Last data update: Jun 24, 2024. (Total: 47078 publications since 2009)
Records 1-7 (of 7 Records) |
Query Trace: Morlock G [original query] |
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Prediction of pyrazinamide resistance in Mycobacterium tuberculosis using structure-based machine-learning approaches
Carter JJ , Walker TM , Walker AS , Whitfield MG , Morlock GP , Lynch CI , Adlard D , Peto TEA , Posey JE , Crook DW , Fowler PW . JAC Antimicrob Resist 2024 6 (2) dlae037 BACKGROUND: Pyrazinamide is one of four first-line antibiotics used to treat tuberculosis; however, antibiotic susceptibility testing for pyrazinamide is challenging. Resistance to pyrazinamide is primarily driven by genetic variation in pncA, encoding an enzyme that converts pyrazinamide into its active form. METHODS: We curated a dataset of 664 non-redundant, missense amino acid mutations in PncA with associated high-confidence phenotypes from published studies and then trained three different machine-learning models to predict pyrazinamide resistance. All models had access to a range of protein structural-, chemical- and sequence-based features. RESULTS: The best model, a gradient-boosted decision tree, achieved a sensitivity of 80.2% and a specificity of 76.9% on the hold-out test dataset. The clinical performance of the models was then estimated by predicting the binary pyrazinamide resistance phenotype of 4027 samples harbouring 367 unique missense mutations in pncA derived from 24 231 clinical isolates. CONCLUSIONS: This work demonstrates how machine learning can enhance the sensitivity/specificity of pyrazinamide resistance prediction in genetics-based clinical microbiology workflows, highlights novel mutations for future biochemical investigation, and is a proof of concept for using this approach in other drugs. |
Optimising pyrazinamide for the treatment of tuberculosis
Zhang N , Savic RM , Boeree MJ , Peloquin C , Weiner M , Heinrich N , Bliven-Sizemore E , Phillips PP , Hoelscher M , Whitworth W , Morlock G , Posey J , Stout JE , Mac Kenzie W , Aarnoutse R , Dooley KE . Eur Respir J 2021 58 (1) Pyrazinamide is a potent sterilising agent that shortens the treatment duration needed to cure tuberculosis. It is synergistic with novel and existing drugs for tuberculosis. The dose of pyrazinamide that optimises efficacy while remaining safe is uncertain, as is its potential role in shortening treatment duration further.Pharmacokinetic data, sputum culture, and safety laboratory results were compiled from TBTC Studies 27 and 28 and PanACEA MAMS-TB, multi-center Phase 2 trials in which participants received rifampicin (range 10-35 mg·kg(-1)), pyrazinamide (range 20-30 mg·kg(-1)), plus two companion drugs. Pyrazinamide pharmacokinetic-pharmacodynamic (PK/PD) and PK-toxicity analyses were performed.In TBTC studies (n=77), higher pyrazinamide maximum concentration (Cmax) was associated with shorter time to culture conversion (TTCC) and higher probability of two-month culture conversion (p-value<0.001). Parametric survival analyses showed that relationships varied geographically, with steeper PK-PD relationships seen among non-African than African participants. In PanACEA MAMS-TB (n=363), TTCC decreased as pyrazinamide Cmax increased and varied by rifampicin Cmax (p-value<0.01). Modeling and simulation suggested that very high doses of pyrazinamide (>4500 mg) or increasing both pyrazinamide and rifampicin would be required to reach targets associated with treatment shortening. Combining all trials, liver toxicity was rare (3.9% with Grade 3 or higher liver function tests, LFT), and no relationship was seen between pyrazinamide Cmax and LFT levels.Pyrazinamide's microbiologic efficacy increases with increasing drug concentrations. Optimising pyrazinamide alone, though, is unlikely to be sufficient to allow tuberculosis treatment shortening; rather, rifampicin dose would need to be increased in parallel. |
Using reduced inoculum densities of Mycobacterium tuberculosis in MGIT Pyrazinamide Susceptibility Testing to prevent false-resistant results and improve accuracy: A multicenter evaluation
Morlock GP , Tyrrell FC , Baynham D , Escuyer VE , Green N , Kim Y , Longley-Olson PA , Parrish N , Pennington C , Tan D , Austin B , Posey JE . Tuberc Res Treat 2017 2017 3748163 The primary platform used for pyrazinamide (PZA) susceptibility testing of Mycobacterium tuberculosis is the MGIT culture system (Becton Dickinson). Since false-resistant results have been associated with the use of this system, we conducted a multicenter evaluation to determine the effect of using a reduced cell density inoculum on the rate of false resistance. Two reduced inoculum densities were compared with that prescribed by the manufacturer (designated as "BD" method). The reduced inoculum methods (designated as "A" and "C") were identical to the manufacturer's protocol in all aspects with the exception of the cell density of the inoculum. Twenty genetically and phenotypically characterized M. tuberculosis isolates were tested in duplicate by ten independent laboratories using the three inoculum methods. False-resistant results declined from 21.1% using the standard "BD" method to 5.7% using the intermediate ("A") inoculum and further declined to 2.8% using the most dilute ("C") inoculum method. The percentages of the resistant results that were false-resistant declined from 55.2% for the "BD" test to 28.8% and 16.0% for the "A" and "C" tests, respectively. These results represent compelling evidence that the occurrence of false-resistant MGIT PZA susceptibility test results can be mitigated through the use of reduced inoculum densities. |
Mycobacterium tuberculosis pncA polymorphisms that do not confer pyrazinamide resistance at a breakpoint concentration of 100 µg/ml in MGIT.
Whitfield MG , Warren RM , Streicher EM , Sampson SL , Sirgel FA , van Helden PD , Mercante A , Willby M , Hughes K , Birkness K , Morlock G , van Rie A , Posey JE . J Clin Microbiol 2015 Sequencing of the Mycobacterium tuberculosis pncA gene allows for pyrazinamide susceptibility testing. We summarize data on pncA polymorphisms which do not confer resistance at a susceptibility breakpoint of 100 mug/ml pyrazinamide in MGIT within a cohort of isolates from South Africa and the US Centre for Disease Control. |
Molecular detection of mutations associated with first- and second-line drug resistance compared with conventional drug susceptibility testing of Mycobacterium tuberculosis.
Campbell PJ , Morlock GP , Sikes RD , Dalton TL , Metchock B , Starks AM , Hooks DP , Cowan LS , Plikaytis BB , Posey JE . Antimicrob Agents Chemother 2011 55 (5) 2032-41 The emergence of multi and extensively drug-resistant tuberculosis is a significant impediment to the control of this disease because treatment becomes more complex and costly. Reliable and timely drug susceptibility testing is critical to ensure patients receive effective treatment and become non-infectious. Molecular methods can provide accurate and rapid drug susceptibility results. We used DNA sequencing to detect resistance to the first-line antituberculosis drugs, isoniazid (INH), rifampin (RIF), pyrazinamide (PZA), and ethambutol (EMB), and the second-line drugs, amikacin (AMK), capreomycin (CAP), kanamycin (KAN), ciprofloxacin, (CIP) and ofloxacin (OFX). Nine loci were sequenced: rpoB for resistance to RIF, katG and inhA (INH), pncA (PZA), embB (EMB), gyrA (CIP and OFX), rrs, eis, and tlyA (KAN, AMK, and CAP). A total of 314 clinical M. tuberculosis complex isolates, representing a variety of antibiotic resistance patterns, genotypes and geographical origins were analyzed. The molecular data were compared to the phenotypic data and the accuracy values were calculated. Sensitivity and specificity values (as percentages) for the first-line drug loci were rpoB (97.1, 93.6), katG (85.4, 100), inhA (16.5, 100), katG and inhA together (90.6, 100) pncA (84.6, 85.8), and embB (78.6, 93.1). The values for the second-line drugs were also calculated. The size and scope of this study, in numbers of loci and isolates examined, and the phenotypic diversity of those isolates, support the use of DNA sequencing to detect drug resistance in the M. tuberculosis complex. Further, the results can be used to design diagnostic tests utilizing other mutation detection technologies. |
Rapid detection of multidrug resistant tuberculosis using real-time PCR and high resolution melt analysis
Ramirez MV , Cowart KC , Campbell PJ , Morlock GP , Sikes D , Winchell JM , Posey JE . J Clin Microbiol 2010 48 (11) 4003-9 The current study describes the development of a unique real-time PCR assay for the detection of mutations conferring drug resistance in Mycobacterium tuberculosis (Mtb). The Rifampicin Resistance Determinant Region (RRDR) of rpoB and specific regions of katG and the inhA promoter were targeted for the detection of rifampicin (RIF) and isoniazid (INH) resistance, respectively. Additionally, this assay was multiplexed to discriminate Mycobacterium tuberculosis complex (MTBC) strains from Nontuberculous Mycobacteria (NTM) strains by targeting the IS6110 insertion element. High resolution melting (HRM) analysis following real-time PCR was used to identify Mtb strains containing mutations at the targeted loci, and locked nucleic acid (LNA) probes were used to enhance the detection of strains containing specific SNP transversion mutations. This method was used to screen 252 Mtb clinical isolates including 154 RIF resistant strains and 174 INH resistant strains based on the agar proportion method of drug susceptibility testing (DST). Of the 154 RIF resistant strains, 148 were also resistant to INH and therefore classified as multidrug resistant (MDR). The assay demonstrated a sensitivity and specificity of 91% and 98%, respectively, for the detection of RIF resistance, and 87% and 100% for the detection of INH resistance. Overall, this assay showed a sensitivity of 85% and a specificity of 98% for the detection of MDR strains. This method provides a rapid, robust, and inexpensive way to detect the dominant mutations known to confer MDR in Mtb strains and offers several advantages over current molecular and culture-based techniques. |
Molecular identification of mutations associated with anti-tuberculosis drug resistance among strains of Mycobacterium tuberculosis
Abbadi SH , Sameaa GA , Morlock G , Cooksey RC . Int J Infect Dis 2009 13 (6) 673-8 BACKGROUND: Understanding the etiologic organism, antimicrobial resistance mechanisms, and transmission of multidrug-resistant tuberculosis (MDR-TB) can be of great value in optimizing strategies to control and prevent its development and transmission. METHODS: One hundred and fifty-five Mycobacterium tuberculosis complex isolates from patients with pulmonary tuberculosis (TB) in Cairo, Egypt were studied. In vitro drug susceptibility testing against rifampin (RIF), isoniazid (INH), streptomycin (SM), ethambutol (EMB), and pyrazinamide (PZA) was performed. Resistance was studied by the standard agar proportion method. Single strand conformation polymorphism (SSCP) and DNA sequence analysis were used to detect mutations in the genes that encode resistance to rpoB, katG, rpsL, and embB. RESULTS: Among 155 consecutive M. tuberculosis isolates, 25 (16.1%) were MDR-TB; 13 of these were from newly diagnosed untreated cases, 12 were from re-treated cases, and none of the MDR-TB isolates had matching IS6110 fingerprints. Among the MDR-TB isolates, rpoB mutations were found in 76% of RIF-resistant isolates, katG mutations were found in 47.1% of INH-resistant isolates, rpsL mutations were found in 55.6% of SM-resistant isolates, and embB mutations were found in 36.4% of EMB-resistant isolates. CONCLUSIONS: No major differences were found in the frequencies of mutations or types of amino acid substitution between newly diagnosed untreated cases and re-treated cases. The high prevalence of MDR-TB at this hospital underscores the need for continuous monitoring of strains and antimicrobial resistance. |
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