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Direct Determination of Pyrazinamide (PZA) Susceptibility by Sputum Microscopic Observation Drug Susceptibility (MODS) Culture at Neutral pH: the MODS-PZA Assay.

ABSTRACT: Pyrazinamide (PZA) is considered the pivot drug in all tuberculosis treatment regimens due to its particular action on the persistent forms of Mycobacterium tuberculosis However, no drug susceptibility test (DST) is considered sufficiently reliable for routine application. Although molecular tests are endorsed, their application is limited to known PZA resistance associated mutations. Microbiological DSTs for PZA have been restricted by technical limitations, especially the necessity for an acidic pH. Here, for the first time, MODS culture at neutral pH was evaluated using high PZA concentrations (400 and 800 μg/ml) to determine PZA susceptibility directly from sputum samples. Sputum samples were cultured with PZA for up to 21 days at 37°C. Plate reading was performed at two time points: R1 (mean, 10 days) and R2 (mean, 13 days) for each PZA concentration. A consensus reference test, composed of MGIT-PZA, pncA sequencing, and the classic Wayne test, was used. A total of 182 samples were evaluated. The sensitivity and specificity for 400 μg/ml ranged from 76.9 to 89.7 and from 93.0 to 97.9%, respectively, and for 800 μg/ml ranged from 71.8 to 82.1 and from 95.8 to 98.6%, respectively. Compared to MGIT-PZA, our test showed a similar turnaround time (medians of 10 and 12 days for PZA-sensitive and -resistant isolates, respectively). In conclusion, MODS-PZA is presented as a fast, simple, and low-cost DST that could complement the MODS assay to evaluate resistance to the principal first-line antituberculosis drugs. Further optimization of test conditions would be useful in order to increase its performance.

SUBMITTER: Alcantara R

PROVIDER: S-EPMC7180241 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Prediction of susceptibility to pyrazinamide (PZA) directly from sputum has been challenging. The MeltPro MTB/PZA assay, based on melting curve analysis, can simultaneously detect Mycobacterium tuberculosis and the resistance to PZA from sputum. We aimed to evaluate the MeltPro MTB/PZA assay to predict PZA resistance among rifampin-resistant tuberculosis (RR-TB) patients. We prospectively enrolled RR-TB patients in the registered trials, and their baseline sputum samples were obtained to perform the assay and culture. DNA sequencing of culture isolates was analyzed and used as the reference standard. Sanger sequencing was performed for samples with discrepant results between next-generation sequencing (NGS) and the investigational assay. The main analysis was conducted in the population of patients with interpretable results by both NGS and the assay. A total of 239 patients with RR-TB were screened, and 220 underwent the MeltPro MTB/PZA assay. The assay provided no information for 25 of 220 patients (11.4%). Among the remaining 195 patients, 13 had negative culture or insufficient raw NGS sequencing data, and 15 had indeterminate assay results. A total of 167 patients were included in the main analysis. Against DNA sequencing, the sensitivity, specificity, and negative predictive value of the assay for detecting resistance to PZA were 91.4% (95% confidence interval [CI], 87.1% to 95.6%), 89.9% (95% CI, 85.3% to 94.5%), and 95.2% (95% CI, 91.9% to 98.4%), respectively. In conclusion, the MeltPro MTB/PZA assay is a fast semiautomatic molecular platform to rapidly predict resistance to PZA from sputum and holds promise as a screening tool with satisfactory sensitivity. IMPORTANCE This study evaluated the accuracy of the MeltPro MTB/PZA assay at detecting the presence of PZA resistance through registered clinical trials. Compared to DNA sequencing, the assay had high sensitivity and negative predictive value, suggesting its potential utility as a screening tool in clinical practice. The assay could serve as an ideal primary screening tool in low PZA-resistant M. tuberculosis prevalence settings and could be used as an additional test to identify PZA resistance rapidly and initially in the RR-TB population.

Project description:New diagnostic tools are urgently needed to interrupt the transmission of tuberculosis and multidrug-resistant tuberculosis. Rapid, sensitive detection of tuberculosis and multidrug-resistant tuberculosis in sputum has been demonstrated in proof-of-principle studies of the microscopic-observation drug-susceptibility (MODS) assay, in which broth cultures are examined microscopically to detect characteristic growth.In an operational setting in Peru, we investigated the performance of the MODS assay for culture and drug-susceptibility testing in three target groups: unselected patients with suspected tuberculosis, prescreened patients at high risk for tuberculosis or multidrug-resistant tuberculosis, and unselected hospitalized patients infected with the human immunodeficiency virus. We compared the MODS assay head-to-head with two reference methods: automated mycobacterial culture and culture on Löwenstein-Jensen medium with the proportion method.Of 3760 sputum samples, 401 (10.7%) yielded cultures positive for Mycobacterium tuberculosis. Sensitivity of detection was 97.8% for MODS culture, 89.0% for automated mycobacterial culture, and 84.0% for Löwenstein-Jensen culture (P<0.001); the median time to culture positivity was 7 days, 13 days, and 26 days, respectively (P<0.001), and the median time to the results of susceptibility tests was 7 days, 22 days, and 68 days, respectively. The incremental benefit of a second MODS culture was minimal, particularly in patients at high risk for tuberculosis or multidrug-resistant tuberculosis. Agreement between MODS and the reference standard for susceptibility was 100% for rifampin, 97% for isoniazid, 99% for rifampin and isoniazid (combined results for multidrug resistance), 95% for ethambutol, and 92% for streptomycin (kappa values, 1.0, 0.89, 0.93, 0.71, and 0.72, respectively).A single MODS culture of a sputum sample offers more rapid and sensitive detection of tuberculosis and multidrug-resistant tuberculosis than the existing gold-standard methods used.

Project description:Universal access to drug susceptibility testing for newly diagnosed tuberculosis patients is recommended. Access to culture-based diagnostics remains limited, and targeted molecular assays are vulnerable to emerging resistance mutations. Improved protocols for direct-from-sputum Mycobacterium tuberculosis sequencing would accelerate access to comprehensive drug susceptibility testing and molecular typing. We assessed a thermo-protection buffer-based direct-from-sample M. tuberculosis whole-genome sequencing protocol. We prospectively analyzed 60 acid-fast bacilli smear-positive clinical sputum samples in India and Madagascar. A diversity of semiquantitative smear positivity-level samples were included. Sequencing was performed using Illumina and MinION (monoplex and multiplex) technologies. We measured the impact of bacterial inoculum and sequencing platforms on genomic read depth, drug susceptibility prediction performance, and typing accuracy. M. tuberculosis was identified by direct sputum sequencing in 45/51 samples using Illumina, 34/38 were identified using MinION-monoplex sequencing, and 20/24 were identified using MinION-multiplex sequencing. The fraction of M. tuberculosis reads from MinION sequencing was lower than from Illumina, but monoplexing grade 3+ samples on MinION produced higher read depth than Illumina (P < 0.05) and MinION multiplexing (P < 0.01). No significant differences in sensitivity and specificity of drug susceptibility predictions were seen across sequencing modalities or within each technology when stratified by smear grade. Illumina sequencing from sputum accurately identified 1/8 (rifampin) and 6/12 (isoniazid) resistant samples, compared to 2/3 (rifampin) and 3/6 (isoniazid) accurately identified with Nanopore monoplex. Lineage agreement levels between direct and culture-based sequencing were 85% (MinION-monoplex), 88% (Illumina), and 100% (MinION-multiplex). M. tuberculosis direct-from-sample whole-genome sequencing remains challenging. Improved and affordable sample treatment protocols are needed prior to clinical deployment.

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Direct Determination of Pyrazinamide (PZA) Susceptibility by Sputum Microscopic Observation Drug Susceptibility (MODS) Culture at Neutral pH: the MODS-PZA Assay.

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