SUNScholar - Browsing by Author "Barnard, Marinus"

Browsing by Author "Barnard, Marinus"

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Diagnostic utility of the line probe assay for the detection of drug resistance in Mycobacterium tuberculosis
(Stellenbosch : Stellenbosch University, 2013-03) Barnard, Marinus; Warren, Robin Mark; Gey van Pittius, Nicolaas C.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Division Molecular Biology and Human Genetics
ENGLISH ABSTRACT: The epidemic levels of drug-resistant tuberculosis (DR-TB) in high-burden countries such as South Africa, which is currently ranked as third highest in the world, is the result of a synergistic relationship between the increased transmission of DR strains, poor patient adherence as well as Human-Immunodeficiency Virus (HIV)-coinfection. The impact of these combined factors on the rise of DR-TB led to an urgent need for the development of new diagnostic tools to rapidly detect TB and its associated drug susceptibility profile. The Foundation for Innovative New Diagnostics (FIND) has taken the onus upon them to ensure that laboratory strengthening becomes a reality by having developed, and still developing, new diagnostic assays in order to improve the laboratory turn-around time (TAT), whereby the transmission of DR-TB strains can be stopped. Laboratory strengthening does not solely rely on new diagnostic assays alone, and thus a Quality Management System, discussed in the dissertation, must be in place to ensure that the rapid result is accurate and reliable. The series of studies encompassed in this dissertation includes methodological validations (both technical and operational) of rapid TB diagnostic assays in order to rapidly and accurately diagnose the disease, and thus reducing the diagnostic delay associated with conventional diagnostic platforms. The studies were conducted “in-house” at the National Health Laboratory Service (NHLS) Reference TB laboratory in Green Point, Cape Town, which is a high-volume public health laboratory. The need to rapidly detect resistance to the first line anti-tubercular drugs Isoniazid and Rifampicin was a priority and thus the performance of a commercial line probe assay (LPA), the GenoType®MTBDRplus Ver1.0 LPA, was assessed for use on smear positive direct patient material. The performance characteristics was superior to that of conventional drug susceptibility testing, where the sensitivity and specificity for the detection of multi-drug resistant TB (MDR-TB) was 98.8 and 100%, respectively, with results in 1-2 days. Based on this study, the World Health Organization (WHO) endorsed the use of molecular LPA for the rapid detection of DR-TB. Furthermore, the need for quality assurance associated with the GenoType®MTBDRplus LPA in the diagnostic laboratory is essential and thus a user manual for the molecular detection of Drug Resistant Tuberculosis in resource-limited settings has also been developed (http://www.finddiagnostics.org/export/sites/default/resource-center/reports brochures/docs/LPA LaboratoryManual22Mar2012.pdf) for which Global Laboratory Initiative (GLI) status is pending. With the outbreak of extensively drug resistant TB (XDR-TB) in Tugela Ferry area in KwaZulu-Natal and the rest of the world, the need to rapidly detect resistance to the second line drugs arose, and thus the performance characteristics of the GenoType®MTBDRsl LPA was assessed for use on smear positive direct patient material. The performance characteristics proved to be excellent once again, with a 93.3% reduction in TAT. The data was scrutinized by the WHO, where it may be used as a triage test to guide treatment, but to date, no final policies on the use thereof has been finalized. The need for rapid point-of-care (POC) testing led to the implementation of the Xpert®MTB/RIF assay in the referral laboratories, for use on both smear positive and smear negative direct patient material. In order to accommodate for laboratories where the LPA has been implemented already, the GenoType®MTBDRplus Ver2.0 LPA was developed, which is aimed for use on all smear types as well. A head-to-head assessment was done between these assays to determine their performance characteristics and it was shown to be equally good. In this study we have shown the utility of molecular diagnostic assays to rapidly diagnose TB and its associated drug susceptibility patterns. This will have a significant impact on diagnostic delay and clinical decision making as well as patient outcome.
Rapid sequencing of the mycobacterium tuberculosis pncA gene for detection of pyrazinamide susceptibility
(American Society for Microbiology, 2014) Streicher, Elizabeth M.; Maharaj, Kashmeel; York, Talita; Van Heerden, Carel; Barnard, Marinus; Diacon, Andreas; Mendel, Carl M.; Bosman, Marlein E.; Hepple, Juli A.; Pym, Alexander S.; Warren, Robin M.; Van Helden, Paul D.
We developed a pyrazinamidase gene DNA-sequencing method to rapidly identify pyrazinamide resistance-causing mutations in GenoLyse-treated, smear-positive sputum specimens. The sensitivity and specificity were 90.9 and 100%, respectively, compared to those of MGIT drug susceptibility testing, after the exclusion of synonymous mutations and nonsynonymous mutations previously associated with susceptibility to pyrazinamide.
Risk factors associated with isoniazid resistance in tuberculosis
(Stellenbosch : University of Stellenbosch, 2005-12) Barnard, Marinus; Victor, Thomas C.; Warren, Rob M.; University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
Tuberculosis (TB) is one of the most serious infectious diseases known to mankind, with devastating outcomes in the poorest countries in the world. Isoniazid is the cornerstone of all first-line anti-TB regimens. Forty-eight percent of all drug resistant TB isolates in the Western Cape are Isoniazid mono-resistant, and the majority of these isolates belong to the Beijing/W strain family. Currently, the known molecular mechanisms which confer Isoniazid resistance in these isolates are attributed to mutations within the katG gene and account for up to 70% of all drug resistant TB isolates. Risk factors for the development of Isoniazid resistance can be attributed to either pathogen or host related factors and may partially account for the other 30% of Isoniazid resistant isolates. In this study, three aspects which may contribute to Isoniazid resistance were investigated: DNA repair in the bacterium, host response to anti-TB treatment and socioeconomic factors. A PCR based dot-blot strategy was used to screen for previously reported missense mutations in the mutT2, Rv3908 and ogt DNA repair genes of different strains of M. tuberculosis. All the Beijing isolates (drug resistant and susceptible), in contrast to the Atypical Beijing strains and other dominant strain families, exhibited missense mutations in all three base excision repair genes. It is therefore speculated that defects in the DNA repair genes (mutator phenotypes) of the Beijing isolates may contribute to the development of drug resistance and hence, may account for the large proportion of isolates that are Isoniazid mono-resistant. A novel method, based on primer extension, was initially developed to screen the NAT2 gene and then used to type individuals into fast, intermediate and slow acetylators of Isoniazid. The newly develop method, which is sensitive and accurate, improves the detection of Single Nucleotide Polymorphisms within the NAT2 gene, in contrast to the traditionally used methods. Utilising this method, it was found that the combination of fast and intermediate acetylators was significantly associated with Isoniazid resistance in the study community. This finding may have an important impact on TB control programmes, since it may allow for the administration of higher dosages of Isoniazid to fast/intermediate acetylators and a lower dose for slow acetylators. Clinical factors (compliance and retreatment after cure) and socio-economic factors (education, employment and income) were found to be significantly associated with the development of INH resistance. Diagnostic delay was also found to be a risk factor, since it may allow for transmission of TB during this period. The HIV prevalence in the study population is low and subsequently HIV status was not associated with the development of INH resistance. This study indicates that a combination of risk factors, both pathogen and host related, are involved in the development of Isoniazid resistance.
The use of digital PCR to improve the application of quantitative molecular diagnostic methods for tuberculosis
(BioMed Central, 2016) Devonshire, Alison S.; O’Sullivan, Denise M.; Honeyborne, Isobella; Jones, Gerwyn; Karczmarczyk, Maria; Pavsic, Jernej; Gutteridge, Alice; Milavec, Mojca; Mendoza, Pablo; Schimmel, Heinz; Van Heuverswyn, Fran; Gorton, Rebecca; Cirillo, Daniela Maria; Borroni, Emanuele; Harris, Kathryn; Barnard, Marinus; Heydenrych, Anthenette; Ndusilo, Norah; Wallis, Carole L.; Pillay, Keshree; Barry, Thomas; Reddington, Kate; Richter, Elvira; Mozioglu, Erkan; Akyurek, Sema; Yalcınkaya, Burhanettin; Akgoz, Muslum; Zel, Jana; Foy, Carole A.; McHugh, Timothy D.; Huggett, Jim F.
Background: Real-time PCR (qPCR) based methods, such as the Xpert MTB/RIF, are increasingly being used to diagnose tuberculosis (TB). While qualitative methods are adequate for diagnosis, the therapeutic monitoring of TB patients requires quantitative methods currently performed using smear microscopy. The potential use of quantitative molecular measurements for therapeutic monitoring has been investigated but findings have been variable and inconclusive. The lack of an adequate reference method and reference materials is a barrier to understanding the source of such disagreement. Digital PCR (dPCR) offers the potential for an accurate method for quantification of specific DNA sequences in reference materials which can be used to evaluate quantitative molecular methods for TB treatment monitoring. Methods: To assess a novel approach for the development of quality assurance materials we used dPCR to quantify specific DNA sequences in a range of prototype reference materials and evaluated accuracy between different laboratories and instruments. The materials were then also used to evaluate the quantitative performance of qPCR and Xpert MTB/RIF in eight clinical testing laboratories. Results: dPCR was found to provide results in good agreement with the other methods tested and to be highly reproducible between laboratories without calibration even when using different instruments. When the reference materials were analysed with qPCR and Xpert MTB/RIF by clinical laboratories, all laboratories were able to correctly rank the reference materials according to concentration, however there was a marked difference in the measured magnitude. Conclusions: TB is a disease where the quantification of the pathogen could lead to better patient management and qPCR methods offer the potential to rapidly perform such analysis. However, our findings suggest that when precisely characterised materials are used to evaluate qPCR methods, the measurement result variation is too high to determine whether molecular quantification of Mycobacterium tuberculosis would provide a clinically useful readout. The methods described in this study provide a means by which the technical performance of quantitative molecular methods can be evaluated independently of clinical variability to improve accuracy of measurement results. These will assist in ultimately increasing the likelihood that such approaches could be used to improve patient management of TB.
Using routinely collected laboratory data to identify high rifampicin-resistant tuberculosis burden communities in the Western Cape Province, South Africa : a retrospective spatiotemporal analysis
(Public Library of Science, 2018) McIntosh, Avery I.; Jenkins, Helen E.; White, Laura F.; Barnard, Marinus; Thomson, Dana R.; Dolby, Tania; Simpson, John; Streicher, Elizabeth M.; Kleinman, Mary B.; Ragan, Elizabeth J.; Van Helden, Paul D.; Murray, Megan B.; Warren, Robin M.; Jacobson, Karen R.
Background: South Africa has the highest tuberculosis incidence globally (781/100,000), with an estimated 4.3% of cases being rifampicin resistant (RR). Control and elimination strategies will require detailed spatial information to understand where drug-resistant We retrospectively identified cases of microbiologically confirmed tuberculosis and RRtuberculosis from all biological samples submitted for tuberculosis testing (n = 2,219,891) to the Western Cape National Health Laboratory Services (NHLS) between January 1, 2008, and June 30, 2013. Because the NHLS database lacks unique patient identifiers, we performed a series of record-linking processes to match specimen records to individual patients. We counted an individual as having a single disease episode if their positive samples came from within two years of each other. Cases were aggregated by clinic location (n = 302) to estimate the percentage of tuberculosis cases with rifampicin resistance per clinic. We used inverse distance weighting (IDW) to produce heatmaps of the RR-tuberculosis percentage across the province. Regression was used to estimate annual changes in the RR- tuberculosis exists tuberculosis percentage by clinic, and estimated average size and direction of change was mapped. We identified 799,779 individuals who had specimens submitted from mappable clinics for testing, of whom 222,735 (27.8%) had microbiologically confirmed tuberculosis. The study population was 43% female, the median age was 36 years (IQR 27±44), and 10,255 (4.6%, 95% CI: 4.6±4.7) cases had documented rifampicin resistance. Among individuals with microbiologically confirmed tuberculosis, 8,947 (4.0%) had more than one disease episode during the study period. The percentage of tuberculosis cases with rifampicin resistance documented among these individuals was 11.4% (95% CI: 10.7±12.0). Overall, the percentage of tuberculosis cases that were RR-tuberculosis was spatially heterogeneous, ranging from 0% to 25% across the province. Our maps reveal significant yearly fluctuations in RR-tuberculosis percentages at several locations. Additionally, the directions of change over time in RR-tuberculosis percentage were not uniform. The main limitation of this study is the lack of unique patient identifiers in the NHLS database, rendering findings to be estimates reliant on the accuracy of the person-matching algorithm. Conclusions: Our maps reveal striking spatial and temporal heterogeneity in RR-tuberculosis percentages across this province. We demonstrate the potential to monitor RR-tuberculosis spatially and temporally with routinely collected laboratory data, enabling improved resource targeting and more rapid locally appropriate interventions. and why it persists in those communities. We demonstrate a method to enable drug-resistant tuberculosis monitoring by identifying high-burden communities in the Western Cape Province using routinely collected laboratory data. Methods and findings:

Browsing by Author "Barnard, Marinus"

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