Multilocus enzyme electrophoresis analysis of rapidly-growing mycobacteria: an alternative tool for identification and typing.

OBJECTIVES: Rapidly growing mycobacteria (RGM) have emerged as important pathogens in clinical settings, associated with esthetic procedures and postsurgical infections, pulmonary infections among cystic fibrosis patients, and other structural pulmonary diseases. Microorganisms belonging to Mycobacterium abscessus-Mycobacterium chelonae and to Mycobacterium fortuitum groups have frequently been associated with outbreaks and various epidemics. In the present study, RGM strains were characterized in order to investigate molecular markers based on proteomic analysis. METHODS: Multilocus enzyme electrophoresis (MLEE) was used for species identification and clonal analysis of RGM recovered from postsurgical wound infections during an epidemic. The study included 30M. abscessus subsp. bolletii clinical isolates, most belonging to the BRA100 clone (epidemic in Rio de Janeiro city), as well as 16 RGM ATCC reference strains. RESULTS: Molecular typing allowed the detection of diversity in the studied population and revealed species-specific isoenzymatic patterns. Additionally, the clonal relationship among M. abscessus subsp. bolletii outbreak isolates, as examined using MLEE, was markedly consistent. CONCLUSIONS: Isoenzymatic characterization was found to be a useful molecular tool to identify RGM species and to determine the relatedness among closely related M. abscessus subsp. bolletii isolates. This may be considered a powerful approach for epidemiological studies on RGM.

Liquid vs Solid Culture Medium to Evaluate Proportion and Time to Change in Management of Suspects of Tuberculosis-A Pragmatic Randomized Trial in Secondary and Tertiary Health Care Units in Brazil.

BACKGROUND: The use of liquid medium (MGIT960) for tuberculosis (TB) diagnosis was recommended by WHO in 2007. However, there has been no evaluation of its effectiveness on clinically important outcomes. METHODS AND FINDINGS: A pragmatic trial was carried out in a tertiary hospital and a secondary health care unit in Rio de Janeiro City, Brazil. Participants were 16 years or older, suspected of having TB. They were excluded if only cerebral spinal fluid or blood specimens were available for analysis. MGIT960 technique was compared with the Lowenstein-Jensen (LJ) method for laboratory diagnosis of active TB. Primary outcome was the proportion of patients who had their initial medical management changed within 2 months after randomisation. Secondary outcomes were: mean time for changing the procedure, patient satisfaction with the overall treatment and adverse events. Data were analysed by intention-to-treat. Between April 2008 and September 2011, 693 patients were enrolled (348 to MGIT, 345 to LJ). Smear and culture results were positive for 10% and 15.7% of participants, respectively. Patients in the MGIT arm had their initial medical management changed more frequently than those in the LJ group (10.1% MGIT vs 3.8% LJ, RR 2.67 95% CI 1.44-.96, p = 0.002, NNT 16, 95% CI 10-39). Mean time for changing the initial procedure was greater in LJ group at both sites: 20.0 and 29.6 days in MGIT group and 52.2 and 64.3 in LJ group (MD 33.5, 95% CI 30.6-36.4, p = 0.0001). No other important differences were observed. CONCLUSIONS: This study suggests that opting for the MGIT960 system for TB diagnosis provides a promising case management model for improving the quality of care and control of TB. TRIAL REGISTRATION: Controlled-Trials.com ISRCTN79888843.

Mycobacterium tuberculosis isolates from Rio de Janeiro reveal unusually low correlation between pyrazinamide resistance and mutations in the pncA gene.

It has been widely accepted, that pyrazinamide (PZA) resistance in Mycobacterium tuberculosis is correlated with mutations in the pncA gene. But since years researchers have been puzzled by the fact that up to 30% of PZA resistant strains do not show any correlation between PZA resistance and mutations in the pncA gene, and thus may vary with geographic area. The objective of the study was to investigate the correlation between PZA susceptibility and mutations in pncA gene in M. tuberculosis isolates from individuals living in a highly endemic area. Therefore we analyzed drug resistant and multidrug resistant (MDR) isolates from patients in Rio de Janeiro, Brazil. From a total of 97 clinical isolates of M. tuberculosis 35 were identified as PZA resistant, 24/35 strains did not show PZase activity and 15/24 (62.5%) strains possess mutation in the pncA gene. This is a low correlation between PZA resistance and PZase activity (68.6%) and even lower correlation between PZA resistance and the presence of mutation in pncA gene (45.7%). Most of the mutations found were conserved near the active site or metal binding site of PZase. The 146A>C mutation was found both in PZA resistant and susceptible isolates, suggesting that this mutation may not fully associated with PZA resistance. Of the mutations found, three have not been previously described. The insertions 192-193 TCCTCGTC and 388-389 AGGTCGATG, although found before, here was found to be a short tandem repeat and in one strain, insertion of the IS6110 was observed 55nt upstream of the gene. All PZA resistant isolates had no mutation in the gene coding ribosomal protein S1 (rpsA), which has recently been proposed as alternate target for pyrazinoic acid (POA). The results show a low association of PZA resistance and pncA gene mutations in a selected patient group from an highly endemic area. Our findings point out that the phenotypic susceptibility testing remains important for the detection of PZA-resistant M. tuberculosis.

Correlation between resistance to pyrazinamide and resistance to other antituberculosis drugs in Mycobacterium tuberculosis strains isolated at a referral hospital.

The correlation between resistance to pyrazinamide (PZA) and resistance to other first-line antituberculosis drugs was investigated in 395 Mycobacterium tuberculosis strains isolated from clinical specimens, representing 14% of the overall number of M. tuberculosis isolates obtained between 2003 and 2008 at the laboratory of a referral university hospital for tuberculosis. A high correlation was found between resistance to PZA and multidrug resistance, as well as between PZA resistance and resistance to rifampin, isoniazid, and ethambutol (p < 0.01 for all). These results highlight the importance of performing PZA susceptibility testing prior to the prescription of this drug in order to treat drug-resistant and multidrug-resistant tuberculosis.

Comparative evaluation under routine conditions of the nitrate reduction assay, the proportion assay and the MGIT 960 assay for drug susceptibility testing of clinical isolates of Mycobacterium tuberculosis.

The performance of the nitrate reductase assay (NRA) was compared with the proportion method (PM) on Lowenstein-Jensen medium and the BACTEC MGIT960 assay under routine conditions using 160 clinical isolates of Mycobacterium tuberculosis with a high proportion of resistant strains. The mean time to obtain results was 8.8 days and the overall agreements between NRA and PM and NRA and M960 were 95% and 94%, respectively. NRA was easy to perform and represents a useful tool for the rapid screening of drug-resistant M. tuberculosis strains in low-resource countries.

Comparative evaluation under routine conditions of the nitrate reduction assay, the proportion assay and the MGIT 960 assay for drug susceptibility testing of clinical isolates of Mycobacterium tuberculosis

The performance of the nitrate reductase assay (NRA) was compared with the proportion method (PM) on Lowenstein-Jensen medium and the BACTEC MGIT960 assay under routine conditions using 160 clinical isolates of Mycobacterium tuberculosis with a high proportion of resistant strains. The mean time to obtain results was 8.8 days and the overall agreements between NRA and PM and NRA and M960 were 95 percent and 94 percent, respectively. NRA was easy to perform and represents a useful tool for the rapid screening of drug-resistant M. tuberculosis strains in low-resource countries.

Identification of Mycobacterium bovis among mycobacterial isolates from human clinical specimens at a university hospital in Rio de Janeiro, Brazil.

In 2005 and 2006, 8,121 clinical specimens submitted to the Mycobacteriology Laboratory of the Clementino Fraga Filho University Hospital/Thoracic Diseases Institute, in the city of Rio de Janeiro, Brazil, were inoculated on Löwenstein-Jensen medium containing glycerol and pyruvate. There were 79 mycobacteria isolates that presented growth only on pyruvate-containing medium, and those isolates were selected for the presumptive identification of Mycobacterium bovis. The selected isolates were screened with biochemical tests, PCR amplification (with the specific primers Rv0577 and Rv1510), and pyrazinamide susceptibility tests. All of the strains isolated showed specific phenotypical and genotypical patterns characteristic of M. tuberculosis, and no M. bovis strains were detected.

Identificação de Mycobacterium bovis em cepas micobacterianas isoladas espécimes clínicos humanos em um complexo hospitalar na cidade do Rio de Janeiro / Identification of Mycobacterium bovis among mycobacterial isolates from human clinical specimens at a university hospital in Rio de Janeiro, Brazil

Entre 2005 e 2006, 8.121 espécimes clínicos enviados ao Laboratório de Micobactérias do Hospital Universitário Clementino Fraga Filho/Instituto de Doenças do Tórax, no Rio de Janeiro, RJ, foram inoculados em meio Löwenstein-Jensen contendo glicerol e piruvato. Desses espécimes, 79 isolados de micobactérias tiveram crescimento somente em meio com piruvato, sendo selecionados para a identificação presuntiva de Mycobacterium bovis. Esses isolados foram submetidos à identificação por testes bioquímicos, amplificação por PCR com primers específicos (Rv0577 e Rv1510) e teste de suscetibilidade à pirazinamida. Todas as cepas apresentaram padrões fenotípicos e genotípicos de M. tuberculosis, não sendo detectado M. bovis.

In 2005 and 2006, 8,121 clinical specimens submitted to the Mycobacteriology Laboratory of the Clementino Fraga Filho University Hospital/Thoracic Diseases Institute, in the city of Rio de Janeiro, Brazil, were inoculated on Löwenstein-Jensen medium containing glycerol and pyruvate. There were 79 mycobacteria isolates that presented growth only on pyruvate-containing medium, and those isolates were selected for the presumptive identification of Mycobacterium bovis. The selected isolates were screened with biochemical tests, PCR amplification (with the specific primers Rv0577 and Rv1510), and pyrazinamide susceptibility tests. All of the strains isolated showed specific phenotypical and genotypical patterns characteristic of M. tuberculosis, and no M. bovis strains were detected.

Identification of nontuberculous mycobacteria isolated from clinical sterile sites in patients at a university hospital in the city of Rio de Janeiro, Brazil.

OBJECTIVE: To identify nontuberculous mycobacteria (NTM) isolated from sterile sites in patients hospitalized between 2001 and 2006 at the Clementino Fraga Filho University Hospital, located in the city of Rio de Janeiro, Brazil. METHODS: During the study period, 34 NTM isolates from sterile sites of 14 patients, most of whom were HIV-positive, were submitted to phenotypic identification and hsp65 PCR-restriction enzyme analysis (PRA). RESULTS: Most isolates were identified as Mycobacterium avium, followed by M. monacense, M. kansasii, and M. abscessus. CONCLUSIONS: The combination of PRA, a relatively simple and inexpensive method, with the evaluation of a few phenotypic characteristics can allow NTM to be accurately identified in the routine of clinical laboratories.

Identificação de micobactérias não tuberculosas isoladas de sítios estéreis em pacientes em um hospital universitário na cidade do Rio de Janeiro / Identification of nontuberculous mycobacteria isolated from clinical sterile sites in patients at a university hospital in the city of Rio de Janeiro, Brazil

OBJETIVO: Identificar micobactérias não tuberculosas (MNT) isoladas de sítios estéreis em pacientes internados no Hospital Universitário Clementino Fraga Filho, Rio de Janeiro (RJ) entre 2001 e 2006. MÉTODOS: Durante o período do estudo, 34 isolados de MNT de sítios estéreis de 14 pacientes, a maioria HIV positivos, foram submetidos a identificação fenotípica e hsp65 PCR-restriction enzyme analysis (PRA, análise por enzimas de restrição por PCR do gene hsp65). RESULTADOS: A maioria dos isolados foi identificada como Mycobacterium avium, seguida por M. monacense, M. kansasii e M. abscessus em menores proporções. CONCLUSÕES: A combinação de PRA, um método relativamente simples e de baixo custo, com algumas características fenotípicas pode fornecer a identificação correta de MNT na rotina de laboratórios clínicos.

OBJECTIVE: To identify nontuberculous mycobacteria (NTM) isolated from sterile sites in patients hospitalized between 2001 and 2006 at the Clementino Fraga Filho University Hospital, located in the city of Rio de Janeiro, Brazil. METHODS: During the study period, 34 NTM isolates from sterile sites of 14 patients, most of whom were HIV-positive, were submitted to phenotypic identification and hsp65 PCR-restriction enzyme analysis (PRA). RESULTS: Most isolates were identified as Mycobacterium avium, followed by M. monacense, M. kansasii, and M. abscessus. CONCLUSIONS: The combination of PRA, a relatively simple and inexpensive method, with the evaluation of a few phenotypic characteristics can allow NTM to be accurately identified in the routine of clinical laboratories.

Epidemic of postsurgical infections caused by Mycobacterium massiliense.

An epidemic of infections after video-assisted surgery (1,051 possible cases) caused by rapidly growing mycobacteria (RGM) and involving 63 hospitals in the state of Rio de Janeiro, Brazil, occurred between August 2006 and July 2007. One hundred ninety-seven cases were confirmed by positive acid-fast staining and/or culture techniques. Thirty-eight hospitals had cases confirmed by mycobacterial culture, with a total of 148 available isolates recovered from 146 patients. Most (n = 144; 97.2%) isolates presented a PRA-hsp65 restriction pattern suggestive of Mycobacterium bolletii or Mycobacterium massiliense. Seventy-four of these isolates were further identified by hsp65 or rpoB partial sequencing, confirming the species identification as M. massiliense. Epidemic isolates showed susceptibility to amikacin (MIC at which 90% of the tested isolates are inhibited [MIC(90)], 8 microg/ml) and clarithromycin (MIC(90), 0.25 microg/ml) but resistance to ciprofloxacin (MIC(90), >or=32 microg/ml), cefoxitin (MIC(90), 128 microg/ml), and doxycycline (MIC(90), >or=64 microg/ml). Representative epidemic M. massiliense isolates that were randomly selected, including at least one isolate from each hospital where confirmed cases were detected, belonged to a single clone, as indicated by the analysis of pulsed-field gel electrophoresis (PFGE) patterns. They also had the same PFGE pattern as that previously observed in two outbreaks that occurred in other Brazilian cities; we designated this clone BRA100. All five BRA100 M. massiliense isolates tested presented consistent tolerance to 2% glutaraldehyde. This is the largest epidemic of postsurgical infections caused by RGM reported in the literature to date in Brazil.

Correlations of mutations in katG, oxyR-ahpC and inhA genes and in vitro susceptibility in Mycobacterium tuberculosis clinical strains segregated by spoligotype families from tuberculosis prevalent countries in South America.

BACKGROUND: Mutations associated with resistance to rifampin or streptomycin have been reported for W/Beijing and Latin American Mediterranean (LAM) strain families of Mycobacterium tuberculosis. A few studies with limited sample sizes have separately evaluated mutations in katG, ahpC and inhA genes that are associated with isoniazid (INH) resistance. Increasing prevalence of INH resistance, especially in high tuberculosis (TB) prevalent countries is worsening the burden of TB control programs, since similar transmission rates are noted for INH susceptible and resistant M. tuberculosis strains. RESULTS: We, therefore, conducted a comprehensive evaluation of INH resistant M. tuberculosis strains (n = 224) from three South American countries with high burden of drug resistant TB to characterize mutations in katG, ahpC and inhA gene loci and correlate with minimal inhibitory concentrations (MIC) levels and spoligotype strain family. Mutations in katG were observed in 181 (80.8%) of the isolates of which 178 (98.3%) was contributed by the katG S315T mutation. Additional mutations seen included oxyR-ahpC; inhA regulatory region and inhA structural gene. The S315T katG mutation was significantly more likely to be associated with MIC for INH >or=2 microg/mL. The S315T katG mutation was also more frequent in Haarlem family strains than LAM (n = 81) and T strain families. CONCLUSION: Our data suggests that genetic screening for the S315T katG mutation may provide rapid information for anti-TB regimen selection, epidemiological monitoring of INH resistance and, possibly, to track transmission of INH resistant strains.

Estudo comparativo de métodos complementares para o diagnóstico da tuberculose bovina em animais reagentes à tuberculinização / Comparative study of complementary diagnostic methods of bovine tuberculosis on skin-test reactive cattle

A tuberculose bovina é uma enfermidade infecciosa de ocorrência mundial. O teste intradérmico é o método padrão para seu diagnóstico da tuberculose, embora possa carecer de sensibilidade e especificidade. O presente estudo teve como objetivo avaliar diferentes métodos complementares para o diagnóstico da tuberculose bovina incluindo os exames macroscópicos de tecidos, histopatológico, bacteriológico e ELISA. Um total de 97 bovinos reagentes à prova de tuberculinização foi testado, e amostras de tecidos e soro foram colhidas no momento do abate. Do total de bovinos examinados, 70 (72,16 por cento) apresentaram lesão macroscópica sugestiva de tuberculose. Na avaliação histopatológica, 63 animais (64,95 por cento) apresentaram lesão granulomatosa característica. Assim, a histopatologia concordou com a avaliação macroscópica em 92,78 por cento das amostras. Em 47 (48,45 por cento) amostras foram visualizados Bacilos Álcool Ácido Resistentes (BAAR), todas positivas à histopatologia ou à avaliação macroscópica.(ACom relação à cultura bacteriológica, foram isolados Mycobacterium bovis em apenas 11 amostras (11,34 por cento). Quanto ao desempenho do teste de ELISA, 33 (34,02 por cento) soros foram reativos. O exame macroscópico detalhado associado ao exame histopatológico, devido à sua alta especificidade, são recomendados como ferramentas complementares e podem ser utilizados para confirmar os casos duvidosos no abatedouro.

Bovine tuberculosis is an infectious disease of world-wide occurrence. The intradermal tuberculin test is the standard test for its detection, but it can lack both sensitivity and specificity. The purpose of this study was to compare the efficiency of different complementary methods for the diagnosis of bovine tuberculosis such as macroscopic, histopathological, bacteriological analysis and Enzyme-linked immunosorbent assay (ELISA). A total of 97 skin-test reactive animals were studied, and tissues and serum samples were collected from all of them at the moment of the slaughter. Seventy animals (72.16 percent) showed macroscopic lesions suggestive of tuberculosis. At the histopathological exam, sixty three (64.95 percent) presented typical granulomatous lesions. Therefore, histopathology agreed with macroscopic inspection in 92.78 percent of the samples. Acid-fast bacilli were observed in tissues samples from 47 (48.45 percent) animals, all of them also positive either at macroscopic inspection or to histopathology. In reference to the bacterial culture, only 11 (11.34 percent) tissues samples yielded Mycobacterium bovis. Thirty three (34.02 percent) serum samples were reactive at ELISA. Detailed visual inspection as well as histopathology, due to its high specificity, are suggested as complementary tools and may be used for confirming doubtful cases of bovine tuberculosis at the slaughterhouse.

Molecular diversity of Mycobacterium tuberculosis strains in a slum area of Rio de Janeiro, Brazil / Diversidade molecular de cepas de Mycobacterium tuberculosis em uma região de favela da cidade do Rio de Janeiro

This retrospective molecular study involving restriction fragment length polymorphism, using insertion sequence 6110 as a marker, was conducted in order to provide an initial insight into the genetic diversity of Mycobacterium tuberculosis strains isolated in the slums of the Complexo de Manguinhos, located in the city of Rio de Janeiro, Brazil. Of the 67 strains evaluated, 23 (34.3 percent) were found to belong to clusters (total clusters, 10). Household and social chains of transmission were associated with clustering, in 20 percent and 60 percent, respectively. Living in the Conjunto Habitacional Programado 2 slum was associated with clustering. Although not significant, it is relevant that 26 percent of the clustered strains presented primary resistance. These findings, although possibly underestimating the prevalence due to the failure to analyze all strains, could help improve the local tuberculosis control program.

Este estudo retrospectivo envolvendo polimorfismo de fragmento de restrição e utilizando como marcador a seqüência de inserção 6110, foi realizado para fornecer informações iniciais quanto à diversidade genética das cepas de Mycobacterium tuberculosis isoladas em favelas do Complexo de Manguinhos, na cidade do Rio de Janeiro. Das 67 cepas isoladas, 23 (34,3 por cento) foram agrupadas em clusters (total de clusters, 10). A transmissão entre comunicantes domiciliares e extra-domicialiares estave associada a 20 por cento e 60 por cento dos clusters, respectivamente. Ser morador do Conjunto Habitacional Programado 2 foi associado à presença de clusters. Embora não significativo, é relevante o fato de que 26 por cento das cepas em cluster apresentaram resistência primária. Estes achados, embora possivelmente subestimados devido à impossibilidade de analisar todas as cepas, fornecem subsídios para a melhoria do programa local de controle da tuberculose.

An alternative for the preadsorption step in the paratuberculosis serodiagnosis: Mycobacterium fortuitum / Mycobacterium fortuitum: uma alternativa para a etapa de pré-adsorção no sorodiagnóstico da paratuberculose

ELISAs for paratuberculosis employ a preadsorption step with Mycobacterium phlei to diminish unspecific reactions As M. fortuitum is one of the most frequent environmental mycobacteria, the purpose of this pilot study was to evaluate its use as an alternative for the preadsorption in ELISAs for paratuberculosis. Results suggest that M. fortuitum can be an alternative instead of or associated to M. phlei with comparable results (κ > 0.8) to conventional ELISAs using M. phlei as a preadsorption antigen.

Ensaios de sorodiagnóstico de paratuberculose (ELISA) utilizam Mycobacterium phlei na etapa de pré-adsorção para diminuir reações inespecíficas. Uma vez que M. fortuitum é uma das micobactérias atípicas mais isoladas no Brasil, o objetivo central deste estudo foi averiguar a possibilidade de sua utilização como antígeno da etapa de pré-adsorção destes testes. Os resultados sugerem que M. fortuitum apresentou resultados comparáveis (κ > 0.8) aos alcançados com M. phlei e que, portanto poderia ser uma alternativa ao invés ou associado a M. phlei na etapa de pré-adsorção de ELISAs para paratuberculose.

Molecular diversity of Mycobacterium tuberculosis strains in a slum area of Rio de Janeiro, Brazil.

This retrospective molecular study involving restriction fragment length polymorphism, using insertion sequence 6110 as a marker, was conducted in order to provide an initial insight into the genetic diversity of Mycobacterium tuberculosis strains isolated in the slums of the Complexo de Manguinhos, located in the city of Rio de Janeiro, Brazil. Of the 67 strains evaluated, 23 (34.3%) were found to belong to clusters (total clusters, 10). Household and social chains of transmission were associated with clustering, in 20% and 60%, respectively. Living in the Conjunto Habitacional Programado 2 slum was associated with clustering. Although not significant, it is relevant that 26% of the clustered strains presented primary resistance. These findings, although possibly underestimating the prevalence due to the failure to analyze all strains, could help improve the local tuberculosis control program.

A retrospective study of the epidemiological aspects of tuberculosis in the Complexo de Manguinhos, an urban slum area in Rio de Janeiro, Brazil, 2000-2002.

To describe some aspects of tuberculosis in a low-income community (the Complexo de Manguinhos, in Rio de Janeiro, Brazil), a retrospective study was carried out. Of the 290 cases reported in the 2000-2002 period, 75.8% were new cases. The annual incidence rates were 157/100,000 (2000), 205/100,000 (2001), and 145/100,000 (2002). Although there was a tendency toward a decrease in the number of cases over the period studied, the difference was not significant, suggesting that tuberculosis continues to be endemic in the area. Therefore, despite the existence of local public health care services, more efficient strategies should be implemented in order to increase the effectiveness of tuberculosis control programs in the area.

Drug resistance in Mycobacterium tuberculosis strains isolated from sputum samples from symptomatic outpatients: Complexo de Manguinhos, Rio de Janeiro, Brazil.

This study aimed to assess drug resistance in Mycobacterium tuberculosis strains isolated from sputum samples. To that end, sputum samples were collected from 263 patients suspected of having tuberculosis. All subjects lived in the Complexo de Manguinhos, which is located in the city of Rio de Janeiro, Brazil. Cultures testing positive between October of 2000 and December of 2002 were tested to determine strain susceptibility to isoniazid, rifampicin, streptomycin, ETHionamide, and ETHambutol. Of the 75 patients diagnosed with tuberculosis, resistance to at least one of the drugs was found in 16 (21.4%). Of those 16 patients, 8 (50%) were new cases, and 8 (50%) had previously been treated. Multidrug-resistant tuberculosis was identified in 8 (10.6%) of the 75 patients, being associated with previous treatment in 6 (8%). The incidence of multidrug-resistant tuberculosis might have been underestimated, since M. tuberculosis was not isolated from all of the samples testing positive for acid-fast bacilli. However, at least, our findings shed some light on the problem.

Resistência a drogas em cepas de mycobacterium tuberculosis isoladas de amostras de escarro de pacientes ambulatoriais sintomáticos: Complexo de Manguinhos, Rio de Janeiro, Brasil / Drug resistance in mycobacterium tuberculosis strains isolated from sputum samples from symptomatic outpatients: Complexo de Manguinhos, Rio de Janeiro, Brazil

Para descrever a resistência a drogas em cepas de Mycobacterium tuberculosis isoladas de amostras de escarro de 263 pacientes suspeitos de tuberculose moradores do Complexo de Manguinhos, Rio de Janeiro, Brasil, as culturas positivas entre outubro de 2000 e dezembro de 2002 foram submetidas a teste de sensibilidade para isoniazida, rifampicina, estreptomicina, etionamida e etambutol. Resistência a qualquer das drogas foi encontrada em 21,4 por cento (16/75) dos pacientes diagnosticados com tuberculose. Destes, 50 por cento (8/16) eram casos novos e 50 por cento (8/16) eram casos com tratamento anterior. A tuberculose multirresistente foi encontrada em 10,6 por cento (8/75) do total de pacientes, estando associada a tratamento anterior em 8 por cento (6/75) deles. Nossos resultados podem ter sido subestimados, pois M. tuberculosis não pôde ser isolado em todas as amostras positivas para bacilos álcool-ácido resistentes. Contudo, eles pelo menos revelam parte do problema.

This study aimed to assess drug resistance in Mycobacterium tuberculosis strains isolated from sputum samples. To that end, sputum samples were collected from 263 patients suspected of having tuberculosis. All subjects lived in the Complexo de Manguinhos, which is located in the city of Rio de Janeiro, Brazil. Cultures testing positive between October of 2000 and December of 2002 were tested to determine strain susceptibility to isoniazid, rifampicin, streptomycin, ETHionamide, and ETHambutol. Of the 75 patients diagnosed with tuberculosis, resistance to at least one of the drugs was found in 16 (21.4 percent). Of those 16 patients, 8 (50 percent) were new cases, and 8 (50 percent) had previously been treated. Multidrug-resistant tuberculosis was identified in 8 (10.6 percent) of the 75 patients, being associated with previous treatment in 6 (8 percent). The incidence of multidrug-resistant tuberculosis might have been underestimated, since M. tuberculosis was not isolated from all of the samples testing positive for acid-fast bacilli. However, at least, our findings shed some light on the problem.

Do DNA extraction methods and Taq polimerase quality improve the double repetitive element (DRE) PCR typing method for Mycobacterium tuberculosis strains?

Double repetitive element (DRE) PCR amplification is a simple Mycobacterium tuberculosis typing method, however amplification failure or poor resolution of bands commit its efficacy. In order to verify if whether or not these features could be minimized by improving DNA extraction procedures or Taq polymerise quality, DRE-PCR was performed on 24 M. tuberculosis DNA samples extracted by heat-shock, mechanical and enzymatic methods applying conventional and hot start Taq pol. We demonstrated that when dealing with the Mycobacterium tuberculosis DRE-PCR typing method, Taq pol of better quality might be more important to improve amplification than the DNA extraction method.

Amplificação de duplo elemento repetido (DRE) por PCR é um método simples para tipagem de Mycobacterium tuberculosis, entretanto falha ou a baixa resolução das bandas na amplificação compromete a eficiência do método. Com o objetivo de verificar se estes problemas podem ou não ser minimizados pela utilização de diferentes procedimentos de extração de DNA ou de qualidades de Taq polimerase, DRE-PCR foi ensaiado em 24 amostras de DNA de M. tuberculosis extraídos pelos métodos de choque-térmico, - mecânico e enzimático utilizando Taq polimerase convencional e hot start Taq pol. Foi demonstrado que a qualidade da Taq pol utilizada talvez seja mais importante para uma melhor amplificação que o método de extração de DNA empregado.