Tuberculosis in a southern Brazilian prison.

The occurrence of tuberculosis (TB) in prisons has been described as an alarming public health problem in many countries, especially in developing nations. The objective of this study was to conduct a survey among prisoners with TB respiratory symptoms in order to estimate the incidence of the disease, to analyze the drug susceptibility profile and genotype the isolates of Mycobacterium tuberculosis in the city of Charqueadas, southern of Brazil. The TB incidence was 55/1,900 inhabitants in the prison; this corresponds to an incidence of 3,789/100,000 inhabitants, with a prevalence of 72/1,900 (4,960/100,000 inhabitants). Drug susceptibility test was performed and, among the analyzed isolates, 85% were susceptible to all drugs tested and 15% were resistant to at least one drug, of which 89% were resistant only to isoniazid (INH) or in combination with another drug. The genotype classification of spoligotyping analysis showed that 40% of the isolates belong to LAM family, 22% to T family, 17.5% to Haarlem family, 12.5% to U family and 3% to X family. The shared international spoligotypes most frequently found were 729 (27%), 50 (9.5%), 42 (8%), 53 (8%) and 863 (8%). In conclusion, it was observed that TB in this specific population had been caused, mostly, by strains that have been transmitted in the last few years, as demonstrated by the large level of genotype clustering. In addition, it was found specific large clusters, which were not often found in the general population from the same period and in the same region.

Colorimetric microwell plate reverse-hybridization assay for Mycobacterium tuberculosis detection.

Direct smear examination using Ziehl-Neelsen staining for pulmonary tuberculosis (PTB) diagnosis is inexpensive and easy to use, but has the major limitation of low sensitivity. Rapid molecular methods are becoming more widely available in centralized laboratories, but they depend on timely reporting of results and strict quality assurance obtainable only from costly commercial kits available in high burden nations. This study describes a pre-commercial colorimetric method, Detect-TB, for detecting Mycobacterium tuberculosis DNA in which an oligonucleotide probe is fixed onto wells of microwell plates and hybridized with biotinylated polymerase chain reaction amplification products derived from clinical samples. The probe is capable of hybridising with the IS6110 insertion element and was used to specifically recognise the M. tuberculosis complex. When combined with an improved silica-based DNA extraction method, the sensitivity of the test was 50 colony-forming units of the M. tuberculosis reference strain H37Rv. The results that were in agreement with reference detection methods were observed in 95.2% (453/476) of samples included in the analysis. Sensitivity and specificity for 301 induced sputum samples and 175 spontaneous sputum samples were 85% and 98%, and 94% and 100%, respectively. This colorimetric method showed similar specificity to that described for commercially available kits and may provide an important contribution for PTB diagnosis.

Prospective study applying spoligotyping directly to DNA from sputum samples of patients suspected of having tuberculosis.

A prospective study was designed to evaluate the clinical usefulness of spoligotyping applied directly to sputum samples. Patients suspected of having tuberculosis were recruited at the Hospital Sanatorio Partenon in Porto Alegre, Brazil. Of the 197 samples included in the analysis, 175 (88.8%) yielded a spoligotyping result that fully matched that obtained from culture. Low bacillary samples presented lower accuracy (50%). From 135 Mycobacterium tuberculosis spoligopatterns, we identified 44 different spoligotypes, of which 21 were shared patterns and 23 were unique. T1 was the most frequent subfamily. The genotyping strategy proposed here presents a short turnaround time and could be helpful in providing rapid information on strain identities in a clinical setting.

Phenotypic and genotypic characterization of drug-resistant Mycobacterium tuberculosis strains.

Of 142 pulmonary tuberculosis patients, 76 were considered high risk for the development of resistance, and 24 were confirmed as resistant strain carriers. Resistant isoniazid strains presented a high frequency of katG and ahpC mutations (90%) correlated with an MIC >4 microg/mL (94%). inhA mutations were not seen. rpoB mutations were identified in 78.6% of rifampicin-resistant strains, usually in codon 531 (72.7%), and 75% had an MIC >16 microg/mL. katG and rpoB mutations recognized 88.2% of multidrug-resistant strains and proved more efficient than the katG and rpoB mutations alone. Seventy percent of resistant pyrazinamide strains had pncA mutations between genes 136 and 188, 62.5% of them with an MIC >900 microg/mL. Pyrazinamidase inactivity was not an efficient resistance marker because 60% of pncA-mutated strains maintained enzymatic activity despite displaying good correlation with high resistance levels. Resistant ethambutol strains had embB mutations in codon 306, with MIC >16 microg/mL.

Evaluation and genotyping of multidrug-resistant cases of tuberculosis in southern Brazil.

Sixty isolates of Mycobacterium tuberculosis identified as multidrug-resistant (MDR) at a reference laboratory in Rio Grande do Sul State during the years 1999 and 2000 were analyzed using the IS6110-restriction fragment length polymorphism (RFLP) technique. We also genotyped 202 susceptible strains to compare the genotyping results, as well as the clinical and demographic data. Spacer oligotyping (spoligotyping) analysis was performed for isolates presenting low IS6110 copy number. Patients with identical DNA pattern strains were considered clustered. From 262 isolates, 94 (36%) belonged to 20 distinct RFLP clusters, and after spoligotyping analysis, 89 of the isolates (34%) remained in cluster. MDR isolates did not differ statistically in clustering proportion from susceptible strains. A significant association between the occurrence of MDR and previous tuberculosis (TB) treatment was observed (p < 0.001), as well as failure on TB treatment (p < 0.001). Human immunodeficiency virus (HIV)-positive patients were associated with susceptible tuberculosis (p = 0.024). We also identified that unmarried patients were more likely to develop TB due to recent transmission than married patients (p < 0.005). The introduction of directly observed therapy short-course (DOTS) strategy will be important in decreasing default and failure rates and avoiding the development of new MDR strains.

Tuberculosis in a southern Brazilian prison

The occurrence of tuberculosis (TB) in prisons has been described as an alarming public health problem in many countries, especially in developing nations. The objective of this study was to conduct a survey among prisoners with TB respiratory symptoms in order to estimate the incidence of the disease, to analyze the drug susceptibility profile and genotype the isolates of Mycobacterium tuberculosis in the city of Charqueadas, southern of Brazil. The TB incidence was 55/1,900 inhabitants in the prison; this corresponds to an incidence of 3,789/100,000 inhabitants, with a prevalence of 72/1,900 (4,960/100,000 inhabitants). Drug susceptibility test was performed and, among the analyzed isolates, 85% were susceptible to all drugs tested and 15% were resistant to at least one drug, of which 89% were resistant only to isoniazid (INH) or in combination with another drug. The genotype classification of spoligotyping analysis showed that 40% of the isolates belong to LAM family, 22% to T family, 17.5% to Haarlem family, 12.5% to U family and 3% to X family. The shared international spoligotypes most frequently found were 729 (27%), 50 (9.5%), 42 (8%), 53 (8%) and 863 (8%). In conclusion, it was observed that TB in this specific population had been caused, mostly, by strains that have been transmitted in the last few years, as demonstrated by the large level of genotype clustering. In addition, it was found specific large clusters, which were not often found in the general population from the same period and in the same region.

Colorimetric microwell plate reverse-hybridization assay for Mycobacterium tuberculosis detection

Direct smear examination using Ziehl-Neelsen staining for pulmonary tuberculosis (PTB) diagnosis is inexpensive and easy to use, but has the major limitation of low sensitivity. Rapid molecular methods are becoming more widely available in centralized laboratories, but they depend on timely reporting of results and strict quality assurance obtainable only from costly commercial kits available in high burden nations. This study describes a pre-commercial colorimetric method, Detect-TB, for detecting Mycobacterium tuberculosis DNA in which an oligonucleotide probe is fixed onto wells of microwell plates and hybridized with biotinylated polymerase chain reaction amplification products derived from clinical samples. The probe is capable of hybridising with the IS6110 insertion element and was used to specifically recognise the M. tuberculosis complex. When combined with an improved silica-based DNA extraction method, the sensitivity of the test was 50 colony-forming units of the M. tuberculosis reference strain H37Rv. The results that were in agreement with reference detection methods were observed in 95.2 percent (453/476) of samples included in the analysis. Sensitivity and specificity for 301 induced sputum samples and 175 spontaneous sputum samples were 85 percent and 98 percent, and 94 percent and 100 percent, respectively. This colorimetric method showed similar specificity to that described for commercially available kits and may provide an important contribution for PTB diagnosis.

Characterization of pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis in Brazil.

In this study the nucleotide sequence of the pncA gene from 59 Mycobacterium tuberculosis clinical isolates was analyzed. Mutations in the pncA gene were identified in 29 of 40 pyrazinamide-resistant isolates, and no pyrazinamidase activity was detected in 39 of them. Twelve mutations found in this work have not been described previously.

Análise molecular de cepas de Mycobacterium tuberculosis provenientes de um centro de saúde ambulatorial em Porto Alegre, (RS) / Molecular analysis of Mycobacterium tuberculosis strains from an outpatient clinic in Porto Alegre, (RS)

INTRODUÇAO: A tuberculose é uma doença antiga que ainda se mantém como um dos maiores males da humanidade no século XXI. Nas últimas décadas, o advento de novas tecnologias utilizando os conhecimentos de biologia molecular tem levado a um aumento na investigação da etiologia, detecção e epidemiologia da tuberculose. OBJETIVO: Avaliar o grau de similaridade entre as cepas de Mycobacterium tuberculosis provenientes do setor de tisiologia do Centro de Saúde Navegantes, de Porto Alegre (RS). MÉTODO: Foi realizado um estudo retrospectivo utilizando 55 amostras de escarro de pacientes atendidos ambulatorialmente no Centro de Saúde Navegantes para realização da técnica de RFLP. Os resultados obtidos pela genotipagem foram correlacionados com os dados gerados a partir da epidemiologia convencional. RESULTADOS: Trinta e nove isolados (70,9 por cento) apresentaram padrão único, enquanto dezesseis isolados (29,1 por cento) apresentaram padrões agrupáveis e formaram 8 clusters, com 2 pacientes em cada. Foi encontrada relação epidemiológica em 6 (37,5 por cento) dos 16 pacientes em cluster. CONCLUSAO: A associação adequada entre epidemiologia convencional e genotipagem de M. tuberculosis contribui para um melhor entendimento da dinâmica de transmissão da tuberculose mesmo quando o estudo é realizado em um único local.