Diversidad de especies de micobacterias no tuberculosas aisladas en ambientes acuáticos de la ciudad de General Pico, La Pampa, Argentina / Species diversity of non-tuberculous mycobacteria isolated from aquatic environments of General Pico city, Province of La Pampa (Argentina)

Las micobacterias no tuberculosas (MNT) no solo se estudian por su importancia como patógenos oportunistas, sino también por sus aplicaciones en biotecnología y biorremediación. Nuestro objetivo fue determinar la presencia de micobacterias en los distintos hábitats acuáticos de la ciudad de General Pico (provincia de La Pampa), así como su diversidad. Los porcentajes de muestras positivas a micobacterias fueron los siguientes: 37,5% en el sistema de distribución de agua de red, 32,6% en el acuífero que abastece dicho sistema, 36,8% en el agua proveniente de las precipitaciones, 53,1% en los humedales del área de influencia, 80% en los natatorios cubiertos y 33,3% en las fuentes decorativas ubicadas en plazas públicas. De los 90 aislamientos de MNT obtenidos el 8,9% no logró ser identificado a nivel de especie con los métodos utilizados, que incluyeron pruebas fenotípicas y métodos moleculares. Las especies más frecuentemente aisladas fueron Mycobacterium fortuitum y Mycobacterium gordonae. Algunas especies identificadas han sido reportadas en casos de micobacteriosis en nuestro país, entre ellas M. fortuitum, M. gordonae, M. intracellulare, M. vaccae, M. lentiflavum y M. nonchromogenicum. No se aislaron MNT en muestras de agua de red con concentraciones de cloro activo residual mayores de 0,8mg/l, mientras que en los natatorios la presencia de hasta 1,5mg/l de cloro activo residual no fue una limitante para la proliferación de estos microorganismos. Se puede considerar que la incidencia de micobacterias en los ambientes acuáticos de General Pico es cercana al 35%, y que la presencia de estos microorganismos y su diversidad se ve afectada por el contacto con el hombre y sus actividades, como así también por la existencia de vida animal.

Non-tuberculous mycobacteria (NTM) are studied not only for their importance as emerging opportunistic pathogens but also for their applications in biotechnology and bioremediation. Our aim was to determine the occurrence and diversity of mycobacteria in different aquatic habitats of General Pico city, Province of La Pampa. The percentage of samples with positive cultures for mycobacteria were the following: 37.5% recovered from the water supply distribution system; 32.6% from the aquifer that supplies water to the distribution system; 36.8% from rain water; 53.1% from the two wetlands in the area of influence; 80% from indoor swimming pools; and 33.3% from water fountains in downtown public squares. Of the 90 NTM isolates, 8.9% could not be identified at the species level with any of the used methods, phenotypic tests and molecular methods. Mycobacterium fortuitum and Mycobacterium gordonae were the most frequently isolated species. Some of the identified species such as, M. fortuitum, M. gordonae, M. intracellulare, M. vaccae, M. lentiflavum and M. nonchromogenicum, have been reported in cases of mycobacteriosis in Argentina. Mycobacteria with values higher than 0.8mg/ml of residual active chlorine were not recovered from the drinking water supply network, whereas in the swimming pools the presence of up to 1.5 mg/l was not a constraint. Based on our results, the presence of mycobacteria in aquatic environments is close to 35% and their occurrence and diversity is affected both by contact with man and his activities as well as by the existence of animal life.

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

Abstract 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.

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.

Mycobacteria mobility shift assay: a method for the rapid identification of Mycobacterium tuberculosis and nontuberculous mycobacteria

The identification of mycobacteria is essential because tuberculosis (TB) and mycobacteriosis are clinically indistinguishable and require different therapeutic regimens. The traditional phenotypic method is time consuming and may last up to 60 days. Indeed, rapid, affordable, specific and easy-to-perform identification methods are needed. We have previously described a polymerase chain reaction-based method called a mycobacteria mobility shift assay (MMSA) that was designed for Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) species identification. The aim of this study was to assess the MMSA for the identification of MTC and NTM clinical isolates and to compare its performance with that of the PRA-hsp65 method. A total of 204 clinical isolates (102 NTM and 102 MTC) were identified by the MMSA and PRA-hsp65. For isolates for which these methods gave discordant results, definitive species identification was obtained by sequencing fragments of the 16S rRNA and hsp65 genes. Both methods correctly identified all MTC isolates. Among the NTM isolates, the MMSA alone assigned 94 (92.2%) to a complex or species, whereas the PRA-hsp65 method assigned 100% to a species. A 91.5% agreement was observed for the 94 NTM isolates identified by both methods. The MMSA provided correct identification for 96.8% of the NTM isolates compared with 94.7% for PRA-hsp65. The MMSA is a suitable auxiliary method for routine use for the rapid identification of mycobacteria.

First Korean Case of Mycobacterium arupense Tenosynovitis

No abstract available.

Rapid detection and differentiation of mycobacterial species using a multiplex PCR system

Introduction The early diagnosis of mycobacterial infections is a critical step for initiating treatment and curing the patient. Molecular analytical methods have led to considerable improvements in the speed and accuracy of mycobacteria detection. Methods The purpose of this study was to evaluate a multiplex polymerase chain reaction system using mycobacterial strains as an auxiliary tool in the differential diagnosis of tuberculosis and diseases caused by nontuberculous mycobacteria (NTM) Results Forty mycobacterial strains isolated from pulmonary and extrapulmonary origin specimens from 37 patients diagnosed with tuberculosis were processed. Using phenotypic and biochemical characteristics of the 40 mycobacteria isolated in LJ medium, 57.5% (n=23) were characterized as the Mycobacterium tuberculosis complex (MTBC) and 20% (n=8) as nontuberculous mycobacteria (NTM), with 22.5% (n=9) of the results being inconclusive. When the results of the phenotypic and biochemical tests in 30 strains of mycobacteria were compared with the results of the multiplex PCR, there was 100% concordance in the identification of the MTBC and NTM species, respectively. A total of 32.5% (n=13) of the samples in multiplex PCR exhibited a molecular pattern consistent with NTM, thus disagreeing with the final diagnosis from the attending physician. Conclusions Multiplex PCR can be used as a differential method for determining TB infections caused by NTM a valuable tool in reducing the time necessary to make clinical diagnoses and begin treatment. It is also useful for identifying species that were previously not identifiable using conventional biochemical and phenotypic techniques. .

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

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

Identification of mycobacteria isolated at University Hospital of Santa Maria, Rio Grande do Sul, Brazil / Identificação de micobactérias isoladas no Hospital Universitário de Santa Maria, Rio Grande do Sul, Brasil

This study evaluated the prevalence of nontuberculous mycobacterium (NTM) in relation to the total number of cases of mycobacterial infections detected in patients admitted at the University Hospital of Santa Maria from 2008 to 2010. From the positive samples for the genus Mycobacterium, 67% belonged to the Mycobacterium tuberculosis complex (MTBC) and 33% of them were classified as NTM. This investigation aims to contribute to the epidemiology of mycobacterioses, inasmuch as patients infected by NTM require distinctive treatment and monitoring in comparison with those infected by MTBC.

Foi avaliada a prevalência de micobactérias não tuberculosas (MNT) em relação ao total de casos de micobacterioses identificadas em pacientes do Hospital Universitário de Santa Maria, entre os anos de 2008 e 2010. Entre as amostras positivas para o gênero Mycobacterium, 67% eram do complexo Mycobacterium tuberculosis (CMTB) e 33% foram classificadas como MNT. Este estudo procura contribuir com a epidemiologia das micobacterioses, uma vez que os pacientes infectados por MNT necessitam de tratamento e acompanhamento diferenciado dos infectados pelo CMTB.

Use of the Ogawa-Kudoh method to isolate mycobacteria in a tuberculosis reference laboratory in northwestern Paraná, Brazil

Culturing is the gold standard method for confirming a diagnosis of tuberculosis (TB). The Brazilian Ministry of Health recently proposed the use of the Ogawa-Kudoh method for sputa cultures to detect Mycobacterium tuberculosis. The aim of the present study was to evaluate 8 years of using the Ogawa-Kudoh method in a TB reference laboratory in northwestern Paraná, Brazil. The present study consisted of a retrospective analysis of sputa cultures records for the detection of mycobacteria using the Ogawa-Kudoh method in the Laboratory of Medical Bacteriology, Laboratory of Teaching and Research in Clinical Analysis (LEPAC), State University of Maringá, from July 2003 to September 2011. The following variables were analyzed: Ziehl Neelsen (Z-N) smears and cultures results and the age and gender of the patients. Sputa samples from 3,231 patients with suspected TB were analyzed. Of these, 67.17% were male with an average age of 45.58 years. Of the total number of Z-N-negative samples (n=2,949), 42 (1.42%) were positive for M. tuberculosis (p >0.05). The Ogawa-Kudoh method is an excellent tool for diagnosing pulmonary TB. It is easy to perform, requires less biosafety equipment than the Petroff method, has a low cost, and has good sensitivity for detecting of M. tuberculosis.

A cultura é o método padrão ouro para confirmação da tuberculose (TB). O Ministério da Saúde Brasileiro propôs, recentemente, a utilização do método de Ogawa-Kudoh para cultura de escarro na detecção de Mycobacterium tuberculosis. O objetivo deste estudo foi avaliar oito anos de utilização do método de Ogawa-Kudoh na rotina de um laboratório de referência na região noroeste do Paraná, Brasil. Realizou-se estudo retrospectivo dos registros das culturas de escarro para a detecção de micobactérias, usando o método Ogawa-Kudoh conduzido no Laboratório de Bacteriologia Médica, Laboratório de ensino e pesquisa em Análises Clínicas (LEPAC) da Universidade Estadual de Maringá (UEM), de Julho de 2003 a Setembro de 2011. As seguintes variáveis foram analisadas: esfregaço Ziehl Neelsen (Z-N), cultura, idade e sexo do paciente. Analisaram-se 3.231 amostras de escarro de pacientes com suspeita de tuberculose. Destes, 67,17% eram do sexo masculino com idade média de 45,58 anos. Do total de amostras Z-N negativas (n=2.949), 42 amostras (42/2949, 1,42%) apresentaram cultura positiva para M. tuberculosis (p>0,05). A utilização do método Ogawa-Kudoh representa excelente ferramenta para o diagnóstico precoce da TB pulmonar. É de fácil execução, requer menos equipamentos de biossegurança do que o método de Petroff, apresenta baixo custo e boa sensibilidade para detecção de M. tuberculosis.

Incidence of tuberculous and non-tuberculous mycobacteria, differentiated by multiplex PCR, in clinical specimens of a large general hospital

OBJECTIVE: To determine the incidence of Mycobacterium tuberculosis complex and non-tuberculous mycobacterial isolates in the routine setting of a large general hospital using an "in-house" multiplex polymerase chain reaction method and to establish a paradigm for the definitive identification of mycobacteria isolated using semi-automated equipment. METHODS: Established tests, including polymerase chain reaction restriction enzyme analysis, PNB, and NAP inhibition tests as the gold standard, showed 100% agreement with an IS6110/hsp65 multiplex polymerase chain reaction when used to identify stock strains (n = 117). RESULTS: In a subsequent study, 8,790 clinical specimens producing 476 isolates were evaluated with multiplex PCR and also showed 100% agreement in identification using PRA-polymerase chain reaction as the gold standard. The application of this technique to routine analysis was demonstrated in this study. A method was established with the initial application of multiplex PCR for all positive liquid cultures and the subsequent identification of non-tuberculous mycobacteria by polymerase chain reaction restriction enzyme analysis. In total, 77% of isolates belonged to the Mycobacterium tuberculosis complex, and 23% were non-tuberculous mycobacteria. CONCLUSIONS: Several non-tuberculous mycobacterial species were identified, primarily M. avium, but other potentially pathogenic species were also frequently observed, including M. fortuitum, M. abscessus, and M. kansasii. The expeditious communication of these data to the clinical staff was fundamental for the diagnosis of clinical cases. Even in settings where tuberculosis is of major importance, the incidence of non-tuberculous mycobacteria infection is substantial.

First Case of Mycobacterium longobardum Infection

Mycobacterium longobardum is a slow-growing, nontuberculous mycobacterium that was first characterized from the M. terrae complex in 2012. We report a case of M. longobardum induced chronic osteomyelitis. A 71-yr-old man presented with inflammation in the left elbow and he underwent a surgery under the suspicion of tuberculous osteomyelitis. The pathologic tissue culture grew M. longobardum which was identified by analysis of the 65-kDa heat shock protein and full-length 16S rRNA genes. The patient was cured with the medication of clarithromycin and ethambutol without further complications. To the best of our knowledge, this is the first report of a M. longobardum infection worldwide.

Laboratory aspects of clinically significant rapidly growing mycobacteria.

The pathogenic potential of the rapidly growing mycobacteria (RGM) has started being recognized. This is due to more sensitive and specific techniques in the laboratory. The RGM are generally defined as nontuberculous species of mycobacteria that show visible growth on agar media within 7 days. RGM are widely distributed in nature and have been isolated from natural water, tap water, and soil. Several biochemical tests, high performance liquid chromatography, and molecular techniques have been developed for rapid identification of these species. The American Thoracic Society and the Infectious Disease Society of America recommend that RGM should be identified to the species level using a recognized acceptable methodology such as polymerase chain reaction restriction enzyme analysis or biochemical testing and routine susceptibility testing of RGM should include amikacin, imipenem, doxycycline, the fluorinated quinolones, a sulphonamide or trimethoprim-sulphamethoxazole, cefoxitin, clarithromycin, linezolid, and tobramycin. The diseases caused by these organisms have varied manifestations. They have been responsible for a number of healthcare-associated outbreaks and pseudo-outbreaks. For recognition of outbreaks, it is important to be familiar with the causative organisms like RGM which are most frequently involved in healthcare-associated outbreaks and pseudo outbreaks. It is essential to intervene as soon as possible to interrupt this transmission. Large gaps still exist in our knowledge of RGM. Unquestionably more studies are required. Through this review, we wish to emphasize that reporting of RGM from clinical settings along with their sensitivity patterns is an absolute need of the hour.

Análise de restrição enzimática do gene hsp65 de isolados clínicos de pacientes com suspeita de tuberculose pulmonar em Teresina, Piauí / Restriction enzyme analysis of the hsp65 gene in clinical isolates from patients suspected of having pulmonary tuberculosis in Teresina, Brazil

OBJETIVO: Identificar as espécies de micobactérias encontradas no escarro de pacientes com suspeita de tuberculose pulmonar e analisar o impacto dessas identificações na abordagem terapêutica. MÉTODOS: Foram avaliados 106 pacientes com suspeita de tuberculose pulmonar encaminhados para o serviço de pneumologia de um hospital público em Teresina, Piauí. Espécimes de escarro matinal foram avaliados quanto à presença de micobactérias por baciloscopia e cultura. Foram utilizadas PCR e análise de restrição enzimática do gene hsp65 (PRA-hsp65) para a identificação das cepas de micobactérias isoladas em cultura. RESULTADOS: Foram analisadas 206 amostras de escarro. A idade dos pacientes variou de 15 a 87 anos, sendo 67 por cento do gênero masculino. Tosse ocorreu em 100 por cento dos casos. O padrão radiográfico predominante foi de lesão moderada, observada em 70 por cento. A positividade no esfregaço foi de 76 por cento, e isolamento em cultura ocorreu em 91 por cento das culturas executadas. Testes tradicionais identificaram micobactérias não tuberculosas (MNT) em 9 por cento dos isolados. O método PRA-hsp65 confirmou esses dados, mostrando sete padrões de bandas capazes de identificar as espécies de MNT isoladas: Mycobacterium kansasii; M. abscessus 1; M. abscessus 2; M. smegmatis; M. flavescens 1; M. gordonae 5 e M. gordonae 7. Todos os pacientes com MNT tinham mais de 60 anos, e observaram-se bronquiectasias em 88 por cento das radiografias. Houve dois casos de reinfecção, identificados inicialmente como infecção por M. abscessus e M. kansasii. CONCLUSÕES: As MNT causam infecção pulmonar em pacientes imunocompetentes, e a identificação das MNT é importante para estabelecer o diagnóstico correto e a decisão terapêutica mais adequada. O método PRA-hsp65 é útil para identificar espécies de MNT e pode ser implantado em laboratórios de biologia molecular não especializados em micobactérias.

OBJECTIVE: To identify mycobacterial species in the sputum of patients suspected of having pulmonary tuberculosis and to determine the impact that the acquisition of this knowledge has on the therapeutic approach. METHODS: We evaluated 106 patients suspected of having pulmonary tuberculosis and referred to the pulmonology department of a public hospital in the city of Teresina, Brazil. Morning sputum specimens were evaluated for the presence of mycobacteria by sputum smear microscopy and culture. We used PCR and restriction enzyme analysis of the hsp65 gene (PRA-hsp65) to identify the strains of mycobacteria isolated in culture. RESULTS: A total of 206 sputum samples were analyzed. Patient ages ranged from 15 to 87 years, and 67 percent were male. There was cough in 100 percent of the cases. The predominant radiographic pattern was moderate disease, observed in 70 percent. Smear positivity was 76 percent, and isolation in culture occurred in 91 percent of the cultures. Traditional tests identified nontuberculous mycobacteria (NTM) in 9 percent of the isolates. The PRA-hsp65 method confirmed these data, showing seven band patterns that were able to identify the isolated species of NTM: Mycobacterium kansasii; M. abscessus 1; M. abscessus 2; M. smegmatis; M. flavescens 1; M. gordonae 5; and M. gordonae 7. All of the patients with NTM were over 60 years of age, and bronchiectasis was seen in 88 percent of the X-rays. There were two cases of reinfection, initially attributed to M. abscessus and M. kansasii. CONCLUSIONS: In immunocompetent patients, NTM can infect the lungs. It is important to identify the specific NTM in order to establish the correct diagnosis and choose the most appropriate therapeutic regimen. The PRA-hsp65 method is useful in identifying NTM species and can be implemented in molecular biology laboratories that do not specialize in the identification of mycobacteria.

Validation of an immunochromatographic assay kit for the identification of the Mycobacterium tuberculosis complex

The performance of the immunochromatographic assay, SD BIOLINE TB Ag MPT64 RAPID®, was evaluated in Madagascar. Using mouse anti-MPT64 monoclonal antibodies for rapid discrimination between the Mycobacterium tuberculosis complex and nontuberculous mycobacteria, the kit was tested on mycobacteria and other pathogens using conventional methods as the gold standard. The results presented here indicate that this kit has excellent sensitivity (100 percent) and specificity (100 percent) compared to standard biochemical detection and can be easily used for the rapid identification of M. tuberculosis complex.

Aplicación de RPC-PLFR en el diagnóstico de micobacterias no tuberculosas / Application of PCR-RFLP in the diagnosis of non-tuberculous mycobacteria

La amplificación por reacción de la polimersa en cadena (RPC) de un fragmento del gen hsp65, seguido del análisis del polimorfismo de la longitud de los fragmentos de restricción (PLFR) por las enzimas BstEll y Haelll, ha demostrado ser muy útil en la identificación de micobacterias no tuberculosas (MNT). En el presente trabajo se les realizó una batería de pruebas bioquímicas así como la RPC-PLFR a un total de 13 cepas de referencia y 46 cepas recibidas en el laboratorio. Los resultados de las pruebas bioquímicas estuvieron disponibles entre 4 a 6 semanas, a diferencia de la RPC-PLFR que requirieron de sólo 48 horas. En ambos métodos, las especies detectadas con mayor frecuencia fueron Mycobacetrium intracellulare, M. kansasii y M. fortuitum. La RPC-PLFR es un método rápido, sencillo y eficaz. Su aplicación en los Laboratorios de Referencia pudiera ser de gran utilidad para el diagnóstico de MNT.

The amplification of a fragment from hsp65 gene by polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) analysis with BstEll and Haelll restriction enzymes has demonstrated to be very useful for identification of Non-Tuberculous Mycobacteria (NTM). The biochemical tests as well as the PCR-RFLP were carried out in 13 reference strains and 46 strains received in the laboratory. The results by biochemical tests were available in 4-6 weeks whereas the PCR-RFLP only required 48 hours. In both methods, Mycobacterium intracellulare, M. kansasii and M. fortuitum were the most frequently detected species. The PCR-RFLP method is fast, cheap and simple. Its application in Reference Laboratories could be very useful for diagnosis of NTM.

Infections with atypical mycobacteria in renal transplant recipients.

Infections due to atypical mycobacteria are infrequent in renal transplant recipients but they cause serious morbidity. These pathogens are common in patients with acquired immune deficiency syndrome (AIDS). We report four proven cases of infections caused with atypical mycobacteriae from 1997 to 2003, by different organisms namely, M. chelonei, M.fortuitum, M. abcessus and M. terrae in renal transplant recipients. Infection with M. terrae documented here is the first occurrence in a renal transplant patient. Histopathological examination of aspirates or biopsy specimens from involved areas and staining and culture for mycobacteriae are essential for diagnosis. Treatment involves antimycobacterial therapy, reduction in immunosuppression and surgery, if indicated. Atypical mycobacterial infections, though currently uncommon, are significant and could prove to be an emerging pathogen in renal transplant recipients in the context of the AIDS epidemic in India.

Identificación de especies micobacterianas en Cuba / Identification of species mycrobacteriums in Cuba

El aumento de las infecciones producidas por micobacterias ambientales u oportunistas (MAO) coincide en muchos casos con el declive de la infección tuberculosa y el incremento de la infección por el virus de inmunodeficiencia humana (VIH). Sobre todo en los países desarrollados donde se está produciendo un aumento global de la incidencia de enfermedad por MAO, y las micobacteriosis principalmente en pacientes inmunodeficientes cada vez son más frecuentes. En este trabajo se estudiaron 80 cepas recibidas en el Laboratorio Nacional de Referencia e Investigaciones de TB y Micobacterias procedentes de diferentes Centros Provinciales de Higiene y Epidemiología, con el fin de conocer el comportamiento en nuestro país de las especies de interés clínico. Encontramos que al clasificar las micobacterias aisladas según los grupos establecidos por Runyon los siguiente: los grupos con mayor frecuencia fueron el Grupo III y el Grupo IV, por especie las de mayor por ciento de aislamiento fueron: Mycobacterium avium-intracellulare, Mycobacterium fortuitum, Mycobacterium chelonae y Mycobacerium malmoense. Estos estudios son de gran utilidad en los laboratorios de Micobacteriología, pues de esta forma se puede llegar a conocer cuales son las especies predominantes en la población y poder establecer una eficaz vigilancia sobre este tipo de infecciones sobre todo en pacientes inmunodeficientes, grupo más sensibles a estas infecciones.

Identificación rápida de micobacterias no tuberculosas mediante análisis de patrones de restricción / Rapid identification of non tuberculous mycobacteria by restriction pattern analysis

Background: The frequency of diseases caused by non tuberculous mycobacteria has increased in the last years. Their clinical diagnosis is difficult, mainly in immunocompromised patients. The identification of these mycobacteria by traditional methods is based on phenotypic characteristics and the results are obtained two to four weeks after their isolation in primary cultures. Aim: To report a new identification method for non tuberculous mycobacteria. Material and methods: The restriction pattern analysis method was implemented. It is based on the amplification, using polymerase chain reaction (PCR), of a polymorphic region of 440 base pairs that codifies Hsp65 protein, followed by a digestion with BstE II and Hae III restriction enzymes. The results were compared with patterns established for each strain. Results: Sixty four strains of mycobacteria obtained from clinical samples and seven reference mycobacteria, were identified using the traditional methods and restriction pattern analysis. The latter method identified the same strain as the former in 87.5% of cases. In the remainder 12.5% of cases there was no agreement between both methods. In these, the sequencing of a fragment of a gene that codifies 16S ribosomal RNA, confirmed the correct identification by restriction patterns. Conclusions: Restriction pattern analysis is a rapid identification method for non tuberculous mycobaterial strains.

Characterization of mycobacteria isolated from bovines by PRA-targetting hsp 65 gene region.

Bovine tuberculosis caused by the bacterium Mycobacterium bovis is a major infectious disease of animals and has zoonotic importance for humans. Even though the incidence is believed to be very low in India, human tuberculosis caused by M. bovis has been increasingly recognized in many other countries of the world. As differentiation of mycobacterial species take long time, a method for the rapid identification of mycobacteria isolated from bovine samples to the species level was used, which is based on polymerase chain reaction (PCR) of the gene encoding for the 65-kD protein followed by restriction analysis. The method involves restriction enzyme analysis of PCR products obtained with primers common to all mycobacteria and generate M. tuberculosis complex specific pattern. PRA was performed on 33 bovine isolates of which 90.9% (30/33) isolates were identified clearly as M. tuberculosis complex, M. fortuitum, M. phlei and M. smegmatis using restriction enzyme Hae III.