Browsing by Author "De Vos, Margaretha"

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    Bedaquiline microheteroresistance after cessation of tuberculosis treatment
    (Massachusetts Medical Society, 2019-05-30) De Vos, Margaretha; Wiggins, Kristin B.; Derendinger, Brigitta; Reuter, Anja; Dolby, Tania; Burns, Scott; Schito, Marco; Engelthaler, David M.; Metcalfe, John; Theron, Grant; Van Rie, Annelies; Posey, James; Warren, Rob; Cox, Helen
    ENGLISH ABSTRACT: Bedaquiline improves survival among persons with multidrug-resistant tuberculosis (MDR-TB).1 We report the case of a 65-year-old South African man who was negative for human immunodeficiency virus and in whom MDR-TB was diagnosed in 2013 (resistant to rifampin and isoniazid; phenotypically susceptible to a fluoroquinolone and amikacin). A baseline radiograph showed changes consistent with bilateral tuberculosis with left apex cavitation. He started standardized treatment that included moxifloxacin, pyrazinamide, kanamycin, ethionamide, isoniazid, and terizidone. After initial sputum culture conversion (at month 3) and clinical improvement, the patient again became culture-positive, and bilateral cavitation developed. After detection of phenotypic resistance to fluoroquinolones (at month 6), his treatment was revised (at month 8) to include high-dose isoniazid, ethambutol, pyrazinamide, terizidone, linezolid, paraaminosalicylic acid, and kanamycin (Figure 1 and the Supplementary Appendix, available with the full text of this letter at NEJM.org). Bedaquiline was added 22 days later and was administered for 6 months.2 The patient remained culture-positive (treatment failure), and treatment was stopped 15 months after revision of the regimen. The patient died 7 months later.
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    Diagnostic accuracy of the FluoroType MTB and MTBDR VER 2.0 assays for the centralized high-throughput detection of Mycobacterium tuberculosis complex DNA and isoniazid and rifampicin resistance
    (Elsevier Ltd, 2021-09) Dippenaar, Anzaan; Derendinger, Brigitta; Dolby, Tania; Beylis, Natalie; Van Helden, Paul D.; Theron, Grant; Warren, Robin M.; De Vos, Margaretha
    Objectives To evaluate the accuracy of two new molecular diagnostic tests for the detection of drug-resistant tuberculosis, the FluoroType MTB and MTBDR VER 2.0 assays, in combination with manual and automated DNA extraction methods. Methods Sputa from 360 Xpert Ultra Mycobacterium tuberculosis complex (MTBC)-positive patients and 250 Xpert Ultra MTBC-negative patients were tested. GenoType MTBDRplus served as reference for MTBC and drug resistance detection. Sanger sequencing was used to resolve discrepancies. Results FluoroType MTB VER 2.0 showed similar MTBC sensitivity compared with FluoroType MTBDR VER 2.0 (manual DNA extraction: 91.6% (294/321) versus 89.8% (291/324); p 0.4); automated DNA extraction: 92.1% (305/331) versus 87.7% (291/332); p 0.05)). FluoroType MTBDR VER2.0 showed comparable diagnostic accuracy to FluoroType MTBDR VER1.0 as previously reported for the detection of MTBC and rifampicin and isoniazid resistance. Conclusions The FluoroType MTB and MTBDR VER 2.0 assays together with an automated DNA extraction and PCR set-up platform may improve laboratory operational efficiency for the diagnosis of MTBC and resistance to rifampicin and isoniazid and show promise for the implementation in a centralized molecular drug susceptibility testing model.
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    Extract from used Xpert MTB/ RIF Ultra cartridges is useful for accurate second-line drug-resistant tuberculosis diagnosis with minimal rpoB-amplicon cross-contamination risk
    (Nature Research (part of Springer Nature), 2020) Venter, Rouxjeane; Minnies, Stephanie; Derendinger, Brigitta; Tshivhula, Happy; De Vos, Margaretha; Dolby, Tania; Ruiters, Ashley; Warren, Robin M.; Theron, Grant
    Xpert MTB/RIF Ultra (Ultra) detects Mycobacterium tuberculosis and rifampicin resistance. Follow-on drug susceptibility testing (DST) requires additional sputum. Extract from the diamond-shaped chamber of the cartridge (dCE) of Ultra’s predecessor, Xpert MTB/RIF (Xpert), is useful for MTBDRsl-based DST but this is unexplored with Ultra. Furthermore, whether CE from non-diamond compartments is useful, the performance of FluoroType MTBDR (FT) on  CE, and rpoB cross-contamination risk associated with the extraction procedure are unknown. We tested MTBDRsl, MTBDRplus, and FT on CEs from chambers from cartridges (Ultra, Xpert) tested on bacilli dilution series. MTBDRsl on Ultra dCE on TB-positive sputa (n = 40) was also evaluated and, separately, rpoB amplicon cross-contamination risk . MTBDRsl on Ultra dCE from dilutions ≥103 CFU/ml (CTmin <25, >“low semi-quantitation”) detected fluoroquinolone (FQ) and second-line injectable (SLID) susceptibility and resistance correctly (some SLIDs-indeterminate). At the same threshold (at which ~85% of Ultra-positives in our setting would be eligible), 35/35 (100%) FQ and 34/35 (97%) SLID results from Ultra dCE were concordant with sputa results. Tests on other chambers were unfeasible. No tubes open during 20 batched extractions had FT-detected rpoB cross-contamination. False-positive Ultra rpoB results was observed when dCE dilutions ≤10−3 were re-tested. MTBDRsl on Ultra dCE is concordant with isolate results. rpoB amplicon cross-contamination is unlikely. These data mitigate additional specimen collection for second-line DST and cross-contamination concerns.
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    Identification of mechanisms regulating the intra cellular concentration of rifampicin in Mycobacterium Tuberculosis
    (Stellenbosch : Stellenbosch University, 2013-03) De Vos, Margaretha; Victor, Thomas Calldo; Warren, Robin Mark; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Division of Molecular Biology and Human Genetics.
    ENGLISH ABSTRACT: Rifampicin resistance in clinical isolates of Mycobacterium tuberculosis develops through selection of bacterial variants harbouring mutations in the rpoB gene. These mutations infer a fitness-cost in the absence of antibiotic pressure, however, fitness-levels of rifampicin-resistant strains can be restored by compensatory mutations in rpoA and rpoC. This study was the first to investigate the epidemiological relevance of these compensatory mutations in clinical M. tuberculosis isolates collected in South Africa. Through targeted DNA sequencing, we demonstrated a strong association between rpoC mutations and transmission, and the rpoB S531L mutation. Our study emphasises the epidemiological relevance of compensatory evolution in response to the emergence of rifampicin resistance, and illustrates how compensatory mutations may be selected as a function of epistatic interactions. Recently a hypothesis has been developed which suggests that the activation of efflux systems through exposure to rifampicin may explain the observed spectrum of rifampicin resistance phenotypes. To elucidate whether rifampicin dependent activation of efflux systems also increases energy production, the RNA expression profiles of candidate energy metabolism genes were investigated. This study demonstrated that rifampicin exposure induced an overall increase in the expression of energy metabolism genes. Our findings suggest that the response to rifampicin is not universal and may depend on other genomic mutations. From these results we conclude that the stress response induced by exposure to rifampicin increases the energy production which fuels efflux activity thereby enabling the cell to extrude rifampicin in an energy dependent manner. This also provides a platform to explain the mechanism by which the newly developed drug, TMC207, increases the rate of culture conversion when used in combination with second-line anti-TB drugs. We propose that inhibition of ATP synthesis by TMC207 will deprive the efflux pumps and transporter genes of energy, which will result in the accumulation of second-line anti-TB drugs within the bacilli, leading to more efficient binding of the second-line drugs to their targets and ultimately to cell death. To identify the genetic basis governing the level of rifampicin resistance, we sequenced the genomes of MDR clinical isolates and in vitro generated rifampicin resistant mutants. Only minor genetic changes in addition to the rpoB mutation were identified in the genomes of in vitro rifampicin resistant mutants which displayed varying levels of resistance. This suggests that these mutants may either use alternative regulatory mechanisms or have acquired SNPs outside the genetic regions investigated in this study to modulate rifampicin resistance levels. In contrast, the genomes of clinical MDR isolates from the Low Copy Clade showed considerable variability in genes involved in cell wall, cellular processes and lipid metabolism, while the genomes from the Beijing Clade displayed variability in genes known to confer drug resistance and compensatory mechanisms. These results suggest that the structure and processes of the cell wall, as well as lipid metabolism plays a critical role in determining the intra-cellular concentration of rifampicin. Finally, this study illustrated the complexity in the physiology of M. tuberculosis resistant to rifampicin, whereby multiple mechanisms are employed by the bacteria to modulate its resistance levels.
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    A landscape of genomic alterations at the root of a near-untreatable tuberculosis epidemic
    (BMC (part of Springer Nature), 2020-02-04) Klopper, Marisa; Heupink, Tim Hermanus; Hill-Cawthorne, Grant; Streicher, Elizabeth M.; Dippenaar, Anzaan; De Vos, Margaretha; Abdallah, Abdallah Musa; Limberis, Jason; Merker, Matthias; Burns, Scott; Niemann, Stefan; Dheda, Keertan; Posey, James; Pain, Arnab; Warren, Robin Mark
    Background: Atypical Beijing genotype Mycobacterium tuberculosis strains are widespread in South Africa and have acquired resistance to up to 13 drugs on multiple occasions. It is puzzling that these strains have retained fitness and transmissibility despite the potential fitness cost associated with drug resistance mutations. Methods: We conducted Illumina sequencing of 211 Beijing genotype M. tuberculosis isolates to facilitate the detection of genomic features that may promote acquisition of drug resistance and restore fitness in highly resistant atypical Beijing forms. Phylogenetic and comparative genomic analysis was done to determine changes that are unique to the resistant strains that also transmit well. Minimum inhibitory concentration (MIC) determination for streptomycin and bedaquiline was done for a limited number of isolates to demonstrate a difference in MIC between isolates with and without certain variants. Results: Phylogenetic analysis confirmed that two clades of atypical Beijing strains have independently developed resistance to virtually all the potent drugs included in standard (pre-bedaquiline) drug-resistant TB treatment regimens. We show that undetected drug resistance in a progenitor strain was likely instrumental in this resistance acquisition. In this cohort, ethionamide (ethA A381P) resistance would be missed in first-line drug-susceptible isolates, and streptomycin (gidB L79S) resistance may be missed due to an MIC close to the critical concentration. Subsequent inadequate treatment historically led to amplification of resistance and facilitated spread of the strains. Bedaquiline resistance was found in a small number of isolates, despite lack of exposure to the drug. The highly resistant clades also carry inhA promoter mutations, which arose after ethA and katG mutations. In these isolates, inhA promoter mutations do not alter drug resistance, suggesting a possible alternative role. Conclusion: The presence of the ethA mutation in otherwise susceptible isolates from ethionamide-naïve patients demonstrates that known exposure is not an adequate indicator of drug susceptibility. Similarly, it is demonstrated that bedaquiline resistance can occur without exposure to the drug. Inappropriate treatment regimens, due to missed resistance, leads to amplification of resistance, and transmission. We put these results into the context of current WHO treatment regimens, underscoring the risks of treatment without knowledge of the full drug resistance profile.
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    Mycobacterial genomic DNA from used Xpert MTB/RIF cartridges can be utilised for accurate second-line genotypic drug susceptibility testing and spoligotyping
    (Nature, 2017) Venter, Rouxjeane; Derendinger, Brigitta; De Vos, Margaretha; Pillay, Samantha; Dolby, Tanya; Simpson, John; Kitchin, Natasha; Ruiters, Ashley; Van Helden, Paul D.; Warren, Robin M.; Theron, Grant
    Xpert MTB/RIF (Xpert) is a widely-used test for tuberculosis (TB) and rifampicin-resistance. Second-line drug susceptibility testing (DST), which is recommended by policymakers, typically requires additional specimen collection that delays effective treatment initiation. We examined whether cartridge extract (CE) from used Xpert TB-positive cartridges was, without downstream DNA extraction or purification, suitable for both genotypic DST (MTBDRplus, MTBDRsl), which may permit patients to rapidly receive a XDR-TB diagnosis from a single specimen, and spoligotyping, which could facilitate routine genotyping. To determine the limit-of-detection and diagnostic accuracy, CEs from dilution series of drug-susceptible and -resistant bacilli were tested (MTBDRplus, MTBDRsl). Xpert TB-positive patient sputa CEs (n = 85) were tested (56 Xpert-rifampicin-susceptible, MTBDRplus and MTBDRsl; 29 Xpert-rifampicin-resistant, MTBDRsl). Spoligotyping was done on CEs from dilution series and patient sputa (n = 10). MTBDRplus had high non-valid result rates. MTBDRsl on CEs from dilutions ≥103CFU/ml (CT ≤ 24, >“low” Xpert semiquantitation category) was accurate, had low indeterminate rates and, on CE from sputa, highly concordant with MTBDRsl isolate results. CE spoligotyping results from dilutions ≥103CFU/ml and sputa were correct. MTBDRsl and spoligotyping on CE are thus highly feasible. These findings reduce the need for additional specimen collection and culture, for which capacity is limited in high-burden countries, and have implications for diagnostic laboratories and TB molecular epidemiology.
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    Mycobacterial genomic DNA from used Xpert MTB/RIF cartridges can be utilised for accurate secondline genotypic drug susceptibility testing and spoligotyping
    (Springer Nature, 2017-11-01) Venter, Rouxjeane; Derendinger, Brigitta; De Vos, Margaretha; Pillay, Samantha; Dolby, Tanya; Simpson, John; Kitchin, Natasha; Ruiters, Ashley; Van Helden, Paul D.; Warren, Robin M.; Theron, Grant
    ENGLISH ABSTRACT: Xpert MTB/RIF (Xpert) is a widely-used test for tuberculosis (TB) and rifampicin-resistance. Second-line drug susceptibility testing (DST), which is recommended by policymakers, typically requires additional specimen collection that delays effective treatment initiation. We examined whether cartridge extract (CE) from used Xpert TB-positive cartridges was, without downstream DNA extraction or purification, suitable for both genotypic DST (MTBDRplus, MTBDRsl), which may permit patients to rapidly receive a XDR-TB diagnosis from a single specimen, and spoligotyping, which could facilitate routine genotyping. To determine the limit-of-detection and diagnostic accuracy, CEs from dilution series of drug-susceptible and -resistant bacilli were tested (MTBDRplus, MTBDRsl). Xpert TB-positive patient sputa CEs (n = 85) were tested (56 Xpert-rifampicin-susceptible, MTBDRplus and MTBDRsl; 29 Xpert-rifampicin-resistant, MTBDRsl). Spoligotyping was done on CEs from dilution series and patient sputa (n = 10). MTBDRplus had high non-valid result rates. MTBDRsl on CEs from dilutions ≥103CFU/ml (CT ≤ 24, >“low” Xpert semiquantitation category) was accurate, had low indeterminate rates and, on CE from sputa, highly concordant with MTBDRsl isolate results. CE spoligotyping results from dilutions ≥103CFU/ml and sputa were correct. MTBDRsl and spoligotyping on CE are thus highly feasible. These findings reduce the need for additional specimen collection and culture, for which capacity is limited in high-burden countries, and have implications for diagnostic laboratories and TB molecular epidemiology.
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    Switching to bedaquiline for treatment of rifampicin-resistant tuberculosis in South Africa : a retrospective cohort analysis
    (Public Library of Science, 2019-10-17) Bouton, Tara C.; De Vos, Margaretha; Ragan, Elizabeth J.; White, Laura F.; Van Zyl, Leonie; Theron, Danie; Horsburgh, C. Robert; Warren, Robin M.; Jacobson, Karen R.
    ENGLISH ABSTRACT: South Africa led the world with guidelines on bedaquiline (BDQ) use as a single drug substitution to manage rifampin resistant tuberculosis regimen toxicity. We examined reasons for giving BDQ in a retrospective cohort: >75% of patients were switched to BDQ for toxicity (ototoxicity or renal dysfunction) rather than drug resistance.