Publications by authors named "Nazir A Ismail"

27 Publications

  • Page 1 of 1

Effectiveness and cardiac safety of bedaquiline-based therapy for drug-resistant tuberculosis: a prospective cohort study.

Clin Infect Dis 2021 Apr 21. Epub 2021 Apr 21.

Wellcome Centre for Infectious Diseases Research in Africa, Institute of Infectious Disease and Molecular Medicine, and Department of Medicine, University of Cape Town, Cape Town, South Africa.

Background: Bedaquiline improves treatment outcomes in patients with rifampin-resistant TB (RR-TB) but prolongs the QT-interval and carries a black-box warning by the U.S. Food and Drug Administration. The World Health Organization recommends that all patients with RR-TB receive a regimen containing bedaquiline, yet a phase 3 clinical trial demonstrating its cardiac safety has not been published.

Methods: We conducted an observational cohort study of RR-TB patients from 3 provinces in South Africa who received regimens containing bedaquiline. We performed rigorous cardiac monitoring, including electrocardiograms (ECGs) performed in triplicate at four time points during bedaquiline therapy. Participants were followed until the end of therapy or 24 months. Outcomes included final tuberculosis treatment outcome and QT-prolongation, defined as any QTcF>500 ms or an absolute change from baseline (△ QTcF) >60 ms.

Results: We enrolled 195 eligible participants, of whom 40% had extensively drug-resistant (XDR) TB. Most participants (97%) received concurrent clofazimine. 74% of participants were cured or successfully completed treatment, and outcomes did not differ by HIV status. QTcF continued to increase throughout bedaquiline therapy, with a mean increase of 23.7 (SD 22.7) ms from baseline to month 6. Four participants experienced a QTcF>500 ms and 19 experienced a △QTcF>60 ms. Older age was independently associated with QT-prolongation. QT-prolongation was neither more common nor severe in participants receiving concurrent lopinavir-ritonavir.

Conclusions: Severe QT-prolongation was uncommon and did not require permanent discontinuation of either bedaquiline or clofazimine. Close QT-monitoring may be advisable in older patients.
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http://dx.doi.org/10.1093/cid/ciab335DOI Listing
April 2021

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

Sci Rep 2020 01 23;10(1):1013. Epub 2020 Jan 23.

National Institute for Communicable Diseases, Centre for Tuberculosis, Johannesburg, South Africa.

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% (ECV) 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 ECV 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 ECV 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.
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http://dx.doi.org/10.1038/s41598-020-57992-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6978314PMC
January 2020

Cryptococcal-related Mortality Despite Fluconazole Preemptive Treatment in a Cryptococcal Antigen Screen-and-Treat Program.

Clin Infect Dis 2020 04;70(8):1683-1690

Institute of Infection & Immunity, St George's University of London, United Kingdom.

Background: Cryptococcal antigen (CrAg) screening and treatment with preemptive fluconazole reduces the incidence of clinically evident cryptococcal meningitis in individuals living with advanced human immunodeficiency virus (HIV) disease. However, mortality remains higher in CrAg-positive than in CrAg-negative patients with similar CD4+ T-lymphocyte counts.

Methods: We conducted a cohort study to investigate causes of morbidity and mortality during 6 months of follow-up among asymptomatic CrAg-positive and CrAg-negative (ratio of 1:2) patients living with HIV with CD4 counts <100 cells/µL attending 2 hospitals in Johannesburg, South Africa. When possible, minimally invasive autopsy (MIA) was performed on participants who died.

Results: Sixty-seven CrAg-positive and 134 CrAg-negative patients were enrolled. Death occurred in 17/67 (25%) CrAg-positive and 12/134 (9%) CrAg-negative participants (hazard ratio for death, adjusted for CD4 count, 3.0; 95% confidence interval, 1.4-6.7; P = .006). Cryptococcal disease was an immediate or contributing cause of death in 12/17 (71%) CrAg-positive participants. Postmortem cryptococcal meningitis and pulmonary cryptococcosis were identified at MIA in all 4 CrAg-positive participants, 3 of whom had negative cerebrospinal fluid CrAg tests from lumbar punctures (LPs) at the time of CrAg screening.

Conclusions: Cryptococcal disease was an important cause of mortality among asymptomatic CrAg-positive participants despite LPs to identify and treat those with subclinical cryptococcal meningitis and preemptive fluconazole for those without meningitis. Thorough investigation for cryptococcal disease with LPs and blood cultures, prompt ART initiation, and more intensive antifungals may reduce mortality among asymptomatic CrAg-positive patients identified through screening.
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http://dx.doi.org/10.1093/cid/ciz485DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7346756PMC
April 2020

Study of Stepwise Acquisition of and Mutations Conferring Bedaquiline Resistance.

Antimicrob Agents Chemother 2019 08 25;63(8). Epub 2019 Jul 25.

Department of Medical Microbiology, Faculty of Health Sciences, University of Pretoria, Prinshof, Gauteng, South Africa

Bedaquiline resistance within may arise through efflux-based () or target-based () pathway mutations. mutant populations from each of five sequential steps in a passaging approach, using a pyrazinamide-resistant ATCC strain, were subjected to MIC determinations and whole-genome sequencing. Exposure to increasing bedaquiline concentrations resulted in increasing phenotypic resistance (up to >2 μg/ml) through MIC determination on solid medium (Middlebrook 7H10). mutations were dynamic, while mutations were fixed, once occurring. We present the following hypothesis for emergence of bedaquiline resistance: mutations may be the first transient step in low-level resistance acquisition, followed by high-level resistance due to fixed mutations.
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http://dx.doi.org/10.1128/AAC.00292-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6658778PMC
August 2019

Whole genome sequencing for drug resistance determination in .

Afr J Lab Med 2019 21;8(1):801. Epub 2019 Feb 21.

Centre for Tuberculosis, World Health Organization TB Supranational Reference Laboratory Network, National Institute for Communicable Diseases, National Health Laboratory Service, Johannesburg, South Africa.

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 treatment in our setting. Whole genome sequencing shows potential as a diagnostic platform for the detection of drug resistance in with the provision of information for several drugs simultaneously.
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http://dx.doi.org/10.4102/ajlm.v8i1.801DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6407317PMC
February 2019

Clofazimine Exposure Selects Efflux Pump Mutants and Bedaquiline Resistance.

Antimicrob Agents Chemother 2019 03 26;63(3). Epub 2019 Feb 26.

Centre for Tuberculosis, National Institute for Communicable Diseases, National Health Laboratory Service, Johannesburg, South Africa.

Six clofazimine-resistant spontaneous mutants obtained from a wild-type or pyrazinamide-resistant ATCC reference strain were selected to evaluate bedaquiline cross-resistance. The reverse was conducted for bedaquiline mutants. All clofazimine mutants harboring an mutation displayed phenotypic cross-resistance. We observed the same for bedaquiline mutants; however, bedaquiline mutants showed no phenotypic cross-resistance. This confirms that upfront clofazimine usage may impact subsequent bedaquiline use due to a shared efflux resistance pathway.
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http://dx.doi.org/10.1128/AAC.02141-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6395909PMC
March 2019

Multidrug-resistant tuberculosis outbreak in South Africa.

Lancet Infect Dis 2019 02 6;19(2):134-135. Epub 2018 Dec 6.

MDR-TB Directorate, National Department of Health, Pretoria, South Africa.

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http://dx.doi.org/10.1016/S1473-3099(18)30715-1DOI Listing
February 2019

Prevalence of drug-resistant tuberculosis in South Africa - Authors' reply.

Lancet Infect Dis 2018 08;18(8):836-837

Medical Research Council, Respiratory and Meningeal Pathogens Research Unit, Johannesburg, South Africa; Department of Science, National Research Foundation, Vaccine Preventable Diseases, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa.

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http://dx.doi.org/10.1016/S1473-3099(18)30422-5DOI Listing
August 2018

Whole-Genome Sequence of a Isolate from a Pediatric Patient in South Africa.

Genome Announc 2018 Jan 18;6(3). Epub 2018 Jan 18.

Centre for Tuberculosis, National Institute for Communicable Diseases, Johannesburg, South Africa

We describe here the draft genome sequence of a 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.
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http://dx.doi.org/10.1128/genomeA.01478-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5773733PMC
January 2018

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

EBioMedicine 2018 Feb 9;28:136-142. Epub 2018 Jan 9.

National Department of Health, Tuberculosis Control and Management Cluster, Pretoria, South Africa.

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 ≤0.125μg/ml and 0.25μg/ml using BMD and ≤1μg/ml and 2μg/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.
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http://dx.doi.org/10.1016/j.ebiom.2018.01.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5835552PMC
February 2018

Draft Genome Sequence of Isolated from an HIV-Positive Patient in South Africa.

Genome Announc 2017 Aug 3;5(31). Epub 2017 Aug 3.

Sequencing Core Facility, National Institute for Communicable Diseases, National Health Laboratory Service, Johannesburg, South Africa.

Here, we report a draft genome sequence of obtained from a sputum sample of a South African HIV-infected patient with suspected pulmonary tuberculosis. The genome described here comprises 6,931,852 bp, revealing 66.2% G+C content, 6,808 coding sequences, and 81 RNA genes.
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http://dx.doi.org/10.1128/genomeA.00759-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5543650PMC
August 2017

The Epidemiology of Meningitis among Adults in a South African Province with a High HIV Prevalence, 2009-2012.

PLoS One 2016;11(9):e0163036. Epub 2016 Sep 26.

National Institute for Communicable Diseases, Johannesburg, South Africa.

Introduction: Meningitis is a major cause of mortality in southern Africa. We aimed to describe the aetiologies and frequencies of laboratory-confirmed fungal and bacterial meningitis among adults in a South African province with an 11% HIV prevalence, over 4 years.

Methods: We conducted a retrospective, observational study of secondary laboratory data, extracted on all cerebrospinal fluid (CSF) specimens submitted to public-sector laboratories in Gauteng province from 2009 through 2012. We calculated cause-specific incidence rates in the general and HIV-infected populations and used Poisson regression to determine if trends were significant.

Results: We identified 11,891 (10.7%) incident cases of meningitis from 110,885 CSF specimens. Cryptococcal meningitis, tuberculous meningitis and pneumococcal meningitis accounted for 62.3% (n = 7,406), 24.6% (n = 2,928) and 10.1% (n = 1,197) of cases over the four-year period. The overall incidence (cases per 100,000 persons) of cryptococcal meningitis declined by 23% from 24.4 in 2009 to 18.7 in 2012 (p <0.001) and decreased by 19% among HIV-infected persons from 178.2 to 144.7 (p <0.001). Tuberculous meningitis decreased by 40% from 11.3 in 2009 to 6.8 in 2012 (p <0.001) and decreased by 36% among HIV-infected persons from 54.4 to 34.9 (p <0.001). Pneumococcal meningitis decreased by 41% from 4.2 in 2009 to 2.5 in 2012 (p <0.001) and decreased by 38% among HIV-infected persons from 28.0 to 17.5 (p <0.001). Among cases of other bacterial meningitis (248/11,891, 2.1%), Neisseria meningitidis (n = 93), Escherichia coli (n = 72) and Haemophilus influenzae (n = 20) were the most common organisms identified.

Conclusions: In this high HIV-prevalence province, cryptococcal meningitis was the leading cause of laboratory-confirmed meningitis among adults. Over a 4-year period, there was a significant decrease in incidence of cryptococcal, tuberculous and pneumococcal meningitis. This coincided with expansion of the national antiretroviral treatment programme, enhanced tuberculosis control programme and routine childhood immunisation with pneumococcal conjugate vaccines.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0163036PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5036788PMC
September 2016

A Multilaboratory, Multicountry Study To Determine MIC Quality Control Ranges for Phenotypic Drug Susceptibility Testing of Selected First-Line Antituberculosis Drugs, Second-Line Injectables, Fluoroquinolones, Clofazimine, and Linezolid.

J Clin Microbiol 2016 12 21;54(12):2963-2968. Epub 2016 Sep 21.

TASK Applied Science, SA MRC Centre for TB Research, DST/NRF Center of Excellence for Biomedical TB Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, South Africa.

Our objective was to establish reference MIC quality control (QC) ranges for drug susceptibility testing of antimycobacterials, including first-line agents, second-line injectables, fluoroquinolones, and World Health Organization category 5 drugs for multidrug-resistant tuberculosis using a 7H9 broth microdilution MIC method. A tier-2 reproducibility study was conducted in eight participating laboratories using Clinical Laboratory and Standards Institute (CLSI) guidelines. Three lots of custom-made frozen 96-well polystyrene microtiter plates were used and prepared with 2× prediluted drugs in 7H9 broth-oleic acid albumin dextrose catalase. The QC reference strain was Mycobacterium tuberculosis H37Rv. MIC frequency, mode, and geometric mean were calculated for each drug. QC ranges were derived based on predefined, strict CLSI criteria. Any data lying outside CLSI criteria resulted in exclusion of the entire laboratory data set. Data from one laboratory were excluded due to higher MIC values than other laboratories. QC ranges were established for 11 drugs: isoniazid (0.03 to 0.12 μg/ml), rifampin (0.03 to 0.25 μg/ml), ethambutol (0.25 to 2 μg/ml), levofloxacin (0.12 to 1 μg/ml), moxifloxacin (0.06 to 0.5 μg/ml), ofloxacin (0.25 to 2 μg/ml), amikacin (0.25 to 2 μg/ml), kanamycin (0.25 to 2 μg/ml), capreomycin (0.5 to 4 μg/ml), linezolid (0.25 to 2 μg/ml), and clofazimine (0.03 to 0.25 μg/ml). QC ranges could not be established for nicotinamide (pyrazinamide surrogate), prothionamide, or ethionamide, which were assay nonperformers. Using strict CLSI criteria, QC ranges against the M. tuberculosis H37Rv reference strain were established for the majority of commonly used antituberculosis drugs, with a convenient 7H9 broth microdilution MIC method suitable for use in resource-limited settings.
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http://dx.doi.org/10.1128/JCM.01138-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5121386PMC
December 2016

A Multilaboratory, Multicountry Study To Determine Bedaquiline MIC Quality Control Ranges for Phenotypic Drug Susceptibility Testing.

J Clin Microbiol 2016 12 21;54(12):2956-2962. Epub 2016 Sep 21.

TASK Applied Science, SA MRC Centre for TB Research, DST/NRF Center of Excellence for Biomedical TB Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, South Africa.

The aim of this study was to establish standardized drug susceptibility testing (DST) methodologies and reference MIC quality control (QC) ranges for bedaquiline, a diarylquinoline antimycobacterial, used in the treatment of adults with multidrug-resistant tuberculosis. Two tier-2 QC reproducibility studies of bedaquiline DST were conducted in eight laboratories using Clinical Laboratory and Standards Institute (CLSI) guidelines. Agar dilution and broth microdilution methods were evaluated. Mycobacterium tuberculosis H37Rv was used as the QC reference strain. Bedaquiline MIC frequency, mode, and geometric mean were calculated. When resulting data occurred outside predefined CLSI criteria, the entire laboratory data set was excluded. For the agar dilution MIC, a 4-dilution QC range (0.015 to 0.12 μg/ml) centered around the geometric mean included 95.8% (7H10 agar dilution; 204/213 observations with one data set excluded) or 95.9% (7H11 agar dilution; 232/242) of bedaquiline MICs. For the 7H9 broth microdilution MIC, a 3-dilution QC range (0.015 to 0.06 μg/ml) centered around the mode included 98.1% (207/211, with one data set excluded) of bedaquiline MICs. Microbiological equivalence was demonstrated for bedaquiline MICs determined using 7H10 agar and 7H11 agar but not for bedaquiline MICs determined using 7H9 broth and 7H10 agar or 7H9 broth and 7H11 agar. Bedaquiline DST methodologies and MIC QC ranges against the H37Rv M. tuberculosis reference strain have been established: 0.015 to 0.12 μg/ml for the 7H10 and 7H11 agar dilution MICs and 0.015 to 0.06 μg/ml for the 7H9 broth microdilution MIC. These methodologies and QC ranges will be submitted to CLSI and EUCAST to inform future research and provide guidance for routine clinical bedaquiline DST in laboratories worldwide.
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http://dx.doi.org/10.1128/JCM.01123-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5121385PMC
December 2016

Evaluation of Semiautomated IS6110-Based Restriction Fragment Length Polymorphism Typing for Mycobacterium tuberculosis in a High-Burden Setting.

J Clin Microbiol 2016 10 3;54(10):2547-52. Epub 2016 Aug 3.

Centre for Tuberculosis, National Institute of Communicable Diseases, Johannesburg, South Africa Department of Medical Microbiology, Faculty of Health Science, University of Pretoria, Pretoria, South Africa.

The manual IS6110-based restriction fragment length polymorphism (RFLP) typing method is highly discriminatory; however, it is laborious and technically demanding, and data exchange remains a challenge. In an effort to improve IS6110-based RFLP to make it a faster format, DuPont Molecular Diagnostics recently introduced the IS6110-PvuII kit for semiautomated typing of Mycobacterium tuberculosis using the RiboPrinter microbial characterization system. This study aimed to evaluate the semiautomated RFLP typing against the standard manual method. A total of 112 isolates collected between 2013 and 2014 were included. All isolates were genotyped using manual and semiautomated RFLP typing methods. Clustering rates and discriminatory indexes were compared between methods. The overall performance of semiautomated RFLP compared to manual typing was excellent, with high discriminatory index (0.990 versus 0.995, respectively) and similar numbers of unique profiles (72 versus 74, respectively), numbers of clustered isolates (33 versus 31, respectively), cluster sizes (2 to 6 and 2 to 5 isolates, respectively), and clustering rates (21.9% and 17.1%, respectively). The semiautomated RFLP system is technically simple and significantly faster than the manual RFLP method (8 h versus 5 days). The analysis is fully automated and generates easily manageable databases of standardized fingerprints that can be easily exchanged between laboratories. Based on its high-throughput processing with minimal human effort, the semiautomated RFLP can be a very useful tool as a first-line method for routine typing of M. tuberculosis isolates, especially where Beijing strains are highly prevalent, followed by manual RFLP typing if resolution is not achieved, thereby saving time and labor.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5035400PMC
http://dx.doi.org/10.1128/JCM.00408-16DOI Listing
October 2016

Field evaluation of a novel preservation medium to transport sputum specimens for molecular detection of Mycobacterium tuberculosis in a rural African setting.

Trop Med Int Health 2016 06 16;21(6):776-82. Epub 2016 May 16.

Department of Medical Microbiology, Faculty of Health Sciences, University of Pretoria, Pretoria, South Africa.

Objectives: To assess the performance of an innovative method of transporting sputum to centralised facilities for molecular detection of Mycobacterium tuberculosis: using a swab to inoculate sputum in a transport medium, PrimeStore(®) Molecular Transport Medium (PS-MTM).

Methods: Two sputum specimens were obtained from suspected patients with tuberculosis (TB) at rural healthcare facilities in South Africa. A swab was taken from each specimen and placed into PS-MTM, prior to it being processed by either liquid culture or Xpert MTB/RIF assay (Xpert).

Results: A total of 141 patients (including 47 with laboratory-confirmed TB) were included in this analysis. M. tuberculosis was detected at 29% by culture and 29% by Xpert, whereas 31% tested positive by IS6110 real-time PCR of PS-MTM from the culture and 36% from the Xpert-paired specimen. Concordance between the method under evaluation with culture was 82% (McNemar, P = 0.55) and 84% (McNemar, P = 0.05) for Xpert. Stratified by culture result, the detection rate by IS6110 real-time PCR of PS-MTM was similar to Xpert for patients with positive culture (P = 0.32), but significantly higher if culture was negative (P = 0.008).

Conclusions: These results suggest that swab collection of sputum into PS-MTM for transport is a promising method for diagnosis of TB in rural healthcare settings, thereby potentially improving the options available for molecular diagnosis of TB in countries incapable of applying decentralised high-tech molecular testing.
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http://dx.doi.org/10.1111/tmi.12701DOI Listing
June 2016

A Novel Molecular Strategy for Surveillance of Multidrug Resistant Tuberculosis in High Burden Settings.

PLoS One 2016 11;11(1):e0146106. Epub 2016 Jan 11.

Centre for Tuberculosis, National Institute of Communicable Diseases, Sandringham, South Africa.

Background: In South Africa and other high prevalence countries, transmission is a significant contributor to rising rates of multidrug resistant tuberculosis (MDR-TB). Thus, there is a need to develop an early detection system for transmission clusters suitable for high burden settings. We have evaluated the discriminatory power and clustering concordance of a novel and simple genotyping approach, combining spoligotyping with pncA sequencing (SpoNC), against two well-established methods: IS6110-RFLP and 24-loci MIRU-VNTR.

Methods: A total of 216 MDR-TB isolates collected from January to June 2010 from the NHLS Central TB referral laboratory in Braamfontein, Johannesburg, representing a diversity of strains from South Africa, were included. The isolates were submitted for genotyping, pncA sequencing and analysis to the Centre for Tuberculosis in South Africa and the Public Health Research Institute Tuberculosis Center at Rutgers University in the United States. Clustering rates, Hunter-Gaston Discriminatory Indexes (HGI) and Wallace coefficients were compared between the methods.

Results: Overall clustering rates were high by both IS6110-RFLP (52.8%) and MIRU-VNTR (45.8%), indicative of on-going transmission. Both 24-loci MIRU-VNTR and IS6110-RFLP had similar HGI (0.972 and 0.973, respectively), with close numbers of unique profiles (87 vs. 70), clustered isolates (129 vs. 146), and cluster sizes (2 to 26 vs. 2 to 25 isolates). Spoligotyping alone was the least discriminatory (80.1% clustering, HGI 0.903), with 28 unique types. However, the discriminatory power of spoligotyping was improved when combined with pncA sequencing using the SpoNC approach (61.8% clustering, HGI 0.958). A high proportion of MDR-TB isolates had mutations in pncA (68%, n = 145), and pncA mutations were significantly associated with clustering (p = 0.007 and p = 0.0013 by 24-loci MIRU-VNTR and IS6110-RFLP, respectively), suggesting high rates of resistance to pyrazinamide among all MDR-TB cases and particularly among clustered cases.

Conclusion: We conclude that SpoNC provides good discrimination for MDR-TB surveillance and early identification of outbreaks in South Africa, with 24-loci MIRU-VNTR applied for pncA wild-type strains as needed.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0146106PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4713439PMC
July 2016

Laboratory evaluation of a specimen transport medium for downstream molecular processing of sputum samples to detect Mycobacterium tuberculosis.

J Microbiol Methods 2015 Oct 14;117:57-63. Epub 2015 Jul 14.

Faculty of Health Sciences, Department of Medical Microbiology, University of Pretoria, Pretoria, South Africa.

Background: Modern molecular-based approaches for the detection of Mycobacterium tuberculosis in sputum samples promise quicker and more accurate detection of cases. However, processing sputum samples at central diagnostic facilities provides a diagnostic approach, but requires a safe and efficient system that is not affected by transport delays and ambient temperature to be feasible. We evaluated the technical properties of PrimeStore®-Molecular Transport Medium (PS-MTM) for its ability to inactivate mycobacteria, ensuring stability of DNA over time at ambient temperatures and to assess the compatibility of the transport medium with DNA extraction systems.

Methods: Assessment of the transport medium for application of sputum samples processed for the detection of M. tuberculosis included the inactivation of M. tuberculosis in spiked sputum samples, compatibility of the medium with three commercial nucleic extraction systems and stability of DNA in the medium at ambient temperature over 28 days. We further performed a clinical laboratory evaluation on 256 sputum specimens sent for tuberculosis investigation.

Results: Complete inactivation of M. tuberculosis occurred within 30 min of exposure at a ratio of 1:3 for sputum to PS-MTM. Sputum specimen in PS-MTM showed very good compatibility with automated bead-based extraction systems, producing high DNA output (estimated lower limits of detection: ~170 CFU/ml). Furthermore, PS-MTM samples remained stable over 28 days at ambient temperature displaying no significant change over time in Ct-values (<5% on a mean starting value of 22.47). Of the 256 clinical sputum specimens, 10.2% were culture positive and 11.0% were positive by real-time PCR of PS-MTM samples.

Conclusions: Collecting and transporting sputum from TB suspects in PS-MTM offer safe transport at ambient temperature, DNA stability for extended periods without cooling and specimens directly suitable for molecular testing. This novel approach may support introduction and further scale-up of molecular diagnostics for TB in resource-limited settings.
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http://dx.doi.org/10.1016/j.mimet.2015.07.010DOI Listing
October 2015

Nationwide and regional incidence of microbiologically confirmed pulmonary tuberculosis in South Africa, 2004-12: a time series analysis.

Lancet Infect Dis 2015 Sep 22;15(9):1066-1076. Epub 2015 Jun 22.

Centre for Tuberculosis, National Institute for Communicable Diseases, Division of National Health Laboratory Service, Sandringham, Johannesburg, South Africa; Medical Research Council, Respiratory and Meningeal Pathogens Research Unit, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa; Department of Science and Technology/National Research Foundation, Vaccine Preventable Diseases, University of the Witwatersrand, Johannesburg, South Africa. Electronic address:

Background: South Africa has the highest incidence of tuberculosis in the world, largely resulting from a high population prevalence of HIV infection. We investigated the incidence of microbiologically confirmed pulmonary tuberculosis, and new cases of pulmonary tuberculosis registered for treatment, nationally and provincially in South Africa from 2004 to 2012, during which time there were changes in antiretroviral therapy (ART) coverage among individuals with HIV infection.

Methods: We identified cases of microbiologically confirmed pulmonary tuberculosis from 2004 to 2012 from the National Health Laboratory Service Corporate Data Warehouse. New cases registered for treatment were identified from National Department of Health electronic registries. A time series analysis, using autoregressive models, was undertaken on incidence of microbiologically confirmed pulmonary disease nationally and provincially; this trend was also examined relative to ART coverage of adults with HIV infection.

Findings: During the 9-year period, 3 523 371 cases of microbiologically confirmed pulmonary tuberculosis were recorded nationally. Annual incidence (per 100 000 population) increased from 650 (95% CI 648-652) in 2004 to 848 (845-850) in 2008, declining to 774 (771-776) by 2012 (9% decrease from 2008 to 2012). Incidence varied by age-group, sex, and province. There was an inverse association between incidence of microbiologically confirmed disease and ART coverage among HIV-infected individuals nationally and provincially. Trends in incidence of tuberculosis cases registered for treatment mirrored those of microbiologically confirmed cases nationally and provincially; however, incidence of microbiologically confirmed cases was consistently higher than cases registered for treatment nationally and in seven of nine provinces.

Interpretation: Since its peak in 2008, the incidence of microbiologically confirmed pulmonary tuberculosis in South Africa had declined by 2012; this decline is associated with an increase in ART coverage. Future integration of registries for microbiologically confirmed cases and new cases registered for treatment would improve the assessment of the burden of pulmonary tuberculosis in South Africa.

Funding: National Institute for Communicable Diseases: Division of the National Health Laboratory Service, South Africa.
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http://dx.doi.org/10.1016/S1473-3099(15)00147-4DOI Listing
September 2015

Performance of a Novel Algorithm Using Automated Digital Microscopy for Diagnosing Tuberculosis.

Am J Respir Crit Care Med 2015 Jun;191(12):1443-9

3 London School of Hygiene and Tropical Medicine, London, United Kingdom.

Rationale: TBDx automated microscopy is a novel technology that processes digital microscopic images to identify acid-fast bacilli (AFB). Use of TBDx as part of a diagnostic algorithm could improve the diagnosis of tuberculosis (TB), but its performance characteristics have not yet been formally tested.

Objectives: To evaluate the performance of the TBDx automated microscopy system in algorithms for diagnosis of TB.

Methods: Prospective samples from patients with presumed TB were processed in parallel with conventional smear microscopy, TBDx microscopy, and liquid culture. All TBDx-positive specimens were also tested with the Xpert MTB/RIF (GXP) assay. We evaluated the sensitivity and specificity of two algorithms-(1) TBDx-GXP (TBDx with positive specimens tested by Xpert MTB/RIF) and (2) TBDx alone-against the gold standard liquid media culture.

Measurements And Main Results: Of 1,210 samples, 1,009 were eligible for evaluation, of which 109 were culture positive for Mycobacterium tuberculosis. The TBDx system identified 70 specimens (68 culture positive) as having 10 or more putative AFB (high positive) and 207 (19 culture positive) as having 1-9 putative AFB (low positive). An algorithm in which "low-positive" results on TBDx were confirmed by GXP had 78% sensitivity (85 of 109) and 99.8% specificity (889 of 900), requiring 21% (207 of 1,009) specimens to be processed by GXP. As a stand-alone test, a "high-positive" result on TBDx had 62% sensitivity and 99.7% specificity.

Conclusions: TBDx used in diagnostic algorithms with GXP provided reasonable sensitivity and high specificity for active TB while dramatically reducing the number GXP tests performed. As a stand-alone microscopy system, its performance was equivalent to that of a highly experienced TB microscopist.
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http://dx.doi.org/10.1164/rccm.201502-0390OCDOI Listing
June 2015

Drug-resistance mechanisms and tuberculosis drugs.

Lancet 2015 Jan;385(9965):305-7

Department of Medicine, Addenbrooke's Hospital, University of Cambridge, Cambridge CB2 0QW, UK; Clinical Microbiology and Public Health Laboratory, Public Health England, Cambridge, UK; Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK.

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http://dx.doi.org/10.1016/S0140-6736(14)62450-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4374148PMC
January 2015

Comparison between the BACTEC MGIT 960 system and the agar proportion method for susceptibility testing of multidrug resistant tuberculosis strains in a high burden setting of South Africa.

BMC Infect Dis 2012 Dec 22;12:369. Epub 2012 Dec 22.

Department of Medical Microbiology, Faculty of Health Science, University of Pretoria, Private bag X323, arcadia, Pretoria, 0007, South Africa.

Background: The increasing problem of multi-drug-resistant (MDR) tuberculosis (TB) [ie resistant to at least isoniazid (INH) and rifampicin (RIF)] is becoming a global problem. Successful treatment outcome for MDR-TB depends on reliable and accurate drug susceptibility testing of first-line and second-line anti-TB drugs.

Method: Consecutive M. tuberculosis isolates identified as MDR-TB during August 2007 to January 2008 using the BACTEC MGIT 960 systems and the agar proportion method were included in this study. Susceptibility testing of MDR-TB isolates against ethambutol (EMB) and streptomycin (STR) as well as two second-line anti-TB drugs, kanamycin (KAN) and ofloxacin (OFX) was performed using the BACTEC MGIT 960 systems at a routine diagnostic laboratory. The results were compared to those obtained by the agar proportion method.

Result: The agreement between the BACTEC MGIT 960 system and the agar proportion method was 44% for EMB, 61% for STR and 89% for both KAN and OFX. The sensitivity and specificity of the BACTEC MGIT 960 system using the agar proportion method as a gold standard was 92% and 37% for EMB, 95% and 37% for STR, 27% and 97% for KAN and 84% and 90% for OFX, respectively.

Conclusions: The BACTEC MGIT 960 system showed acceptable sensitivity for EMB, STR, and OFX; however, the BACTEC MGIT 960 system was less specific for EMB and STR and demonstrated a low sensitivity for KAN. The lower agreement found between the two methods suggests the unreliability of the BACTEC MGIT 960 system for the drugs tested. The reasons for the lower agreement between the two methods need to be investigated and further studies are needed in this setting to confirm the study finding.
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http://dx.doi.org/10.1186/1471-2334-12-369DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3543708PMC
December 2012

Next-generation ion torrent sequencing of drug resistance mutations in Mycobacterium tuberculosis strains.

J Clin Microbiol 2012 Dec 12;50(12):3831-7. Epub 2012 Sep 12.

Longhorn Vaccines & Diagnostics, San Antonio, Texas, USA.

A novel protocol for full-length Mycobacterium tuberculosis gene analysis of first- and second-line drug resistance was developed using the Ion Torrent Personal Genome Machine (PGM). Five genes-rpoB (rifampin), katG (isoniazid), pncA (pyrazinamide), gyrA (ofloxacin/fluoroquinolone), and rrs (aminoglycosides)-were amplified and sequenced, and results were compared to those obtained by genotypic Hain line probe assay (LPA) and phenotypic Bactec MGIT 960 analysis using 26 geographically diverse South African clinical isolates collected between July and November 2011. Ion Torrent sequencing exhibited 100% (26/26) concordance to phenotypic resistance obtained by MGIT 960 culture and genotypic rpoB and katG results by LPA. In several rifampin-resistant isolates, Ion Torrent sequencing revealed uncommon substitutions (H526R and D516G) that did not have a defined mutation by LPA. Importantly, previously uncharacterized mutations in rpoB (V194I), rrs (G878A), and pncA (Q122Stop) genes were observed. Ion Torrent sequencing may facilitate tracking and monitoring geographically diverse multidrug-resistant and extensively drug-resistant strains and could potentially be integrated into selected regional and reference settings throughout Africa, India, and China.
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http://dx.doi.org/10.1128/JCM.01893-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3502959PMC
December 2012

Molecular characterization and second-line antituberculosis drug resistance patterns of multidrug-resistant Mycobacterium tuberculosis isolates from the northern region of South Africa.

J Clin Microbiol 2012 Sep 30;50(9):2857-62. Epub 2012 May 30.

Department of Medical Microbiology, Faculty of Health Sciences, University of Pretoria, Pretoria, South Africa.

Despite South Africa being one of the high-burden multidrug-resistant tuberculosis (MDR-TB) countries, information regarding the population structure of drug-resistant Mycobacterium tuberculosis strains is limited from many regions of South Africa. This study investigated the population structure and transmission patterns of drug-resistant M. tuberculosis isolates in a high-burden setting of South Africa as well as the possible association of genotypes with drug resistance and demographic characteristics. A total of 336 consecutive MDR-TB isolates from four provinces of South Africa were genotyped using spoligotyping and mycobacterial interspersed repetitive-unit-variable number tandem repeat (MIRU-VNTR) typing. Drug susceptibility testing for ofloxacin, kanamycin, and capreomycin was performed using the agar proportion method. The results showed that 4.8% of MDR-TB isolates were resistant to ofloxacin, 2.7% were resistant to kanamycin, and 4.5% were resistant to capreomycin, while 7.1% were extensively drug resistant (XDR), and the remaining 83.6% were susceptible to all of the second-line drugs tested. Spoligotyping grouped 90.8% of the isolates into 25 clusters, while 9.2% isolates were unclustered. Ninety-one percent of the 336 isolates were assigned to 21 previously described shared types, with the Beijing family being the predominant genotype in the North-West and Limpopo Provinces, while the EAI1_SOM family was the predominant genotype in the Gauteng and Mpumalanga Provinces. No association was found between genotypes and specific drug resistance patterns or demographic information. The high level of diversity and the geographical distribution of the drug-resistant M. tuberculosis isolates in this study suggest that the transmission of TB in the study settings is not caused by the clonal spread of a specific M. tuberculosis strain.
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http://dx.doi.org/10.1128/JCM.00358-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3421812PMC
September 2012

Analytical performance of the Roche LightCycler® Mycobacterium Detection Kit for the diagnosis of clinically important mycobacterial species.

PLoS One 2011 22;6(9):e24789. Epub 2011 Sep 22.

Department of Medical Microbiology, University of Pretoria, Pretoria, South Africa.

Background: The LightCycler® Mycobacterium Detection Kit based on real-time PCR technology for the detection of Mycobacterium tuberculosis, Mycobacterium avium and Mycobacterium kansasii was recently developed. This study evaluated its analytical sensitivity, specificity and reproducibility.

Methodology/principal Findings: Plasmid standards were prepared and used to determine the limit of detection. The assay was also performed against organisms other than mycobacteria, other mycobacterial strains and interfering substances to exclude cross-reactivity and interference. Reference standards were prepared and tested to assess the assay's reproducibility. All PCR assays were performed using the LightCycler® 2.0 Instrument. The detection limit for M. tuberculosis was 28 copies per microlitre. Neither cross-reactivity nor interference occurred with non-mycobacterial organisms and substances tested. Overall reproducibility for consecutive measurements, run-to-run, lot-to-lot, day-to-day and laboratory-to-laboratory achieved a coefficient of variance of less than two percent.

Significance: The LightCycler® Mycobacterium Detection kit has shown to be a robust and accurate assay with the potential to be used as a rapid TB diagnostic test.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0024789PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3178549PMC
March 2012

Comparison of the VersaTREK blood culture system against the Bactec9240 system in patients with suspected bloodstream infections.

Ann Clin Microbiol Antimicrob 2011 Feb 5;10. Epub 2011 Feb 5.

Department of Medical Microbiology, University of Pretoria and Tshwane Academic Division of the National Health Laboratory Service (NHLS), Pretoria, South Africa.

Background: To evaluate the VersaTREK (TREK Diagnostic Systems, Cleveland, Ohio) blood culture system against the Bactec9240 (BD Microbiology, Cockeysville, MD), for the recovery of bloodstream pathogens.

Methods: Venous blood from patients with suspected bacterial sepsis was evenly distributed into bottles of each system. Positive signals were recorded and bottles processed onto standard media for organism recovery. False positive signals were regarded if no organisms were seen on Gram stain and no growth was observed.

Results: 177 bottles were available for analysis; the Bactec9240 system yielded 43 positive, 134 negative results and no false positive signals. The VersaTREK system had 58 positive signals with 14 being false positives.

Conclusions: In our setting with high background burden of immuno-compromised patients, the VersaTREK system compared favourably with the Bactec9240 in recovering blood stream aerobic and facultative anaerobic pathogens from patients with suspected bacterial sepsis. A concern is the high false positivity rate. Due to its versatility to accommodate small and large workloads as well as using smaller volumes of blood, this system may establish itself as a useful alternative for the recovery of bloodstream pathogens.
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http://dx.doi.org/10.1186/1476-0711-10-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3042901PMC
February 2011