Publications by authors named "Margaretha de Vos"

17 Publications

  • Page 1 of 1

Antimycobacterial Activity, Synergism, and Mechanism of Action Evaluation of Novel Polycyclic Amines against .

Adv Pharmacol Pharm Sci 2021 11;2021:5583342. Epub 2021 Jun 11.

School of Pharmacy, Faculty of Natural Sciences, University of the Western Cape, Cape Town, South Africa.

has developed extensive resistance to numerous antimycobacterial agents used in the treatment of tuberculosis. Insufficient intracellular accumulation of active moieties allows for selective survival of mycobacteria with drug resistance mutations and accordingly promotes the development of microbial drug resistance. Discovery of compounds with new mechanisms of action and physicochemical properties that promote intracellular accumulation, or compounds that act synergistically with other antimycobacterial drugs, has the potential to reduce and prevent further drug resistance. To this end, antimycobacterial activity, mechanism of action, and synergism in combination therapy were investigated for a series of polycyclic amine derivatives. Compound selection was based on the presence of moieties with possible antimycobacterial activity, the inclusion of bulky lipophilic carriers to promote intracellular accumulation, and previously demonstrated bioactivity that potentially support inhibition of efflux pump activity. The most potent antimycobacterial demonstrated a minimum inhibitory concentration (MIC) of 9.6 M against H37Rv. Genotoxicity and inhibition of the cytochrome respiratory complex were excluded as mechanisms of action for all compounds. Inhibition of cell wall synthesis was identified as a likely mechanism of action for the two most active compounds ( and ). Compounds and demonstrated synergistic activity with the known Rv1258c efflux pump substrate, spectinomycin, pointing to possible efflux pump inhibition. For this series, the nature of the side chain, rather than the type of polycyclic carrier, seems to play a determining role in the antimycobacterial activity and cytotoxicity of the compounds. Contrariwise, the nature of the polycyclic carrier, particularly the azapentacycloundecane cage, appears to promote synergistic activity. Results point to the possibility of combining an azapentacycloundecane carrier with a side chain that promotes antimycobacterial activity to develop dual acting molecules for the treatment of
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http://dx.doi.org/10.1155/2021/5583342DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8238621PMC
June 2021

Diagnostic accuracy of Panbio rapid antigen tests on oropharyngeal swabs for detection of SARS-CoV-2.

PLoS One 2021 24;16(6):e0253321. Epub 2021 Jun 24.

Infectious Disease Division, Geneva University Hospitals, Geneva, Switzerland.

Background: Antigen-detecting rapid diagnostic tests (Ag-RDTs) for the detection of SARS-CoV-2 offer new opportunities for testing in the context of the COVID-19 pandemic. Nasopharyngeal swabs (NPS) are the reference sample type, but oropharyngeal swabs (OPS) may be a more acceptable sample type in some patients.

Methods: We conducted a prospective study in a single screening center to assess the diagnostic performance of the Panbio™ COVID-19 Ag Rapid Test (Abbott) on OPS compared with reverse-transcription quantitative PCR (RT-qPCR) using NPS during the second pandemic wave in Switzerland.

Results: 402 outpatients were enrolled in a COVID-19 screening center, of whom 168 (41.8%) had a positive RT-qPCR test. The oropharyngeal Ag-RDT clinical sensitivity compared to nasopharyngeal RT-qPCR was 81% (95%CI: 74.2-86.6). Two false positives were noted out of the 234 RT-qPCR negative individuals, which resulted in a clinical specificity of 99.1% (95%CI: 96.9-99.9) for the Ag-RDT. For cycle threshold values ≤ 26.7 (≥ 1E6 SARS-CoV-2 genomes copies/mL, a presumed cut-off for infectious virus), 96.3% sensitivity (95%CI: 90.7-99.0%) was obtained with the Ag-RDT using OPS.

Interpretation: Based on our findings, the diagnostic performance of the Panbio™ Covid-19 RDT with OPS samples, if taken by a trained person and high requirements regarding quality of the specimen, meet the criteria required by the WHO for Ag-RDTs (sensitivity ≥80% and specificity ≥97%) in a high incidence setting in symptomatic individuals.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0253321PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8224876PMC
July 2021

Analytical performance of the Xpert MTB/XDR® assay for tuberculosis and expanded resistance detection.

Diagn Microbiol Infect Dis 2021 Apr 20;101(1):115397. Epub 2021 Apr 20.

FIND, Geneva, Switzerland; Division of Tropical Medicine, Center of Infectious Diseases, University Hospital of Heidelberg, Heidelberg, Germany.

In a manufacturer-independent laboratory validation study, the Xpert MTB/XDR® assay demonstrated equivalent limit of detection to Xpert MTB/RIF®, detected 100% of tested resistance mutations and showed some utility for resistance detection in strain mixtures. The Xpert MTB/XDR assay is a reliable, sensitive assay for tuberculosis and expanded resistance detection.
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http://dx.doi.org/10.1016/j.diagmicrobio.2021.115397DOI Listing
April 2021

The Abbott PanBio WHO emergency use listed, rapid, antigen-detecting point-of-care diagnostic test for SARS-CoV-2-Evaluation of the accuracy and ease-of-use.

PLoS One 2021 27;16(5):e0247918. Epub 2021 May 27.

Division of Clinical Tropical Medicine, Heidelberg University Hospital, Heidelberg, Germany.

Objectives: Diagnostics are essential for controlling the pandemic. Identifying a reliable and fast diagnostic device is needed for effective testing. We assessed performance and ease-of-use of the Abbott PanBio antigen-detecting rapid diagnostic test (Ag-RDT).

Methods: This prospective, multi-centre diagnostic accuracy study enrolled at two sites in Germany. Following routine testing with reverse-transcriptase polymerase chain reaction (RT-PCR), a second study-exclusive swab was performed for Ag-RDT testing. Routine swabs were nasopharyngeal (NP) or combined NP/oropharyngeal (OP) whereas the study-exclusive swabs were NP. To evaluate performance, sensitivity and specificity were assessed overall and in predefined sub-analyses accordingly to cycle-threshold values, days after symptom onset, disease severity and study site. Additionally, an ease-of-use assessment (EoU) and System Usability Scale (SUS) were performed.

Results: 1108 participants were enrolled between Sept 28 and Oct 30, 2020. Of these, 106 (9.6%) were PCR-positive. The Abbott PanBio detected 92/106 PCR-positive participants with a sensitivity of 86.8% (95% CI: 79.0% - 92.0%) and a specificity of 99.9% (95% CI: 99.4%-100%). The sub-analyses indicated that sensitivity was 95.8% in Ct-values <25 and within the first seven days from symptom onset. The test was characterized as easy to use (SUS: 86/100) and considered suitable for point-of-care settings.

Conclusion: The Abbott PanBio Ag-RDT performs well for SARS-CoV-2 testing in this large manufacturer independent study, confirming its WHO recommendation for Emergency Use in settings with limited resources.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0247918PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8158996PMC
June 2021

Head-to-head performance comparison of self-collected nasal versus professional-collected nasopharyngeal swab for a WHO-listed SARS-CoV-2 antigen-detecting rapid diagnostic test.

Med Microbiol Immunol 2021 Aug 24;210(4):181-186. Epub 2021 May 24.

Division of Clinical Tropical Medicine, Centre of Infectious Diseases, Heidelberg University Hospital, Im Neuenheimer Feld 324, 69120, Heidelberg, Germany.

In 2020, the World Health Organization (WHO) recommended two SARS-CoV-2 lateral flow antigen-detecting rapid diagnostics tests (Ag-RDTs), both initially with nasopharyngeal (NP) sample collection. Independent head-to-head studies are necessary for SARS-CoV-2 Ag-RDT nasal sampling to demonstrate comparability of performance with nasopharyngeal (NP) sampling. We conducted a head-to-head comparison study of a supervised, self-collected nasal mid-turbinate (NMT) swab and a professional-collected NP swab, using the Panbio™ Ag-RDT (distributed by Abbott). We calculated positive and negative percent agreement between the sampling methods as well as sensitivity and specificity for both sampling techniques compared to the reference standard reverse transcription polymerase chain reaction (RT-PCR). A SARS-CoV-2 infection could be diagnosed by RT-PCR in 45 of 290 participants (15.5%). Comparing the NMT and NP sampling the positive percent agreement of the Ag-RDT was 88.1% (37/42 PCR positives detected; CI 75.0-94.8%). The negative percent agreement was 98.8% (245/248; CI 96.5-99.6%). The overall sensitivity of Panbio with NMT sampling was 84.4% (38/45; CI 71.2-92.3%) and 88.9% (40/45; CI 76.5-95.5%) with NP sampling. Specificity was 99.2% (243/245; CI 97.1-99.8%) for both, NP and NMT sampling. The sensitivity of the Panbio test in participants with high viral load (> 7 log SARS-CoV-2 RNA copies/mL) was 96.3% (CI 81.7-99.8%) for both, NMT and NP sampling. For the Panbio supervised NMT self-sampling yields comparable results to NP sampling. This suggests that nasal self-sampling could be used for to enable scaled-up population testing.Clinical Trial DRKS00021220.
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http://dx.doi.org/10.1007/s00430-021-00710-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8142294PMC
August 2021

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.

Clin Microbiol Infect 2021 Apr 30. Epub 2021 Apr 30.

Department of Science and Innovation-National Research Foundation Centre for Excellence for Biomedical Tuberculosis Research, SAMRC Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, South Africa. Electronic address:

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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http://dx.doi.org/10.1016/j.cmi.2021.04.022DOI Listing
April 2021

Melting the : Nondetection of Kanamycin Resistance Markers by Routine Diagnostic Tests and Identification of New Promoter Variants.

Antimicrob Agents Chemother 2021 06 17;65(7):e0250220. Epub 2021 Jun 17.

DSI-NRF Centre of Excellence for Biomedical Tuberculosis Research, South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa.

promoter mutations can confer reduced Mycobacterium tuberculosis kanamycin susceptibility. GenoType MTBDR a widely used assay evaluating this region, wrongly classified 17/410 isolates as promoter wild type. Six out of seventeen isolates harbored mutations known to confer kanamycin resistance, and the remainder harbored either novel promoter mutations (7/11) or disputed mutations (4/11). GenoType MTBDR can miss established and new variants that cause reduced susceptibility. These data highlight the importance of reflex phenotypic kanamycin testing.
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http://dx.doi.org/10.1128/AAC.02502-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8218670PMC
June 2021

Diagnostic accuracy of two commercial SARS-CoV-2 antigen-detecting rapid tests at the point of care in community-based testing centers.

PLoS One 2021 31;16(3):e0248921. Epub 2021 Mar 31.

Division of Infectious Disease, Geneva University Hospitals, Geneva, Switzerland.

Objectives: Determine the diagnostic accuracy of two antigen-detecting rapid diagnostic tests (Ag-RDT) for SARS-CoV-2 at the point of care and define individuals' characteristics providing best performance.

Methods: We performed a prospective, single-center, point of care validation of two Ag-RDT in comparison to RT-PCR on nasopharyngeal swabs.

Results: Between October 9th and 23rd, 2020, 1064 participants were enrolled. The PanbioTM Covid-19 Ag Rapid Test device (Abbott) was validated in 535 participants, with 106 positive Ag-RDT results out of 124 positive RT-PCR individuals, yielding a sensitivity of 85.5% (95% CI: 78.0-91.2). Specificity was 100.0% (95% CI: 99.1-100) in 411 RT-PCR negative individuals. The Standard Q Ag-RDT (SD Biosensor, Roche) was validated in 529 participants, with 170 positive Ag-RDT results out of 191 positive RT-PCR individuals, yielding a sensitivity of 89.0% (95%CI: 83.7-93.1). One false positive result was obtained in 338 RT-PCR negative individuals, yielding a specificity of 99.7% (95%CI: 98.4-100). For individuals presenting with fever 1-5 days post symptom onset, combined Ag-RDT sensitivity was above 95%. Lower sensitivity of 88.2% was seen on the same day of symptom development (day 0).

Conclusions: We provide an independent validation of two widely available commercial Ag-RDTs, both meeting WHO criteria of ≥80% sensitivity and ≥97% specificity. Although less sensitive than RT-PCR, these assays could be beneficial due to their rapid results, ease of use, and independence from existing laboratory structures. Testing criteria focusing on patients with typical symptoms in their early symptomatic period onset could further increase diagnostic value.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0248921PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8011749PMC
April 2021

Comparative Analytical Evaluation of Four Centralized Platforms for the Detection of Mycobacterium tuberculosis Complex and Resistance to Rifampicin and Isoniazid.

J Clin Microbiol 2021 02 18;59(3). Epub 2021 Feb 18.

Foundation for Innovative New Diagnostics, Geneva, Switzerland.

Failure to rapidly identify drug-resistant tuberculosis (TB) increases the risk of patient mismanagement, the amplification of drug resistance, and ongoing transmission. We generated comparative analytical data for four automated assays for the detection of TB and multidrug-resistant TB (MDR-TB): Abbott RealTie MTB and MTB RIF/INH (Abbott), Hain Lifescience FluoroType MTBDR (Hain), BD Max MDR-TB (BD), and Roche cobas MTB and MTB-RIF/INH (Roche). We included Xpert MTB/RIF (Xpert) and GenoType MTBDR as comparators for TB and drug resistance detection, respectively. We assessed analytical sensitivity for the detection of the complex using inactivated strains ( H37Rv and ) spiked into TB-negative sputa and computed the 95% limits of detection (LOD). We assessed the accuracy of rifampicin and isoniazid resistance detection using well-characterized strains with high-confidence mutations accounting for >85% of first-line resistance mechanisms globally. For H37Rv and , we measured LOD values of 3,781 and 2,926 (Xpert), 322 and 2,182 (Abbott), 826 and 4,301 (BD), 10,398 and 23,139 (Hain), and 2,416 and 2,136 (Roche) genomes/ml, respectively. Assays targeting multicopy genes or targets (Abbott, BD, and Roche) showed increased analytical sensitivity compared to Xpert. Quantification of the panel by quantitative real-time PCR prevents the determination of absolute values, and results reported here can be interpreted for comparison purposes only. All assays showed accuracy comparable to that of Genotype MTBDR for the detection of rifampicin and isoniazid resistance. The data from this analytical study suggest that the assays may have clinical performances similar to those of WHO-recommended molecular TB and MDR-TB assays.
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http://dx.doi.org/10.1128/JCM.02168-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8106716PMC
February 2021

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.

Sci Rep 2020 02 14;10(1):2633. Epub 2020 Feb 14.

DST/NRF Centre of Excellence for Biomedical Tuberculosis Research, SA MRC Centre for Molecular and Cellular Biology, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa.

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 ≥10 CFU/ml (C <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 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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http://dx.doi.org/10.1038/s41598-020-59164-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7021780PMC
February 2020

A landscape of genomic alterations at the root of a near-untreatable tuberculosis epidemic.

BMC Med 2020 02 4;18(1):24. Epub 2020 Feb 4.

South African Medical Research Council Centre for Tuberculosis Research, DST NRF Centre of Excellence for Biomedical Tuberculosis research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa.

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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http://dx.doi.org/10.1186/s12916-019-1487-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6998097PMC
February 2020

Switching to bedaquiline for treatment of rifampicin-resistant tuberculosis in South Africa: A retrospective cohort analysis.

PLoS One 2019 17;14(10):e0223308. Epub 2019 Oct 17.

Section of Infectious Diseases, Boston University School of Medicine, Boston, MA, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0223308PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6797261PMC
March 2020

Whole genome sequencing provides additional insights into recurrent tuberculosis classified as endogenous reactivation by IS6110 DNA fingerprinting.

Infect Genet Evol 2019 11 2;75:103948. Epub 2019 Jul 2.

NRF/DST Centre of Excellence for Biomedical Tuberculosis Research, South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa.

Recurrent tuberculosis (TB) after successful TB treatment occurs due to endogenous reactivation (relapse) or exogenous reinfection. We revisited the conclusions of relapse in a high TB incidence setting that were drawn on the basis of IS6110 restriction fragment length polymorphism (RFLP) analysis in a large retrospective cohort study in suburban Cape Town, South Africa. Using whole genome sequencing (WGS), we undertook pair-wise genome comparison of Mycobacterium tuberculosis strains cultured from diagnostic sputum samples collected at the index and recurrent TB episode for 25 recurrent TB cases who had been classified as relapse based on identical DNA fingerprint patterns in the earlier study. We found that paired strain genome sequences were identical or showed minimal variant differences in 22 of 25 recurrent TB cases, consistent with relapse. One showed 20 variant differences, suggestive of exogenous reinfection. Two of the 25 had mixed infections, each with the index episode strain detected as the dominant strain at recurrence in one of these patients, the minority strain harboured drug-resistance conferring mutations (rpoB, katG). In conclusion, our study highlights the additional value of WGS for investigating recurrent TB in settings with high infection pressure and closely related circulating strains, where the extent of re- and mixed infection may be underestimated.
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http://dx.doi.org/10.1016/j.meegid.2019.103948DOI Listing
November 2019

Whole genome sequencing of Mycobacterium tuberculosis: current standards and open issues.

Nat Rev Microbiol 2019 09;17(9):533-545

Foundation for Innovative New Diagnostics, Geneva, Switzerland.

Whole genome sequencing (WGS) of Mycobacterium tuberculosis has rapidly progressed from a research tool to a clinical application for the diagnosis and management of tuberculosis and in public health surveillance. This development has been facilitated by drastic drops in cost, advances in technology and concerted efforts to translate sequencing data into actionable information. There is, however, a risk that, in the absence of a consensus and international standards, the widespread use of WGS technology may result in data and processes that lack harmonization, comparability and validation. In this Review, we outline the current landscape of WGS pipelines and applications, and set out best practices for M. tuberculosis WGS, including standards for bioinformatics pipelines, curated repositories of resistance-causing variants, phylogenetic analyses, quality control and standardized reporting.
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http://dx.doi.org/10.1038/s41579-019-0214-5DOI Listing
September 2019

Diagnostic Accuracy and Utility of FluoroType MTBDR, a New Molecular Assay for Multidrug-Resistant Tuberculosis.

J Clin Microbiol 2018 09 27;56(9). Epub 2018 Aug 27.

DST-NRF Centre of Excellence for Biomedical Tuberculosis Research/South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, South Africa

Most cases of multidrug-resistant (MDR) tuberculosis (TB) are never diagnosed (328,300 of the ∼490,000 cases in 2016 were missed). The Xpert MTB/RIF assay detects resistance only to rifampin, despite ∼20% of rifampin-resistant cases being susceptible to isoniazid (a critical first-line drug). Consequently, many countries require further testing with the GenoType MTBDR assay. However, MTBDR is not recommended for use on smear-negative specimens, and thus, many specimens require culture-based drug susceptibility testing. Furthermore, MTBDR requires specialized expertise, lengthy hands-on time, and significant laboratory infrastructure and interpretation is not automated. To address these gaps, we evaluated the accuracy of the FluoroType MTBDR (FluoroType) assay. Sputa from 244 smear-positive and 204 smear-negative patients with presumptive TB (Xpert MTB positive, = 343) were tested. Culture and MTBDR on isolates served as reference standards (for active TB and MDR-TB, respectively). Sanger sequencing and MTBDR, both of which were performed on sputa, were used to resolve discrepancies. The sensitivity of FluoroType for the detection of complex was 98% (95% confidence interval [CI], 95 to 99%) and 92% (95% CI, 84 to 96%) for smear-positive and smear-negative specimens, respectively (232/237 versus 90/98 specimens; < 0.009). The sensitivity and specificity for smear-negative specimens were 100% and 97%, respectively, for rifampin resistance; 100% and 98%, respectively, for isoniazid resistance; and 100% and 100%, respectively, for MDR-TB. FluoroType identified 98%, 97%, and 97% of the , , and promoter mutations, respectively. FluoroType has excellent sensitivity with sputa equivalent to that of MTBDR with the isolates and can provide rapid drug susceptibility testing for rifampin and isoniazid. In addition, the capacity of FluoroType to simultaneously identify virtually all mutations in the , , and promoter may be useful for individualized treatment regimens.
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http://dx.doi.org/10.1128/JCM.00531-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6113470PMC
September 2018

Mycobacterial genomic DNA from used Xpert MTB/RIF cartridges can be utilised for accurate second-line genotypic drug susceptibility testing and spoligotyping.

Sci Rep 2017 11 1;7(1):14854. Epub 2017 Nov 1.

DST/NRF Centre of Excellence for Biomedical Tuberculosis Research, SA MRC Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa.

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 ≥10CFU/ml (C ≤ 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 ≥10CFU/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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http://dx.doi.org/10.1038/s41598-017-14385-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5666021PMC
November 2017