MAC from M. bovis-infected individuals. Three trials were performed with naturally MAC-infected swine, experimentally MAC-infected or experimentally M. bovis-infected animals. The skin test readings positive or negative were, respectively, correlated to the presence or absence of gross lesions and histological alterations in lymph nodes at slaughter age. Reaction with both antigens were observed in MAC and M. bovis-inoculated animals as well. Reactions wider then 0.5cm in diameter or showing skin thickness increasing more then 0.2cm in the time frame between inoculation and reading were considered to be positive, as compared to the average results of the group. Herds with MAC-infected swine could be detected with either avian or bovine PPD. Both diameter and skin thickness were efficient in detecting MAC-infected swine, however, reading the diameter of the reaction was shown to be more easily and economically performed.

Evaluation of Mycobacterium avium subsp. paratuberculosis faecal culture protocols and media

A paratuberculose é uma importante enterite de ruminantes causada por Mycobacterium avium subsp. paratuberculosis (Map). A enfermidade é oficialmente considerada exótica no Brasil, mas inquéritos sorológicos recentes e o isolamento do agente etiológico sugerem que ela pode estar presente em nossos rebanhos. O objetivo do estudo foi avaliar três diferentes fórmulas do Ágar gema de ovo de Herrold suplementado com micobactina J (HEYM) e quatro protocolos de cultura fecal quanto ao crescimento de Map, bem como custo e facilidade de implementação. Três fórmulas de HEYM foram inoculadas com duas suspensões de Map. Fezes contaminadas artificialmente e naturalmente com Map foram tratadas pelos quatro protocolos de cultura fecal. O protocolo da centrifugação e a fórmula de HEYM recomendada pela OIE demonstraram os melhores resultados quanto à recuperação de Map.

Mycobacterium avium restriction fragment lenght polymorphism-IS IS1245 and the simple double repetitive element polymerase chain reaction typing method to screen genetic diversity in Brazilian strains

Simple double repetitive element polymerase chain reaction (MaDRE-PCR) and Pvu II-IS1245 restriction fragment length polymorphism (RFLP) typing methods were used to type 41 Mycobacterium avium isolates obtained from 14 Aids inpatients and 10 environment and animals specimens identified among 53 mycobacteria isolated from 237 food, chicken, and pig. All environmental and animals strains showed orphan patterns by both methods. By MaDRE-PCR four patients, with multiple isolates, showed different patterns, suggesting polyclonal infection that was confirmed by RFLP in two of them. This first evaluation of MaDRE-PCR on Brazilian M. avium strains demonstrated that the method seems to be useful as simple and less expensive typing method for screening genetic diversity in M. avium strains on selected epidemiological studies, although with limitation on analysis identical patterns except for one band.