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.

Comparison of two laboratory-developed PCR methods for the diagnosis of pulmonary tuberculosis in Brazilian patients with and without HIV infection.

BACKGROUND: Direct smear examination with Ziehl-Neelsen (ZN) staining for the diagnosis of pulmonary tuberculosis (PTB) is cheap and easy to use, but its low sensitivity is a major drawback, particularly in HIV seropositive patients. As such, new tools for laboratory diagnosis are urgently needed to improve the case detection rate, especially in regions with a high prevalence of TB and HIV. OBJECTIVE: To evaluate the performance of two in house PCR (Polymerase Chain Reaction): PCR dot-blot methodology (PCR dot-blot) and PCR agarose gel electrophoresis (PCR-AG) for the diagnosis of Pulmonary Tuberculosis (PTB) in HIV seropositive and HIV seronegative patients. METHODS: A prospective study was conducted (from May 2003 to May 2004) in a TB/HIV reference hospital. Sputum specimens from 277 PTB suspects were tested by Acid Fast Bacilli (AFB) smear, Culture and in house PCR assays (PCR dot-blot and PCR-AG) and their performances evaluated. Positive cultures combined with the definition of clinical pulmonary TB were employed as the gold standard. RESULTS: The overall prevalence of PTB was 46% (128/277); in HIV+, prevalence was 54.0% (40/74). The sensitivity and specificity of PCR dot-blot were 74% (CI 95%; 66.1%-81.2%) and 85% (CI 95%; 78.8%-90.3%); and of PCR-AG were 43% (CI 95%; 34.5%-51.6%) and 76% (CI 95%; 69.2%-82.8%), respectively. For HIV seropositive and HIV seronegative samples, sensitivities of PCR dot-blot (72% vs 75%; p=0.46) and PCR-AG (42% vs 43%; p=0.54) were similar. Among HIV seronegative patients and PTB suspects, ROC analysis presented the following values for the AFB smear (0.837), Culture (0.926), PCR dot-blot (0.801) and PCR-AG (0.599). In HIV seropositive patients, these area values were (0.713), (0.900), (0.789) and (0.595), respectively. CONCLUSION: Results of this study demonstrate that the in house PCR dot blot may be an improvement for ruling out PTB diagnosis in PTB suspects assisted at hospitals with a high prevalence of TB/HIV.

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.

In house colorimetric reverse hybridisation assay for detection of the mutation most frequently associated with resistance to isoniazid in Mycobacterium tuberculosis.

Mutations in the katG gene have been identified and correlated with isoniazid (INH) resistance in Mycobacterium tuberculosis isolates. The mutation AGC-->ACC (Ser-->Thr) at katG315 has been reported to be the most frequent and is associated with transmission and multidrug resistance. Rapid detection of this mutation could therefore improve the choice of an adequate anti-tuberculosis regimen, the epidemiological monitoring of INH resistance and, possibly, the tracking of transmission of resistant strains. An in house reverse hybridisation assay was designed in our laboratory and evaluated with 180 isolates of M. tuberculosis. It could successfully characterise the katG315 mutation in 100% of the samples as compared to DNA sequencing. The test is efficient and is a promising alternative for the rapid identification of INH resistance in regions with a high prevalence of katG315 mutants.

Genetic diversity of Mycobacterium tuberculosis isoniazid monoresistant and multidrug-resistant in Rio Grande do Sul, a tuberculosis high-burden state in Brazil.

Tuberculosis (TB) remains a major public health problem in the world and Brazil is among the countries with the highest incidence and prevalence rates, and Rio Grande do Sul, a Brazilian state, occupy a prominent position. Multidrug-resistant Mycobacterium tuberculosis (MDR-TB) further aggravates this scenario, making it more difficult to treat and control the disease. Isoniazid monoresistance (IMR) may increase the risk of progression to MDR-TB and treatment failure. However, most drug resistance molecular tests only focus on detecting rifampicin (RIF) resistance.In the present study, we characterized a total of 63 drug resistant isolates of M. tuberculosis (35 MDR, 26 IMR and two isolates monoresistant to rifampicin [RMR]) of the Rio Grande do Sul state by MIRU-VNTR (24 loci), spoligotyping, presence of RDRio, fbpC103, pks15/1 and sequencing of the katG, rpoB and inhA genes. We observed a higher proportion of the LAM family 30/63 (47.61%). In IMR, mutations were found in the katG gene (98% at codon 315) in 72.5%, and mutations in the promoter region of the inhA gene in 6.25% of the isolates. In MDR-TB and RMR-TB isolates, 92.1% had mutations in the rpoB gene (57% at codon 531). The presence of a 12 bp insertion between codons 516 and 517 of the rpoB gene in MDR-TB isolates was found in five isolates. In conclusion, we observed that the highest frequency of IMR-TB and MDR-TB strains belong to the LAM and Haarlem genotypes in Rio Grande do Sul state. A significant number of isolates previously characterized as Mycobacterium pinnipedi2 through spoligotyping were found to belong to the M. tuberculosis LAM family. This was responsible for a number of significant cases and the molecular profile of this strain and the pattern of mutations related to drug resistance were analyzed. These findings may contribute to a better understanding about the spread of M. tuberculosis resistant in southern of Brazil.

PCR colorimetric dot-blot assay and clinical pretest probability for diagnosis of Pulmonary Tuberculosis in smear-negative patients.

BACKGROUND: Smear-negative pulmonary tuberculosis (SNPTB) accounts for 30% of Pulmonary Tuberculosis (PTB) cases reported annually in developing nations. Polymerase chain reaction (PCR) may provide an alternative for the rapid detection of Mycobacterium tuberculosis (MTB); however little data are available regarding the clinical utility of PCR in SNPTB, in a setting with a high burden of TB/HIV co-infection. METHODS: To evaluate the performance of the PCR dot-blot in parallel with pretest probability (Clinical Suspicion) in patients suspected of having SNPTB, a prospective study of 213 individuals with clinical and radiological suspicion of SNPTB was carried out from May 2003 to May 2004, in a TB/HIV reference hospital. Respiratory specialists estimated the pretest probability of active disease into high, intermediate, low categories. Expectorated sputum was examined by direct microscopy (Ziehl-Neelsen staining), culture (Lowenstein Jensen) and PCR dot-blot. Gold standard was based on culture positivity combined with the clinical definition of PTB. RESULTS: In smear-negative and HIV subjects, active PTB was diagnosed in 28.4% (43/151) and 42.2% (19/45), respectively. In the high, intermediate and low pretest probability categories active PTB was diagnosed in 67.4% (31/46), 24% (6/25), 7.5% (6/80), respectively. PCR had sensitivity of 65% (CI 95%: 50%-78%) and specificity of 83% (CI 95%: 75%-89%). There was no difference in the sensitivity of PCR in relation to HIV status. PCR sensitivity and specificity among non-previously TB treated and those treated in the past were, respectively: 69%, 43%, 85% and 80%. The high pretest probability, when used as a diagnostic test, had sensitivity of 72% (CI 95%:57%-84%) and specificity of 86% (CI 95%:78%-92%). Using the PCR dot-blot in parallel with high pretest probability as a diagnostic test, sensitivity, specificity, positive and negative predictive values were: 90%, 71%, 75%, and 88%, respectively. Among non-previously TB treated and HIV subjects, this approach had sensitivity, specificity, positive and negative predictive values of 91%, 79%, 81%, 90%, and 90%, 65%, 72%, 88%, respectively. CONCLUSION: PCR dot-blot associated with a high clinical suspicion may provide an important contribution to the diagnosis of SNPTB mainly in patients that have not been previously treated attended at a TB/HIV reference hospital.

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: a study of 111 cases in an area of high prevalence in the extreme south of Brazil.

Tuberculosis is an increasingly important public health problem in developing countries. We studied 111 tuberculosis patients confirmed by mycobacteria isolation between 1998 and 2000 in Rio Grande, in south Brazil. One-hundred-thirteen pulmonary and extrapulmonary clinical materials were examined through culture by the Ogawa-Kudoh method and through smear examination by the Ziehl-Nielsen or Kinyoun methods. The identification of Mycobacterium tuberculosis was done through the usual phenotypical methods. The proportion method (indirect technique) was used to determine the resistance of isolates. The man/woman ratio was 2.6:1, 75% were white, and the group had a mean age of 39.7+/-12.7. Risk factors were present in 38.7% (34 men and 9 women, p=0.2), with a mean age of 35 (p=0.002); infection by HIV was the most frequent (28.8%), followed by alcoholism (16.2%) and injected drug use (15.3%). The frequency of M. tuberculosis isolates was 99.1%. Pulmonary disease occurred in 88.3% of the cases, extrapulmonary in 9.9% and 1.8% in both. There was an association between extrapulmonary tuberculosis and the presence of risk factors (p=0.0001). Resistance to isoniazid was found in 4% of the isolates and to isoniazid and rifampin in 2%, all being patients with some risk factors. The profile of tuberculosis in the population of this study followed the pattern described for developing regions of the world.

First detection of Mycobacterium triplex in Latin America.

In this study we describe the first isolation of Mycobacterium triplex in Latin America. This species causes infections in humans, with very few reports from around the world. We isolated two sputum specimens of a patient with a 6-year history of human immunodeficiency and tuberculosis treatment failure. All tests used confirmed M. triplex and the patient responded well to drug therapy for 18months.

Drug-resistant tuberculosis in subjects included in the Second National Survey on Antituberculosis Drug Resistance in Porto Alegre, Brazil.

OBJECTIVE: To describe the prevalence of multidrug-resistant tuberculosis (MDR-TB) among tuberculosis patients in a major Brazilian city, evaluated via the Second National Survey on Antituberculosis Drug Resistance, as well as the social, demographic, and clinical characteristics of those patients. METHODS: Clinical samples were collected from tuberculosis patients seen between 2006 to 2007 at three hospitals and five primary health care clinics participating in the survey in the city of Porto Alegre, Brazil. The samples were subjected to drug susceptibility testing. The species of mycobacteria was confirmed using biochemical methods. RESULTS: Of the 299 patients included, 221 (73.9%) were men and 77 (27.3%) had a history of tuberculosis. The mean age was 36 years. Of the 252 patients who underwent HIV testing, 66 (26.2%) tested positive. The prevalence of MDR-TB in the sample as a whole was 4.7% (95% CI: 2.3-7.1), whereas it was 2.2% (95% CI: 0.3-4.2) among the new cases of tuberculosis and 12.0% (95% CI: 4.5-19.5) among the patients with a history of tuberculosis treatment. The multivariate analysis showed that a history of tuberculosis and a longer time to diagnosis were both associated with MDR-TB. CONCLUSIONS: If our results are corroborated by other studies conducted in Brazil, a history of tuberculosis treatment and a longer time to diagnosis could be used as predictors of MDR-TB.

Epidemic of surgical-site infections by a single clone of rapidly growing mycobacteria in Brazil.

AIM: Our aim is to investigate if the clusters of postsurgical mycobacterial infections, reported between 2004 and 2008 in seven geographically distant states in Brazil, were caused by a single mycobacterial strain. MATERIALS & METHODS: Available information from 929 surgical patients was obtained from local health authorities. A total of 152 isolates from surgical patients were identified by PCR restriction enzyme analysis of the hsp65 gene (PRA-hsp65) and sequencing of the rpoB gene. Isolates were typed by pulsed-field gel electrophoresis (PFGE) using two restriction enzymes, DraI and AseI. A total of 15 isolates not related to surgical cases were analyzed for comparison. RESULTS: All isolates were identified as Mycobacterium abscessus ssp. massiliense. Isolates from surgical patients and one sputum isolate grouped in a single PFGE cluster, composed of two closely related patterns, with one band difference. A total of 14 other isolates unrelated to surgical cases showed distinctive PFGE patterns. CONCLUSION: A particular strain of M. abscessus ssp. massiliense was associated with a prolonged epidemic of postsurgical infections in seven Brazilian states, suggesting that this strain may be distributed in Brazilian territory and better adapted to cause surgical-site infections.

Drug-resistant tuberculosis in subjects included in the Second National Survey on Antituberculosis Drug Resistance in Porto Alegre, Brazil / Tuberculose resistente em pacientes incluídos no II Inquérito Nacional de Resistência aos Fármacos Antituberculose realizado em Porto Alegre, Brasil

OBJECTIVE: To describe the prevalence of multidrug-resistant tuberculosis (MDR-TB) among tuberculosis patients in a major Brazilian city, evaluated via the Second National Survey on Antituberculosis Drug Resistance, as well as the social, demographic, and clinical characteristics of those patients. METHODS: Clinical samples were collected from tuberculosis patients seen between 2006 to 2007 at three hospitals and five primary health care clinics participating in the survey in the city of Porto Alegre, Brazil. The samples were subjected to drug susceptibility testing. The species of mycobacteria was confirmed using biochemical methods. RESULTS: Of the 299 patients included, 221 (73.9%) were men and 77 (27.3%) had a history of tuberculosis. The mean age was 36 years. Of the 252 patients who underwent HIV testing, 66 (26.2%) tested positive. The prevalence of MDR-TB in the sample as a whole was 4.7% (95% CI: 2.3-7.1), whereas it was 2.2% (95% CI: 0.3-4.2) among the new cases of tuberculosis and 12.0% (95% CI: 4.5-19.5) among the patients with a history of tuberculosis treatment. The multivariate analysis showed that a history of tuberculosis and a longer time to diagnosis were both associated with MDR-TB. CONCLUSIONS: If our results are corroborated by other studies conducted in Brazil, a history of tuberculosis treatment and a longer time to diagnosis could be used as predictors of MDR-TB. .

OBJETIVO: Descrever a prevalência de tuberculose multirresistente (TBMR) em pacientes com tuberculose em uma importante cidade brasileira através do II Inquérito Nacional de Resistência aos Fármacos Antituberculose, assim como as características sociais, demográficas e clínicas desses pacientes. MÉTODOS: De 2006 a 2007, amostras clínicas de pacientes de três hospitais e das cinco unidades básicas de saúde participantes do inquérito realizado em Porto Alegre foram coletadas e submetidas ao teste de sensibilidade aos fármacos. A confirmação das espécies de micobactérias ocorreu por métodos bioquímicos. RESULTADOS: Foram incluídos 299 pacientes. Desses, 221 (73,9%) eram homens e 77 (27,3%) tinham história de tuberculose. A idade média foi de 36 anos. Dos 252 pacientes testados para HIV, 66 (26,2%) estavam infectados. A prevalência da TBMR na amostra geral foi de 4,7% (IC95%: 2,3-7,1); enquanto essa foi de 2,2% (IC95%: 0,3-4,2) nos pacientes virgens de tratamento e de 12,0% (IC 95%: 4,5-19,5) naqueles com história de tratamento antituberculose. A análise multivariada mostrou que história de tuberculose e maior tempo para o diagnóstico associaram-se a TBMR. CONCLUSÕES: Caso esses resultados sejam confirmados em outros estudos no Brasil, a história de tratamento antituberculose e o maior tempo para o diagnóstico poderão ser utilizados como preditores de TBMR. .

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.

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.

Epidemic of surgical-site infections by a single clone of rapidly growing mycobacteria in Brazil

Aim: Our aim is to investigate if the clusters of postsurgical mycobacterialinfections, reported between 2004 and 2008 in seven geographically distantstates in Brazil, were caused by a single mycobacterial strain.MATERIALS & METHODS: Available information from 929 surgical patients was obtained from local health authorities. A total of 152 isolates from surgical patients were identified by PCR restriction enzyme analysis of the hsp65 gene (PRA-hsp65) and sequencing of the rpoB gene. Isolates were typed by pulsed-field gel electrophoresis (PFGE) using two restriction enzymes, DraI and AseI. A total of 15 isolates not related to surgical cases were analyzed for comparison.RESULTS: All isolates were identified as Mycobacterium abscessus ssp. massiliense. Isolates from surgical patients and one sputum isolate grouped in a single PFGE cluster, composed of two closely related patterns, with one band difference. A total of 14 other isolates unrelated to surgical cases showed distinctive PFGE patterns.CONCLUSION: A particular strain of M. abscessus ssp. massiliense was associated with a prolonged epidemic of postsurgical infections in seven Brazilian states, suggesting that this strain may be distributed in Brazilian territory and better adapted to cause surgical-site infections.AU

Detection of Mycobacterium avium in blood samples of patients with AIDS by using PCR.

Sixty-nine blood samples from 47 patients infected with human immunodeficiency virus were analyzed by using PCR to detect Mycobacterium avium. The sensitivity can be up to 95.7%, depending on the detection method used and the number of blood samples analyzed from each patient. The procedure can be helpful in the diagnosis of mycobacterial disease.

In house colorimetric reverse hybridisation assay for detection of the mutation most frequently associated with resistance to isoniazid in Mycobacterium tuberculosis

Mutations in the katG gene have been identified and correlated with isoniazid (INH) resistance in Mycobacterium tuberculosis isolates. The mutation AGC→ACC (Ser→Thr) at katG315 has been reported to be the most frequent and is associated with transmission and multidrug resistance. Rapid detection of this mutation could therefore improve the choice of an adequate anti-tuberculosis regimen, the epidemiological monitoring of INH resistance and, possibly, the tracking of transmission of resistant strains. An in house reverse hybridisation assay was designed in our laboratory and evaluated with 180 isolates of M. tuberculosis. It could successfully characterise the katG315 mutation in 100 percent of the samples as compared to DNA sequencing. The test is efficient and is a promising alternative for the rapid identification of INH resistance in regions with a high prevalence of katG315 mutants.

Tuberculosis: a study of 111 cases in an area of high prevalence in the extreme south of Brazil

Tuberculosis is an increasingly important public health problem in developing countries. We studied 111 tuberculosis patients confirmed by mycobacteria isolation between 1998 and 2000 in Rio Grande, in south Brazil. One-hundred-thirteen pulmonary and extrapulmonary clinical materials were examined through culture by the Ogawa-Kudoh method and through smear examination by the Ziehl-Nielsen or Kinyoun methods. The identification of Mycobacterium tuberculosis was done through the usual phenotypical methods. The proportion method (indirect technique) was used to determine the resistance of isolates. The man/woman ratio was 2.6:1, 75 percent were white, and the group had a mean age of 39.7±12.7. Risk factors were present in 38.7 percent (34 men and 9 women, p=0.2), with a mean age of 35 (p=0.002); infection by HIV was the most frequent (28.8 percent), followed by alcoholism (16.2 percent) and injected drug use (15.3 percent). The frequency of M. tuberculosis isolates was 99.1 percent. Pulmonary disease occurred in 88.3 percent of the cases, extrapulmonary in 9.9 percent and 1.8 percent in both. There was an association between extrapulmonary tuberculosis and the presence of risk factors (p=0.0001). Resistance to isoniazid was found in 4 percent of the isolates and to isoniazid and rifampin in 2 percent, all being patients with some risk factors. The profile of tuberculosis in the population of this study followed the pattern described for developing regions of the world.