Detection of non-tuberculosus mycobacteria (NTMs) in lung samples using 16S rRNA.

BACKGROUND: Non-tuberculous mycobacteria (NTMs) cause diseases known as mycobacteriosis and are an important cause of morbidity and mortality. The diagnosis of pulmonary disease caused by NTM is hampered by its clinical similarity with tuberculosis (TB) and by the lack of an accurate and rapid laboratory diagnosis. OBJECTIVES: Detect DNA from NTMs directly from lung samples using real-time polymerase chain reaction (qPCR) for amplification of 16S rRNA. Additionally, DNA sequencing (hsp65 and rpoB genes) was used to identify the species of MNTs. METHODS: A total of 68 sputum samples (54 with suspected NTMs and 14 with TB) from patients treated at a referral hospital were used. FINDINGS: Of these, 27/54 (50%) were qPCR positive for NTMs and 14/14 TB patients (controls) were qPCR negative with an almost perfect concordance (Kappa of 0.93) with the Mycobacterium spp. culture. Sequencing confirmed the presence of NTM in all positive samples. The most common species was Mycobacterium gordonae (33%), followed by Mycobacterium abscessus (26%), Mycobacterium fortuitum (22%), Mycobacterium avium (15%) and Mycobacterium peregrinum (4%). MAIN CONCLUSIONS: The qPCR technique for detecting NTMs targeting 16S rRNA has the potential to detect NTMs and rapidly differentiate from Mycobacterium tuberculosis. However, it is necessary to identify the species to help in the differential diagnosis between disease and contamination, and to guide the choice of the therapeutic scheme.

Performance of a molecular assay to detect Mycobacterium tuberculosis complex DNA in clinical specimens: multicenter study in Brazil.

BACKGROUND: In high tuberculosis (TB) burden countries, there are few data on the performance of new molecular commercialised assays developed locally. OBJECTIVE: To evaluate the performance of a new molecular commercialised assay for TB diagnosis (Detect-TB) in three laboratories. METHODS: A total of 302 sputum samples from an equal number of patients with presumptive diagnosis of pulmonary tuberculosis (PTB) were submitted for routine smear microscopy, culture, and Detect-TB assay at three different sites in Brazil (the cities of Caxias do Sul, São Paulo and Canoas). FINDINGS: Seventy four (24.7%) TB cases were diagnosed (65 bacteriologically confirmed). When compared to smear microscopy/culture results, the overall sensitivity and specificity of Detect-TB assay was 84.6% (CI 95%; 73.7-91.6) and 93.1% (CI 95%; 89.1-95.8), respectively. When compared to bacteriological and clinical diagnostic criteria, the sensitivity and specificity of Detect-TB assay was 74.3% (CI 95%; 63.3-82.9) and 92.9% (CI 95%; 88.7-95.6), respectively. Among the three sites - Caxias do Sul, São Paulo and Canoas - the sensitivity and specificity were respectively 94.7% and 97.8%; 71.4% and 93.9%, 82.1% and 88.9%. MAIN CONCLUSIONS: These findings suggest that the Detect-TB assay could be applied routinely in reference laboratories across different regions in Brazil.

Smear plus Detect-TB for a sensitive diagnosis of pulmonary tuberculosis: a cost-effectiveness analysis in an incarcerated population.

BACKGROUND: Prison conditions can favor the spread of tuberculosis (TB). This study aimed to evaluate in a Brazilian prison: the performance and accuracy of smear, culture and Detect-TB; performance of smear plus culture and smear plus Detect-TB, according to different TB prevalence rates; and the cost-effectiveness of these procedures for pulmonary tuberculosis (PTB) diagnosis. METHODS: This paper describes a cost-effectiveness study. A decision analytic model was developed to estimate the costs and cost-effectiveness of five routine diagnostic procedures for diagnosis of PTB using sputum specimens: a) Smear alone, b) Culture alone, c) Detect-TB alone, d) Smear plus culture and e) Smear plus Detect-TB. The cost-effectiveness ratio of costs were evaluated per correctly diagnosed TB case and all procedures costs were attributed based on the procedure costs adopted by the Brazilian Public Health System. RESULTS: A total of 294 spontaneous sputum specimens from patients suspected of having TB were analyzed. The sensibility and specificity were calculated to be 47% and 100% for smear; 93% and 100%, for culture; 74% and 95%, for Detect-TB; 96% and 100%, for smear plus culture; and 86% and 95%, for smear plus Detect-TB. The negative and positive predictive values for smear plus Detect-TB, according to different TB prevalence rates, ranged from 83 to 99% and 48 to 96%, respectively. In a cost-effectiveness analysis, smear was both less costly and less effective than the other strategies. Culture and smear plus culture were more effective but more costly than the other strategies. Smear plus Detect-TB was the most cost-effective method. CONCLUSIONS: The Detect-TB evinced to be sensitive and effective for the PTB diagnosis when applied with smear microscopy. Diagnostic methods should be improved to increase TB case detection. To support rational decisions about the implementation of such techniques, cost-effectiveness studies are essential, including in prisons, which are known for health care assessment problems.

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.

Molecular characterization of nontuberculous Mycobacteria in a tuberculosis and HIV reference unit in the State of Amazonas, Brazil.

BACKGROUND: In recent years, the prevalence of nontuberculous mycobacterial (NTM) infections has increased in different regions of the world. The American Thoracic Society (ATS) recommends standardized identification criteria, reinforcing the need for faster and less complicated clinical and laboratory techniques. METHODS: In this retrospective study, NTM species isolated from pulmonary, extrapulmonary, and disseminated samples from patients treated at a TB/HIV reference unit in the State of Amazonas from 2011 to 2014 were identified through a combination of molecular techniques. RESULTS: To identify the molecular technique, 50 cryopreserved NTM cultures were recovered and subcultivated in culture medium. The potentially pathogenic NTM species identified were M. avium, M. intracellulare, M. kansasii, M. chelonae, M. abscessus, M. fortuitum, and M. peregrinum. Results of GenoType® showed moderate agreement with those of genomic sequencing (kappa = 0.60), whereas the results obtained by the PRA-hsp65 technique disagreed with the results obtained by sequencing (kappa = 0.49). CONCLUSIONS: Our findings highlight that GenoType CM is a good method for the identification of NTM, as well as the need for the application of standardized criteria, such as those set forth by the ATS.

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.

Polymerase chain reaction test in induced sputum of patients with pulmonary tuberculosis.

INTRODUCTION: Induced sputum (IS) is an alternative method of obtaining sputum, but IS smears are frequently negative. Culture is more time consuming in its results, and less useful to guide the diagnosis. Polymerase chain reaction (PCR) is the most common methodology for rapid diagnosis of tuberculosis (TB), and few studies evaluated its role in IS samples. OBJECTIVES: The objective of this study is to determine the diagnostic yield of PCR for TB compared with culture in IS samples. MATERIALS AND METHODS: Prospective study. Inpatients and outpatients of >18 years with respiratory symptoms suggestive of PTB were invited to participate. The subjects were interviewed using a standardized questionnaire, and collected IS. Three samples were obtained for AFB smear and culture. A fourth sample was obtained for PCR test. RESULTS: A total of 116 IS samples were evaluated. The sensitivity, specificity, positive predictive value and negative predictive values of PCR were 95.2%, 48.4%, 29.0% and 97.9%, respectively. The area under the receiver operating characteristic curve was .72 for the PCR test (P < .0001). CONCLUSIONS: Although the PCR specificity could be underestimated, if we consider PCR to be more sensitive than the culture method used, we believed that these PCR-positive tests mean false positives. The results of PCR should always be interpreted carefully in conjunction with clinical information.

Detection of non-tuberculosus mycobacteria (NTMs) in lung samples using 16S rRNA

BACKGROUND Non-tuberculous mycobacteria (NTMs) cause diseases known as mycobacteriosis and are an important cause of morbidity and mortality. The diagnosis of pulmonary disease caused by NTM is hampered by its clinical similarity with tuberculosis (TB) and by the lack of an accurate and rapid laboratory diagnosis. OBJECTIVES Detect DNA from NTMs directly from lung samples using real-time polymerase chain reaction (qPCR) for amplification of 16S rRNA. Additionally, DNA sequencing (hsp65 and rpoB genes) was used to identify the species of MNTs. METHODS A total of 68 sputum samples (54 with suspected NTMs and 14 with TB) from patients treated at a referral hospital were used. FINDINGS Of these, 27/54 (50%) were qPCR positive for NTMs and 14/14 TB patients (controls) were qPCR negative with an almost perfect concordance (Kappa of 0.93) with the Mycobacterium spp. culture. Sequencing confirmed the presence of NTM in all positive samples. The most common species was Mycobacterium gordonae (33%), followed by Mycobacterium abscessus (26%), Mycobacterium fortuitum (22%), Mycobacterium avium (15%) and Mycobacterium peregrinum (4%). MAIN CONCLUSIONS The qPCR technique for detecting NTMs targeting 16S rRNA has the potential to detect NTMs and rapidly differentiate from Mycobacterium tuberculosis. However, it is necessary to identify the species to help in the differential diagnosis between disease and contamination, and to guide the choice of the therapeutic scheme.

Molecular characterization of nontuberculous Mycobacteria in a tuberculosis and HIV reference unit in the State of Amazonas, Brazil

ABSTRACT Background: In recent years, the prevalence of nontuberculous mycobacterial (NTM) infections has increased in different regions of the world. The American Thoracic Society (ATS) recommends standardized identification criteria, reinforcing the need for faster and less complicated clinical and laboratory techniques. Methods: In this retrospective study, NTM species isolated from pulmonary, extrapulmonary, and disseminated samples from patients treated at a TB/HIV reference unit in the State of Amazonas from 2011 to 2014 were identified through a combination of molecular techniques. Results: To identify the molecular technique, 50 cryopreserved NTM cultures were recovered and subcultivated in culture medium. The potentially pathogenic NTM species identified were M. avium, M. intracellulare, M. kansasii, M. chelonae, M. abscessus, M. fortuitum, and M. peregrinum. Results of GenoType® showed moderate agreement with those of genomic sequencing (kappa = 0.60), whereas the results obtained by the PRA-hsp65 technique disagreed with the results obtained by sequencing (kappa = 0.49). Conclusions: Our findings highlight that GenoType CM is a good method for the identification of NTM, as well as the need for the application of standardized criteria, such as those set forth by the ATS.

Performance of a molecular assay to detect Mycobacterium tuberculosis complex DNA in clinical specimens: multicenter study in Brazil

BACKGROUND In high tuberculosis (TB) burden countries, there are few data on the performance of new molecular commercialised assays developed locally. OBJECTIVE To evaluate the performance of a new molecular commercialised assay for TB diagnosis (Detect-TB) in three laboratories. METHODS A total of 302 sputum samples from an equal number of patients with presumptive diagnosis of pulmonary tuberculosis (PTB) were submitted for routine smear microscopy, culture, and Detect-TB assay at three different sites in Brazil (the cities of Caxias do Sul, São Paulo and Canoas). FINDINGS Seventy four (24.7%) TB cases were diagnosed (65 bacteriologically confirmed). When compared to smear microscopy/culture results, the overall sensitivity and specificity of Detect-TB assay was 84.6% (CI 95%; 73.7-91.6) and 93.1% (CI 95%; 89.1-95.8), respectively. When compared to bacteriological and clinical diagnostic criteria, the sensitivity and specificity of Detect-TB assay was 74.3% (CI 95%; 63.3-82.9) and 92.9% (CI 95%; 88.7-95.6), respectively. Among the three sites - Caxias do Sul, São Paulo and Canoas - the sensitivity and specificity were respectively 94.7% and 97.8%; 71.4% and 93.9%, 82.1% and 88.9%. MAIN CONCLUSIONS These findings suggest that the Detect-TB assay could be applied routinely in reference laboratories across different regions in Brazil.

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.