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.

Evaluation of four molecular methods for the diagnosis of tuberculosis in pulmonary and blood samples from immunocompromised patients.

The present study analysed the concordance among four different molecular diagnostic methods for tuberculosis (TB) in pulmonary and blood samples from immunocompromised patients. A total of 165 blood and 194 sputum samples were collected from 181 human immunodeficiency virus (HIV)-infected patients with upper respiratory complaints, regardless of suspicious for TB. The samples were submitted for smear microscopy, culture and molecular tests: a laboratory-developed conventional polymerase chain reaction (PCR) and real-time quantitative PCR (qPCR) and the Gen-Probe and Detect-TB Ampligenix kits. The samples were handled blindly by all the technicians involved, from sample processing to results analysis. For sputum, the sensitivity and specificity were 100% and 96.7% for qPCR, 81.8% and 94.5% for Gen-Probe and 100% and 66.3% for Detect-TB, respectively. qPCR presented the best concordance with sputum culture [kappa (k) = 0.864)], followed by Gen-Probe (k = 0.682). For blood samples, qPCR showed 100% sensitivity and 92.3% specificity, with a substantial correlation with sputum culture (k = 0.754) and with the qPCR results obtained from sputum of the corresponding patient (k = 0.630). Conventional PCR demonstrated the worst results for sputa and blood, with a sensitivity of 100% vs. 88.9% and a specificity of 46.3% vs. 32%, respectively. Commercial or laboratory-developed molecular assays can overcome the difficulties in the diagnosis of TB in paucibacillary patients using conventional methods available in most laboratories.

Detection of rifampin-resistant genotypes in Mycobacterium tuberculosis by reverse hybridization assay.

We used a colorimetric reverse dot blot hybridization (CRDH) assay to detect the presence of mutations in a specific region of the rpoB gene, associated with rifampin (RIF) resistance, in a panel of 156 DNAs extracted from 103 RIF-sensitive and 53 RIF-resistant cultures of Mycobacterium tuberculosis. When compared with the antimicrobial susceptibility test (AST), the sensitivity and specificity of the CRDH were 92.3% and 98.1%, respectively. When compared with sequencing, the sensitivity and specificity of the CRDH were 90.6% and 100%, respectively. To evaluate the performance of the assay directly in clinical specimens, 30 samples from tuberculosis patients were used. For these samples, the results of the CRDH were 100% consistent with the results of the AST and sequencing. These results indicate that the rate of concordance of the CRDH is high when compared to conventional methods and sequencing data. The CRDH can be successfully applied when a rapid test is required for the identification of RIF resistance in M. tuberculosis.

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.

Genomic characterization of variants on mycolic acid metabolism genes in Mycobacterium tuberculosis isolates from Santa Catarina, Southern Brazil.

Mycobacterium tuberculosis has a complex cell wall containing mycolic acids (MA), which play an important role in pathogenesis, virulence, and survival by protecting the cell against harsh environments. Studies have shown that genes encoding enzymes involved in MA synthesis are essential to mycobacterial functionality. Here, we used whole-genome sequencing to evaluate mutations in genes related to MA metabolism in M. tuberculosis isolates from pulmonary tuberculosis patients of the Florianópolis Metropolitan Area, Santa Catarina, Brazil, and assessed associations with clinical, epidemiological, and genotypic data. The mutations Rv3057c Asp112Ala (104/151), Rv3720 His70Arg (104/151), and Rv3802c Val50Phe (105/151) were identified in about 69% of the isolates and were related to the LAM lineage. SIT 216/LAM5 (13.2%, 20/151) had the highest frequency and presented the mutations accD2 Lys23Glu, kasA Gly269Ser, mmaA4 Asn165Ser, otsB1 Asp617Asn, Rv3057c Asp112Ala, Rv3720 His70Arg, Rv3802c Val50Phe, and tgs4 Ala216Glu. All SIT 73/T isolates (6.6%, 10/151) showed a characteristic and exclusive gene mutation pattern: amiD Rv3376 3790075G > A, fbpA-aftB 4266941G > A, echA11 Asn220fs, and otsB2 Ser110Arg. SITs 20/LAM1, 64/LAM6, 50/H3, 137/X2, and 119/X1 were also related to specific mutations. SITs from the LAM lineage differed in mutation profile from those of the T, Haarlem, and X lineages. Isolates from patients who had treatment failure showed mutations that do not seem to have a pattern related to this outcome. It was possible to identify a broad repertoire of single-nucleotide polymorphisms in genes related to MA metabolism in M. tuberculosis isolates. This study also described, for the first time, the variability between different SITs/sublineages of Lineage 4 circulating in Florianópolis Metropolitan Area.

Genomic epidemiology of Mycobacterium tuberculosis in Santa Catarina, Southern Brazil.

Mycobacterium tuberculosis (M.tb), the pathogen responsible for tuberculosis (TB) poses as the major cause of death among infectious diseases. The knowledge about the molecular diversity of M.tb enables the implementation of more effective surveillance and control measures and, nowadays, Whole Genome Sequencing (WGS) holds the potential to produce high-resolution epidemiological data in a high-throughput manner. Florianópolis, the state capital of Santa Catarina (SC) in south Brazil, shows a high TB incidence (46.0/100,000). Here we carried out a WGS-based evaluation of the M.tb strain diversity, drug-resistance and ongoing transmission in the capital metropolitan region. Resistance to isoniazid, rifampicin, streptomycin was identified respectively in 4.0% (n = 6), 2.0% (n = 3) and 1.3% (n = 2) of the 151 studied strains by WGS. Besides, resistance to pyrazinamide and ethambutol was detected in 0.7% (n = 1) and reistance to ethionamide and fluoroquinolone (FQ) in 1.3% (n = 2), while a single (0.7%) multidrug-resistant (MDR) strain was identified. SNP-based typing classified all isolates into M.tb Lineage 4, with high proportion of sublineages LAM (60.3%), T (16.4%) and Haarlem (7.9%). The average core-genome distance between isolates was 420.3 SNPs, with 43.7% of all isolates grouped across 22 genomic clusters thereby showing the presence of important ongoing TB transmission events. Most clusters were geographically distributed across the study setting which highlights the need for an urgent interruption of these large transmission chains. The data conveyed by this study shows the presence of important and uncontrolled TB transmission in the metropolitan area and provides precise data to support TB control measures in this region.

Author Correction: Genomic epidemiology of Mycobacterium tuberculosis in Santa Catarina, Southern Brazil.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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.

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.

Evaluation of real-time PCR of patient pleural effusion for diagnosis of tuberculosis.

BACKGROUND: Pleural tuberculosis (TB) diagnosis often requires invasive procedures such as pleural biopsy. The aim of this study was to evaluate the role of real-time polymerase chain reaction (PCR) for the IS6110 sequence of M. tuberculosis in pleural fluid specimens as a rapid and non-invasive test for pleural TB diagnosis. FINDINGS: For this cross-sectional study, 150 consecutive patients with pleural effusion diagnosed by chest radiography, who were referred for diagnostic thoracocentesis and pleural biopsy and met eligibility criteria, had a pleural fluid specimen submitted for real-time PCR testing. Overall, 98 patients had pleural TB and 52 had pleural effusion secondary to other disease. TB diagnosis was obtained using acid-fast bacilli (AFB) smear or culture for mycobacteria and/or histopathologic examination in 94 cases and by clinical findings in 4 cases. Sensitivity, specificity, positive and negative predictive values of PCR testing for pleural TB diagnosis were 42.8% (95% CI 38.4 - 44.8), 94.2% (95% CI 85.8 - 98.0), 93.3% (95% CI 83.6 - 97.7), and 48.5% (95% CI 44.2 - 50.4), respectively. The real-time PCR test improved TB detection from 30.6% to 42.9% when compared to AFB smear and culture methods performed on pleural fluid specimens, although the best sensitivity was achieved by combining the results of culture and histopathology of pleural tissue specimens. CONCLUSION: The real-time PCR test of pleural fluid specimens is a useful and non-invasive additional assay for fast diagnosis of pleural TB.

Evaluation of a novel microplate colorimetric hybridization genotyping assay for human papillomavirus.

Persistent infection with high-risk human papillomavirus (HR-HPV) has been associated with cervical cancer. Developing assays for the identification of these viral types is of great importance for monitoring patients and controlling strategies. The development of the MCHA (microplate colorimetric hybridization assay), a PCR-based method for identifying six of the most common HR-HPV types (HPV 16, 18, 31, 33, 39 and 45) is described. The MCHA combines the amplification with the GP5+/GP6+ consensus primers followed by PCR reverse hybridization with specific probes and detection through a colorimetric assay. The performance of the MCHA was evaluated using 108 DNA samples typed previously by the PapilloCheck(®). The agreement between both methods was 69.4% for HPV 16; 79.1% for HPV 45; 82.4% for HPV 18; 93.6% for HPV 31; 87.9% for HPV 33, and 17.6% for HPV 39. The assay had higher sensitivity than the Papillocheck(®), particularly for identifying HPV 16 and 18. The MCHA seemed to be sensitive and specific for the identification of the most prevalent HPV types in invasive cervical cancer, HPV 16, 18, 45, 33 and 31. It requires low-cost reagents and common laboratory apparatus.

Evaluation of four molecular methods for the diagnosis of tuberculosis in pulmonary and blood samples from immunocompromised patients

The present study analysed the concordance among four different molecular diagnostic methods for tuberculosis (TB) in pulmonary and blood samples from immunocompromised patients. A total of 165 blood and 194 sputum samples were collected from 181 human immunodeficiency virus (HIV)-infected patients with upper respiratory complaints, regardless of suspicious for TB. The samples were submitted for smear microscopy, culture and molecular tests: a laboratory-developed conventional polymerase chain reaction (PCR) and real-time quantitative PCR (qPCR) and the Gen-Probe and Detect-TB Ampligenix kits. The samples were handled blindly by all the technicians involved, from sample processing to results analysis. For sputum, the sensitivity and specificity were 100% and 96.7% for qPCR, 81.8% and 94.5% for Gen-Probe and 100% and 66.3% for Detect-TB, respectively. qPCR presented the best concordance with sputum culture [kappa (k) = 0.864)], followed by Gen-Probe (k = 0.682). For blood samples, qPCR showed 100% sensitivity and 92.3% specificity, with a substantial correlation with sputum culture (k = 0.754) and with the qPCR results obtained from sputum of the corresponding patient (k = 0.630). Conventional PCR demonstrated the worst results for sputa and blood, with a sensitivity of 100% vs. 88.9% and a specificity of 46.3% vs. 32%, respectively. Commercial or laboratory-developed molecular assays can overcome the difficulties in the diagnosis of TB in paucibacillary patients using conventional methods available in most laboratories.

Detection of rifampin-resistant genotypes in Mycobacterium tuberculosis by reverse hybridization assay

We used a colorimetric reverse dot blot hybridization (CRDH) assay to detect the presence of mutations in a specific region of the rpoB gene, associated with rifampin (RIF) resistance, in a panel of 156 DNAs extracted from 103 RIF-sensitive and 53 RIF-resistant cultures of Mycobacterium tuberculosis. When compared with the antimicrobial susceptibility test (AST), the sensitivity and specificity of the CRDH were 92.3 percent and 98.1 percent, respectively. When compared with sequencing, the sensitivity and specificity of the CRDH were 90.6 percent and 100 percent, respectively. To evaluate the performance of the assay directly in clinical specimens, 30 samples from tuberculosis patients were used. For these samples, the results of the CRDH were 100 percent consistent with the results of the AST and sequencing. These results indicate that the rate of concordance of the CRDH is high when compared to conventional methods and sequencing data. The CRDH can be successfully applied when a rapid test is required for the identification of RIF resistance in M. tuberculosis.

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.

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.