Mutation frequency in drug-susceptible clinical isolates of Mycobacterium tuberculosis under aerobic and anaerobic conditions.

M. Med. Sc. University of KwaZulu-Natal, Durban 2015.Abstract not available

Co-warm B2

In this 21st century is where are the technologies are in advance technological development especially in electrics and electronics. Most of the vending machine can cool and warm the drinks separately. In personal used, the consumers need to buy two different products in cooling and warming the beverages. Practically, this contributes to the high-cost lifestyle and not user-friendly. This is more troubling if there are going for a travel or doing outdoor activities. Based on the current practical and applied technology in electronic device now, the power source of this proposed product uses the USB connection as its main source. Therefore, the target of this project to deliver the portable electronic device can be achieved

The Integrity and Yield of Genomic DNA Isolated from Whole Blood Following Long-Term Storage at -30°C.

Long-term storage of whole blood can affect the integrity of DNA if it is not done under optimal conditions. The aim of this study was to determine whether long-term storage (2-19 years) of whole blood samples at -30°C had a negative effect on the quality or quantity of genomic DNA that could be recovered at extraction. Genomic DNA was isolated from 2758 whole blood samples collected in 4 mL EDTA vacutainers from 1997 to 2012. DNA was extracted using the Qiagen® FlexiGene® DNA kit. The average storage duration at -30°C was 12 years. The quality and quantity of the isolated DNA were assessed using spectrophotometry (NanoDrop™), a fluorometric assay for double-stranded DNA (Qubit™), and agarose gel electrophoresis. The mean DNA yield per sample was found to be 114 μg from whole blood volumes that ranged from 0.5 to 4 mL. The mean A260/280 ratio and median A260/280 ratios were both 1.8. No correlation was found between the duration of storage and the total yield or the quality of DNA extracted. These data suggest that high-quality DNA can be extracted from whole blood samples that are stored at -30°C for up to 19 years

Whole genome sequencing for drug resistance determination in Mycobacterium tuberculosis

South Africa remains challenged with a high tuberculosis burden accompanied by an increase in drug resistant cases. We assessed the use of the Illumina MiSeq, a next-generation sequencing platform for whole genome sequencing, followed by bioinformatic analysis using a commercial software package to determine resistance to selected drugs used for Mycobacterium tuberculosis treatment in our setting. Whole genome sequencing shows potential as a diagnostic platform for the detection of drug resistance in Mycobacterium tuberculosis with the provision of information for several drugs simultaneously

Whole genome sequencing for drug resistance determination in Mycobacterium tuberculosis

South Africa remains challenged with a high tuberculosis burden accompanied by an increase in drug resistant cases. We assessed the use of the Illumina MiSeq, a next-generation sequencing platform for whole genome sequencing, followed by bioinformatic analysis using a commercial software package to determine resistance to selected drugs used for Mycobacterium tuberculosis treatment in our setting. Whole genome sequencing shows potential as a diagnostic platform for the detection of drug resistance in Mycobacterium tuberculosis with the provision of information for several drugs simultaneously.Funding was provided by the National Institute for Communicable Diseases, a division of the National Health Laboratory Service, South Africa.The National Institute for Communicable Diseases, a division of the National Health Laboratory Service, South Africahttp://www.ajlmonline.orgam2020Medical Microbiolog

Whole-genome sequence of a Mycobacterium goodii isolate from a pediatric patient in South Africa

We describe here the draft genome sequence of a Mycobacterium goodii isolate from a pediatric patient in Western Cape, South Africa. To our knowledge, this is the second reported genome of this rapidly growing nontuberculous mycobacterial species.http://genomea.asm.orgam2018Medical Microbiolog

Assessment of epidemiological and genetic characteristics and clinical outcomes of resistance to bedaquiline in patients treated for rifampicin-resistant tuberculosis : a cross-sectional and longitudinal study

BACKGROUND : Bedaquiline improves outcomes of patients with rifampicin-resistant and multidrug-resistant (MDR) tuberculosis; however, emerging resistance threatens this success. We did a cross-sectional and longitudinal analysis evaluating the epidemiology, genetic basis, and treatment outcomes associated with bedaquiline resistance, using data from South Africa (2015–19). METHODS : Patients with drug-resistant tuberculosis starting bedaquiline-based treatment had surveillance samples submitted at baseline, month 2, and month 6, along with demographic information. Culture-positive baseline and post-baseline isolates had phenotypic resistance determined. Eligible patients were aged 12 years or older with a positive culture sample at baseline or, if the sample was invalid or negative, a sample within 30 days of the baseline sample submitted for bedaquiline drug susceptibility testing. For the longitudinal study, the first surveillance sample had to be phenotypically susceptible to bedaquiline for inclusion. Whole-genome sequencing was done on bedaquiline-resistant isolates and a subset of bedaquiline-susceptible isolates. The National Institute for Communicable Diseases tuberculosis reference laboratory, and national tuberculosis surveillance databases were matched to the Electronic Drug-Resistant Tuberculosis Register. We assessed baseline resistance prevalence, mutations, transmission, cumulative resistance incidence, and odds ratios (ORs) associating risk factors for resistance with patient outcomes. FINDINGS : Between Jan 1, 2015, and July 31, 2019, 8041 patients had surveillance samples submitted, of whom 2023 were included in the cross-sectional analysis and 695 in the longitudinal analysis. Baseline bedaquiline resistance prevalence was 3·8% (76 of 2023 patients; 95% CI 2·9–4·6), and it was associated with previous exposure to bedaquiline or clofazimine (OR 7·1, 95% CI 2·3–21·9) and with rifampicin-resistant or MDR tuberculosis with additional resistance to either fluoroquinolones or injectable drugs (pre-extensively-drug resistant [XDR] tuberculosis: 4·2, 1·7–10·5) or to both (XDR tuberculosis: 4·8, 2·0–11·7). Rv0678 mutations were the sole genetic basis of phenotypic resistance. Baseline resistance could be attributed to previous bedaquiline or clofazimine exposure in four (5·3%) of 76 patients and to primary transmission in six (7·9%). Odds of successful treatment outcomes were lower in patients with baseline bedaquiline resistance (0·5, 0·3–1). Resistance during treatment developed in 16 (2·3%) of 695 patients, at a median of 90 days (IQR 62–195), with 12 of these 16 having pre-XDR or XDR. INTERPRETATION : Bedaquiline resistance was associated with poorer treatment outcomes. Rapid assessment of bedaquiline resistance, especially when patients were previously exposed to bedaquiline or clofazimine, should be prioritised at baseline or if patients remain culture-positive after 2 months of treatment. Preventing resistance by use of novel combination therapies, current treatment optimisation, and patient support is essential.National Institute for Communicable Diseases of South Africa.http://www.thelancet.com/infectionhj2023Medical Microbiolog

The 2021 WHO catalogue of Mycobacterium tuberculosis complex mutations associated with drug resistance: a genotypic analysis.

Background: Molecular diagnostics are considered the most promising route to achievement of rapid, universal drug susceptibility testing for Mycobacterium tuberculosis complex (MTBC). We aimed to generate a WHO-endorsed catalogue of mutations to serve as a global standard for interpreting molecular information for drug resistance prediction. Methods: In this systematic analysis, we used a candidate gene approach to identify mutations associated with resistance or consistent with susceptibility for 13 WHO-endorsed antituberculosis drugs. We collected existing worldwide MTBC whole-genome sequencing data and phenotypic data from academic groups and consortia, reference laboratories, public health organisations, and published literature. We categorised phenotypes as follows: methods and critical concentrations currently endorsed by WHO (category 1); critical concentrations previously endorsed by WHO for those methods (category 2); methods or critical concentrations not currently endorsed by WHO (category 3). For each mutation, we used a contingency table of binary phenotypes and presence or absence of the mutation to compute positive predictive value, and we used Fisher's exact tests to generate odds ratios and Benjamini-Hochberg corrected p values. Mutations were graded as associated with resistance if present in at least five isolates, if the odds ratio was more than 1 with a statistically significant corrected p value, and if the lower bound of the 95% CI on the positive predictive value for phenotypic resistance was greater than 25%. A series of expert rules were applied for final confidence grading of each mutation. Findings: We analysed 41 137 MTBC isolates with phenotypic and whole-genome sequencing data from 45 countries. 38 215 MTBC isolates passed quality control steps and were included in the final analysis. 15 667 associations were computed for 13 211 unique mutations linked to one or more drugs. 1149 (7·3%) of 15 667 mutations were classified as associated with phenotypic resistance and 107 (0·7%) were deemed consistent with susceptibility. For rifampicin, isoniazid, ethambutol, fluoroquinolones, and streptomycin, the mutations' pooled sensitivity was more than 80%. Specificity was over 95% for all drugs except ethionamide (91·4%), moxifloxacin (91·6%) and ethambutol (93·3%). Only two resistance mutations were identified for bedaquiline, delamanid, clofazimine, and linezolid as prevalence of phenotypic resistance was low for these drugs. Interpretation: We present the first WHO-endorsed catalogue of molecular targets for MTBC drug susceptibility testing, which is intended to provide a global standard for resistance interpretation. The existence of this catalogue should encourage the implementation of molecular diagnostics by national tuberculosis programmes. Funding: Unitaid, Wellcome Trust, UK Medical Research Council, and Bill and Melinda Gates Foundation

Epidemiological cut-offs for Sensititre susceptibility testing of Mycobacterium tuberculosis : interpretive criteria cross validated with whole genome sequencing

Universal drug susceptibility testing (DST) is an important requirement of the End TB Strategy. The Sensititre broth micro-dilution assay (BMD) tests multiple drugs quantitatively. We defined interpretive criteria for this assay and analysed genotypic-phenotypic relationships. 385 Mycobacterium tuberculosis clinical isolates were processed for BMD and whole genome sequencing. The epidemiological cut-off value 99% (ECV99) amongst genotypically wild type (gWT) strains defined susceptibility. Minimum inhibitory concentration distributions of the resistance-associated variants (RAVs) for each drug were analysed. Susceptibility (μg/mL) criteria were determined as follows: rifampicin (≤0.125), isoniazid (≤0.25), ethambutol (≤2.0), moxifloxacin (≤0.5), levofloxacin (≤1.0), amikacin (≤2.0), kanamycin (≤8.0), capreomycin (≤4.0), clofazimine (≤0.25) and linezolid (≤2.0). Most drugs showed clear separation between gWT and RAV. Isoniazid showed a tri-modal pattern with 14/17 strains at ECV99 harbouring a fabG1 c. -15C > T RAV. Ethambutol RAVs at embB codons 306, 405 and 497 were responsible for resistance and showed differential distributions. Moxifloxacin RAVs (gyrA codon 90) were a dilution or two higher than the ECV99 while gyrB RAVs were uncommon and showed drug specific resistance propensity. Interpretive criteria established were robust facilitating progress towards universal DST and individualised precision medicine. This study demonstrates the value of quantitative DST to accurately interpret mutation data.Janssen Pharmaceuticalshttp://www.nature.com/srepam2021Medical Microbiolog

Defining Bedaquiline Susceptibility, Resistance, Cross-Resistance and Associated Genetic Determinants: A Retrospective Cohort Study

Background: Bedaquiline (BDQ) is a novel agent approved for use in combination treatment of multi-drug resistant tuberculosis (MDR-TB). We sought to determine BDQ epidemiological cut-off values (ECVs), define and assess interpretive criteria against putative resistance associated variants (RAVs), microbiological outcomes and cross resistance with clofazimine (CFZ). Methods: A retrospective cohort study was conducted. Minimal inhibitory concentrations (MIC) to BDQ were determined using 7H9 broth microdilution (BMD) and MGIT960. RAVs were genetically characterised using whole genome sequencing. BDQ ECVs were determined using ECOFFinder and compared with 6-month culture conversion status and CFZ MICs. Findings: A total of 391 isolates were analysed. Susceptible and intermediate categories were determined to have MICs of ≤0.125 μg/ml and 0.25 μg/ml using BMD and ≤1 μg/ml and 2 μg/ml using MGIT960 respectively. Microbiological failures occurred among BDQ exposed patients with a non-susceptible BDQ MIC, an Rv0678 mutation and ≤2 active drug classes. The Rv0678 RAVs were not the dominant mechanism of CFZ resistance and cross resistance was limited to isolates with an Rv0678 mutation. Interpretation: Criteria for BDQ susceptibility are defined and will facilitate improved early detection of resistance. Cross- resistance between BDQ and CFZ is an emerging concern but in this study was primarily among those with an Rv0678 mutation