Publications by authors named "Grant Theron"

84 Publications

Anaerobe-enriched gut microbiota predicts pro-inflammatory responses in pulmonary tuberculosis.

EBioMedicine 2021 May 8;67:103374. Epub 2021 May 8.

DSI-NRF Centre of Excellence for Biomedical Tuberculosis Research, and SAMRC Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, Cape Town, South Africa. Electronic address:

Background: The relationship between tuberculosis (TB), one of the leading infectious causes of death worldwide, and the microbiome, which is critical for health, is poorly understood.

Methods: To identify potential microbiome-host interactions, profiling of the oral, sputum and stool microbiota [n = 58 cases, n = 47 culture-negative symptomatic controls (SCs)] and whole blood transcriptome were done in pre-treatment presumptive pulmonary TB patients. This was a cross-sectional study. Microbiota were also characterised in close contacts of cases (CCCs, n = 73) and close contacts of SCs (CCSCs, n = 82) without active TB.

Findings: Cases and SCs each had similar α- and β-diversities in oral washes and sputum, however, β-diversity differed in stool (PERMANOVA p = 0•035). Cases were enriched with anaerobes in oral washes, sputum (Paludibacter, Lautropia in both) and stool (Erysipelotrichaceae, Blautia, Anaerostipes) and their stools enriched in microbial genes annotated as amino acid and carbohydrate metabolic pathways. In pairwise comparisons with their CCCs, cases had Megasphaera-enriched oral and sputum microbiota and Bifidobacterium-, Roseburia-, and Dorea-depleted stools. Compared to their CCSCs, SCs had reduced α-diversities and many differential taxa per specimen type. Cases differed transcriptionally from SCs in peripheral blood (PERMANOVA p = 0•001). A co-occurrence network analysis showed stool taxa, Erysipelotrichaceae and Blautia, to negatively co-correlate with enriched "death receptor" and "EIF2 signalling" pathways whereas Anaerostipes positively correlated with enriched "interferon signalling", "Nur77 signalling" and "inflammasome" pathways; all of which are host pathways associated with disease severity. In contrast, none of the taxa enriched in SCs correlated with host pathways.

Interpretation: TB-specific microbial relationships were identified in oral washes, induced sputum, and stool from cases before the confounding effects of antibiotics. Specific anaerobes in cases' stool predict upregulation of pro-inflammatory immunological pathways, supporting the gut microbiota's role in TB.

Funding: European & Developing Countries Clinical Trials Partnership, South African-Medical Research Council, National Institute of Allergy and Infectious Diseases.
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http://dx.doi.org/10.1016/j.ebiom.2021.103374DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8122180PMC
May 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 Eis: non-detection of kanamycin resistance markers by routine diagnostic tests and identification of new -promoter variants.

Antimicrob Agents Chemother 2021 Apr 26. Epub 2021 Apr 26.

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.

-promoter mutations can confer reduced kanamycin susceptibility. GenoType MTBDR a widely used assay evaluating this region, wrongly classified 17/410 isolates as -promoter wildtype. 6/17 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
April 2021

The child ecosystem and childhood pulmonary tuberculosis: A South African perspective.

Pediatr Pulmonol 2021 Mar 25. Epub 2021 Mar 25.

Department of Paediatrics and Child Health, Red Cross Childrens Hospital and SA-MRC Unit on Child & Adolescent Health, University of Cape Town, Cape Town, Western Cape, South Africa.

Introduction: This study investigates drivers of childhood pulmonary tuberculosis (PTB) using a childhood ecosystem approach in South Africa. An ecosystem approach toward identifying risk factors for PTB may identify targeted interventions.

Methods: Data were collected as part of a prospective cohort study of children presenting at a primary care facility or tertiary hospital with possible TB. Characterization of the childhood ecosystem included proximal, medial, and distal determinants. Proximal determinants included child characteristics that could impact PTB outcomes. Medial determinants included relational factors, such as caregiver health, which might impact interactions with the child. Distal determinants included macro-level determinants of disease, such as socioeconomic status and food insecurity. Children who started on TB treatment were followed for up to 6 months. Multivariate regression models tested independent associations between factors associated with PTB in children.

Results: Of 1202 children enrolled, 242 (20%) of children had confirmed PTB, 756 (63%) were started on TB treatment, and 444 (37%) had respiratory conditions other than TB. In univariate analyses, childhood malnutrition and caregiver smoking were associated with treated or confirmed PTB. In multivariate analyses, proximal factors, such as male gender and hospitalization, as well as low socioeconomic status as a distal factor, were associated with PTB.

Conclusions: Interventions may need to target subgroups of children and families with elevated proximal, medial, and distal risk factors for PTB. Screening for risk factors, such as caregiver's health, may guide targeting. The provision of social protection programs to bolster economic security may be an important intervention for attenuating childhood exposure to risk factors.
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http://dx.doi.org/10.1002/ppul.25369DOI Listing
March 2021

Face masks in the post-COVID-19 era: a silver lining for the damaged tuberculosis public health response?

Lancet Respir Med 2021 04 22;9(4):340-342. Epub 2021 Jan 22.

DSI-NRF Centre of Excellence for Biomedical Tuberculosis Research and SA/MRC Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town 7550, South Africa. Electronic address:

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http://dx.doi.org/10.1016/S2213-2600(21)00020-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7826055PMC
April 2021

"I got tested at home, the help came to me": acceptability and feasibility of home-based TB testing of household contacts using portable molecular diagnostics in South Africa.

Trop Med Int Health 2021 Mar 4;26(3):343-354. Epub 2021 Jan 4.

Charles R. Drew University of Medicine and Science, Los Angeles, CA, USA.

Objective: The effectiveness of household contact investigations is limited by low referral uptake for clinic-based TB testing by symptomatic household contacts. We qualitatively investigated the acceptability and perceived benefits of home-based TB testing using a portable GeneXpert-I instrument (GX-I) in an urban South African township.

Methods: In-depth interviews were conducted with household contacts tested and those that observed testing. Semi-structured interviews explored household contact's understanding of TB, perceptions of the GX-I device and testing procedures, confidentiality, willingness to refer others, and views on home- vs. clinic-based testing. Focus group discussions with home-based TB testing implementing staff assessed operational considerations for scale-up. Data were analysed using a constant comparison approach to qualitatively evaluate the acceptability and feasibility of home-based TB testing.

Results: Thirty in-depth interviews and two focus group discussions were conducted. Observing one's own sputum being tested resulted in an emergent trust in home-based TB testing, the GX-I device and one's test results. Home-based TB testing was considered convenient, helped to overcome apathy towards testing and mitigated barriers to clinic-based testing. Perceptions that home-based TB testing contributes to improved household and community health resulted in an emergent theme of alleviation of health insecurities. Operational concerns regarding inadvertent disclosure of one's diagnosis to household members and time spent in people's homes were identified.

Conclusions: Home-based TB testing was acceptable and feasible. Individuals expressed belief in the machine by being able to witness the testing process. Though most themes mirrored qualitative studies of home-based HIV testing, the alleviation of health insecurities theme is unique to home-based TB testing. Future research must evaluate the impact of home-based TB testing on case finding yield, time-to-treatment initiation and household outcomes.
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http://dx.doi.org/10.1111/tmi.13533DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8060432PMC
March 2021

Cost-effectiveness of post-treatment follow-up examinations and secondary prevention of tuberculosis in a high-incidence setting: a model-based analysis.

Lancet Glob Health 2020 09;8(9):e1223-e1233

Department of Health Policy and Management, Yale School of Public Health, New Haven, CT, USA.

Background: In settings of high tuberculosis incidence, previously treated individuals remain at high risk of recurrent tuberculosis and contribute substantially to overall disease burden. Whether tuberculosis case finding and preventive interventions among previously treated people are cost-effective has not been established. We aimed to estimate costs and health benefits of annual post-treatment follow-up examinations and secondary preventive therapy for tuberculosis in a tuberculosis-endemic setting.

Methods: We developed a transmission-dynamic mathematical model and calibrated it to data from two high-incidence communities of approximately 40 000 people in suburban Cape Town, South Africa. We used the model to estimate overall cost and disability-adjusted life-years (DALYs) associated with annual follow-up examinations and secondary isoniazid preventive therapy (IPT), alone and in combination, among individuals completing tuberculosis treatment. We investigated scenarios under which these interventions were restricted to the first year after treatment completion, or extended indefinitely. For each intervention scenario, we projected health system costs and DALYs averted with respect to the current status quo of tuberculosis control. All estimates represent mean values derived from 1000 epidemic trajectories simulated over a 10-year period (2019-28), with 95% uncertainty intervals (UIs) calculated as the 2·5th and 97·5th percentile values.

Findings: We estimated that a single follow-up examination at the end of the first year after treatment completion combined with 12 months of secondary IPT would avert 2472 DALYs (95% UI -888 to 7801) over a 10-year period and is expected to be cost-saving compared with current control efforts. Sustained annual follow-up and continuous secondary IPT beyond the first year after treatment would avert an additional 1179 DALYs (-1769 to 4377) over 10 years at an expected additional cost of US$18·2 per DALY averted. Strategies of follow-up without secondary IPT were dominated (ie, expected to result in lower health impact at higher costs) by strategies that included secondary IPT.

Interpretation: In this high-incidence setting, post-treatment follow-up and secondary preventive therapy can accelerate declines in tuberculosis incidence and potentially save resources for tuberculosis control. Empirical trials to assess the feasibility of these interventions in settings most severely affected by tuberculosis are needed.

Funding: National Institutes of Health, Günther Labes Foundation, Oskar Helene Heim Foundation.
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http://dx.doi.org/10.1016/S2214-109X(20)30227-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7549318PMC
September 2020

Bacterial and host determinants of cough aerosol culture positivity in patients with drug-resistant versus drug-susceptible tuberculosis.

Nat Med 2020 09 29;26(9):1435-1443. Epub 2020 Jun 29.

Centre for Lung Infection and Immunity, Division of Pulmonology, Department of Medicine and UCT Lung Institute & South African MRC/UCT Centre for the Study of Antimicrobial Resistance, University of Cape Town, Cape Town, South Africa.

A burgeoning epidemic of drug-resistant tuberculosis (TB) threatens to derail global control efforts. Although the mechanisms remain poorly clarified, drug-resistant strains are widely believed to be less infectious than drug-susceptible strains. Consequently, we hypothesized that lower proportions of patients with drug-resistant TB would have culturable Mycobacterium tuberculosis from respirable, cough-generated aerosols compared to patients with drug-susceptible TB, and that multiple factors, including mycobacterial genomic variation, would predict culturable cough aerosol production. We enumerated the colony forming units in aerosols (≤10 µm) from 452 patients with TB (227 with drug resistance), compared clinical characteristics, and performed mycobacterial whole-genome sequencing, dormancy phenotyping and drug-susceptibility analyses on M. tuberculosis from sputum. After considering treatment duration, we found that almost half of the patients with drug-resistant TB were cough aerosol culture-positive. Surprisingly, neither mycobacterial genomic variants, lineage, nor dormancy status predicted cough aerosol culture positivity. However, mycobacterial sputum bacillary load and clinical characteristics, including a lower symptom score and stronger cough, were strongly predictive, thereby supporting targeted transmission-limiting interventions. Effective treatment largely abrogated cough aerosol culture positivity; however, this was not always rapid. These data question current paradigms, inform public health strategies and suggest the need to redirect TB transmission-associated research efforts toward host-pathogen interactions.
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http://dx.doi.org/10.1038/s41591-020-0940-2DOI Listing
September 2020

Xpert MTB/RIF-detected Rifampicin Resistance is a Sub-Optimal Surrogate for Multidrug Resistant Tuberculosis in Eastern Democratic Republic of the Congo: Diagnostic and Clinical Implications.

Clin Infect Dis 2020 Jun 26. Epub 2020 Jun 26.

Laboratoire de Recherche Biomédicale Professeur André Lurhuma, Université Catholique de Bukavu (UCB), Bukavu, DRC.

Background: Rifampicin (RIF) resistance is highly correlated with isoniazid (INH) resistance and used as proxy for multidrugresistant tuberculosis (MDR-TB). Using MTBDRplus as a comparator, we evaluated the predictive value of Xpert® MTB/RIF (Xpert)detected RIF resistance for MDR-TB in eastern Democratic Republic of the Congo (DRC).

Methods: We conducted a cross-sectional study involving data from new or retreatment pulmonary adult TB cases evaluated between July 2013 and December 2016. Separate, paired sputa for smear microscopy and MTBDRplus were collected. Xpert testing was performed subject to the availability of Xpert cartridges on sample remnants after microscopy.

Results: Among 353 patients, 193 (54.7%) were previously treated and 224 (63.5%) were MTBDRplus TB-positive. Of the 224, 43 (19.2%) were RIFmono-resistant, 11 (4.9%) were INHmono-resistant, 53 (23.7%) had MDR-TB, and 117 (52.2%) were RIF- and INH-susceptible. Overall, among the 96 samples detected by MTBDRplus as RIF-resistant, 53 (55.2%) had MDR-TB. Xpert testing was performed in 179 (50.7%) specimens, and amongst these, 163 (91.1%) were TB-positive and 73 (44.8%) RIF-resistant. Only 45/73 (61.6%) Xpertidentified RIF-resistant isolates had concomitant MTBDRplus-detected INH resistance. Xpert had a sensitivity of 100.0% (95% CI; 92.1-100.0) for detecting RIFresistance but a positive predictive value of only 61.6% (95% CI; 49.5-72.8) for MDR-TB. The most frequent mutations associated with RIF and INH resistance were S531L and S315T1, respectively.

Conclusions: In this high-risk MDR-TB study population, Xpert had low positive predictive value for the presence of MDR-TB. Comprehensive resistance testing for both INH and RIF should be performed in this setting.
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http://dx.doi.org/10.1093/cid/ciaa873DOI Listing
June 2020

Systematic review of mutations associated with resistance to the new and repurposed Mycobacterium tuberculosis drugs bedaquiline, clofazimine, linezolid, delamanid and pretomanid.

J Antimicrob Chemother 2020 08;75(8):2031-2043

Department of Biomedical Informatics, Harvard Medical School, 10 Shattuck Street, Boston, MA 02115, USA.

Background: Improved genetic understanding of Mycobacterium tuberculosis (MTB) resistance to novel and repurposed anti-tubercular agents can aid the development of rapid molecular diagnostics.

Methods: Adhering to PRISMA guidelines, in March 2018, we performed a systematic review of studies implicating mutations in resistance through sequencing and phenotyping before and/or after spontaneous resistance evolution, as well as allelic exchange experiments. We focused on the novel drugs bedaquiline, delamanid, pretomanid and the repurposed drugs clofazimine and linezolid. A database of 1373 diverse control MTB whole genomes, isolated from patients not exposed to these drugs, was used to further assess genotype-phenotype associations.

Results: Of 2112 papers, 54 met the inclusion criteria. These studies characterized 277 mutations in the genes atpE, mmpR, pepQ, Rv1979c, fgd1, fbiABC and ddn and their association with resistance to one or more of the five drugs. The most frequent mutations for bedaquiline, clofazimine, linezolid, delamanid and pretomanid resistance were atpE A63P, mmpR frameshifts at nucleotides 192-198, rplC C154R, ddn W88* and ddn S11*, respectively. Frameshifts in the mmpR homopolymer region nucleotides 192-198 were identified in 52/1373 (4%) of the control isolates without prior exposure to bedaquiline or clofazimine. Of isolates resistant to one or more of the five drugs, 59/519 (11%) lacked a mutation explaining phenotypic resistance.

Conclusions: This systematic review supports the use of molecular methods for linezolid resistance detection. Resistance mechanisms involving non-essential genes show a diversity of mutations that will challenge molecular diagnosis of bedaquiline and nitroimidazole resistance. Combined phenotypic and genotypic surveillance is needed for these drugs in the short term.
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http://dx.doi.org/10.1093/jac/dkaa136DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7825472PMC
August 2020

Differential RD-1-specific IFN-γ host responses to diverse Mycobacterium tuberculosis strains in HIV-uninfected persons may be explained by genotypic variation in the ESX-1 region.

Int J Infect Dis 2020 Jul 24;96:240-243. Epub 2020 Apr 24.

Centre for Lung Infection and Immunity, Division of Pulmonology, Department of Medicine and UCT Lung Institute & South African MRC/UCT Centre for the Study of Antimicrobial Resistance, University of Cape Town, Cape Town, South Africa; Institute of Infectious Diseases and Molecular Medicine, University of Cape Town, Cape Town, South Africa; Faculty of Infectious and Tropical Diseases, Department of Infection Biology, London School of Hygiene and Tropical Medicine, London, UK. Electronic address:

Objectives: Between-person variability in T-cell-specific interferon-gamma release assay (IGRA) responses and discordance between IGRA test formats are poorly understood.

Methods: We evaluated the IFN-γ responses (QuantiFERON-TB Gold-In-Tube [QFT-GIT] and TSPOT-TB) stratified according to the Mycobacterium tuberculosis spoligotype of the culture isolate obtained from the same patients with confirmed active tuberculosis (n = 91). We further analysed differences within the RD-1-encoding ESX-1 region between the different strain types using whole genome sequencing.

Results: In HIV-uninfected patients, TSPOT.TB and QFT-GIT IFN-γ responses were 5-fold (p < 0.01) and 2-fold higher (p < 0.05) for those infected with family 33 compared to the LAM strain (additionally, TSPOT.TB responses were 5.6-fold [p < 0.05] and 2.6-fold higher [p < 0.05] for the patients infected with the family 33 versus the X strain and Beijing versus the LAM strain, respectively). Multivariate analysis revealed that strain type (determined by spoligotyping) was independently associated with the magnitude of the IGRA response (varied by IGRA test type) and this is likely explained by variability in the ESX-1 region of Mycobacteriumtuberculosis (determined by next-generation sequencing).

Conclusions: These data have implications for the understanding of between-person heterogeneity in IGRA responses, Mycobateriumtuberculosis-specific host immunity, and the discordance between different IGRA test formats.
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http://dx.doi.org/10.1016/j.ijid.2020.04.053DOI Listing
July 2020

Blood transcriptional biomarkers for active pulmonary tuberculosis in a high-burden setting: a prospective, observational, diagnostic accuracy study.

Lancet Respir Med 2020 04 13;8(4):407-419. Epub 2020 Mar 13.

Division of Infection and Immunity, University College London, London, UK. Electronic address:

Background: Blood transcriptional signatures are candidates for non-sputum triage or confirmatory tests of tuberculosis. Prospective head-to-head comparisons of their diagnostic accuracy in real-world settings are necessary to assess their clinical use. We aimed to compare the diagnostic accuracy of candidate transcriptional signatures identified by systematic review, in a setting with a high burden of tuberculosis and HIV.

Methods: We did a prospective observational study nested within a diagnostic accuracy study of sputum Xpert MTB/RIF (Xpert) and Xpert MTB/RIF Ultra (Ultra) tests for pulmonary tuberculosis. We recruited consecutive symptomatic adults aged 18 years or older self-presenting to a tuberculosis clinic in Cape Town, South Africa. Participants provided blood for RNA sequencing, and sputum samples for liquid culture and molecular testing using Xpert and Ultra. We assessed the diagnostic accuracy of candidate blood transcriptional signatures for active tuberculosis (including those intended to distinguish active tuberculosis from other diseases) identified by systematic review, compared with culture or Xpert MTB/RIF positivity as the standard reference. In our primary analysis, patients with tuberculosis were defined as those with either a positive liquid culture or Xpert result. Patients with missing blood RNA or sputum results were excluded. Our primary objective was to benchmark the diagnostic accuracy of candidate transcriptional signatures against the WHO target product profile (TPP) for a tuberculosis triage test.

Findings: Between Feb 12, 2016, and July 18, 2017, we obtained paired sputum and RNA sequencing data from 181 participants, 54 (30%) of whom had confirmed pulmonary tuberculosis. Of 27 eligible signatures identified by systematic review, four achieved the highest diagnostic accuracy with similar area under the receiver operating characteristic curves (Sweeney3: 90·6% [95% CI 85·6-95·6]; Kaforou25: 86·9% [80·9-92·9]; Roe3: 86·9% [80·3-93·5]; and BATF2: 86·8% [80·6-93·1]), independent of age, sex, HIV status, previous tuberculosis, or sputum smear result. At test thresholds that gave 70% specificity (the minimum WHO TPP specificity for a triage test), these four signatures achieved sensitivities between 83·3% (95% CI 71·3-91·0) and 90·7% (80·1-96·0). No signature met the optimum criteria, of 95% sensitivity and 80% specificity proposed by WHO for a triage test, or the minimum criteria (of 65% sensitivity and 98% specificity) for a confirmatory test, but all four correctly identified Ultra-positive, culture-negative patients.

Interpretation: Selected blood transcriptional signatures met the minimum WHO benchmarks for a tuberculosis triage test but not for a confirmatory test. Further development of the signatures is warranted to investigate their possible effects on clinical and health economic outcomes as part of a triage strategy, or when used as add-on confirmatory test in conjunction with the highly sensitive Ultra test for Mycobacterium tuberculosis DNA.

Funding: Royal Society Newton Advanced Fellowship, Wellcome Trust, National Institute of Health Research, and UK Medical Research Council.
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http://dx.doi.org/10.1016/S2213-2600(19)30469-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7113842PMC
April 2020

Xpert MTB/RIF Ultra and Xpert MTB/RIF for diagnosis of tuberculosis in an HIV-endemic setting with a high burden of previous tuberculosis: a two-cohort diagnostic accuracy study.

Lancet Respir Med 2020 04 14;8(4):368-382. Epub 2020 Feb 14.

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, Stellenbosch University, Cape Town, South Africa. Electronic address:

Background: Xpert MTB/RIF Ultra (Ultra) is a new test for tuberculosis undergoing global roll-out. We assessed the performance of Ultra compared with Xpert MTB/RIF (Xpert) in an HIV-endemic setting where previous tuberculosis is frequent and current test performance is suboptimal.

Methods: In this two-cohort diagnostic accuracy study, we used sputum samples from patients in South Africa to evaluate the accuracy of Ultra and Xpert against a single culture reference standard. For the first cohort (cohort A), we recruited adults (aged ≥18 years) with symptoms of presumptive tuberculosis at Scottsdene clinic in Cape Town, South Africa. We collected three sputum samples from each patient in cohort A, two at the first visit of which one was tested using Xpert and the other was tested using culture, and one sample the next morning which was tested using Ultra. In a separate cohort of patients with presumptive tuberculosis and recent previous tuberculosis (≤2 years) who had submitted sputum samples to the National Health Laboratory Services (cohort B), decontaminated sediments were, after processing, randomly allocated (1:1) for testing with Ultra or Xpert. For both cohorts we calculated the sensitivity and specificity of Ultra and Xpert and evaluated the effects of different methods of interpreting Ultra trace results.

Findings: Between Feb 6, 2016, and Feb 2, 2018, we recruited 302 people into cohort A, all of whom provided sputum samples and 239 were included in the head-to-head analyses of Ultra and Xpert. For cohort B, we collected sputum samples from eligible patients who had submitted samples between Dec 6, 2016, and Dec 21, 2017, to give a cohort of 831 samples, of which 352 were eligible for inclusion in analyses and randomly assigned to Ultra (n=173) or Xpert (n=179). In cohort A, Ultra gave more non-actionable results (not positive or negative) than did Xpert (28 [10%] 275 vs 14 [5%] 301; p=0·011). In the head-to-head analysis, in smear-negative patients, sensitivity of Ultra was 80% (95% CI 64-90) and of Xpert was 73% (57-85; p=0·45). Overall, specificity of Ultra was lower than that of Xpert (90% [84-94] vs 99% [95-100]; p=0·001). In cohort B, overall sensitivity was 92% (81-98) for Xpert versus 86% (73-95; p=0·36) for Ultra and overall specificity was 69% (60-77) for Ultra versus 84% (78-91; p=0·005) for Xpert. Ultra specificity estimates improved after reclassification of results with the lowest Ultra-positive semiquantitation category (trace) to negative (15% [8-22]). In cohort A, the positive predictive value (PPV) for Ultra was 78% (67-87) and for Xpert was 96% (87-99; p=0·004); in cohort B, the PPV for Ultra was 50% (43-57) and for Xpert was 70% (61-78; p=0·014). Ultra PPV estimates in previously treated patients were low: at 15% tuberculosis prevalence, half of Ultra-positive patients with presumptive tuberculosis would be culture negative, increasing to approximately 70% in patients with recent previous tuberculosis. In cohort B, 21 (28%) of 76 samples that were Ultra positive were rifampicin indeterminate (all trace) and, like cohort A, most were culture negative (19 [90%] of 21).

Interpretation: In a setting with a high burden of previous tuberculosis, Ultra generated more non-actionable results and had diminished specificity compared with Xpert. In patients with recent previous tuberculosis, a quarter of Ultra-positive samples were indeterminate for rifampicin resistance and culture negative, suggesting that additional drug-resistance testing will probably be unsuccessful. Our data have implications for the handling of Ultra-positive results in patients with previous tuberculosis in high burden settings.

Funding: South African Medical Research Council, the EDCTP2 program, and the Faculty of Medicine and Health Sciences, Stellenbosch University.
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http://dx.doi.org/10.1016/S2213-2600(19)30370-4DOI Listing
April 2020

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

Guidance for Studies Evaluating the Accuracy of Sputum-Based Tests to Diagnose Tuberculosis.

J Infect Dis 2019 10;220(220 Suppl 3):S99-S107

World Health Organization, Geneva, Switzerland.

Tests that can replace sputum smear microscopy have been identified as a top priority diagnostic need for tuberculosis by the World Health Organization. High-quality evidence on diagnostic accuracy for tests that may meet this need is an essential requirement to inform decisions about policy and scale-up. However, test accuracy studies are often of low and inconsistent quality and poorly reported, leading to uncertainty about true test performance. Here we provide guidance for the design of diagnostic test accuracy studies of sputum smear-replacement tests. Such studies should have a cross-sectional or cohort design, enrolling either a consecutive series or a random sample of patients who require evaluation for tuberculosis. Adults with respiratory symptoms are the target population. The reference standard should at a minimum be a single, automated, liquid culture, but additional cultures, follow-up, clinical case definition, and specific measures to understand discordant results should also be included. Inclusion of smear microscopy and Xpert MTB/RIF (or MTB/RIF Ultra) as comparators is critical to allow broader comparability and generalizability of results, because disease spectrum can vary between studies and affects relative test performance. Given the complex nature of sputum (the primary specimen type used for pulmonary TB), careful design and reporting of the specimen flow is essential. Test characteristics other than accuracy (such as feasibility, implementation considerations, and data on impact on patient, population and health systems outcomes) are also important aspects.
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http://dx.doi.org/10.1093/infdis/jiz258DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6782025PMC
October 2019

Still dying in plain sight: missed and misclassified deaths due to tuberculosis in hospitals.

Eur Respir J 2019 Sep 1;54(3). Epub 2019 Oct 1.

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

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http://dx.doi.org/10.1183/13993003.01578-2019DOI Listing
September 2019

Investigation of Preanalytical Variables Impacting Pathogen Cell-Free DNA in Blood and Urine.

J Clin Microbiol 2019 11 23;57(11). Epub 2019 Oct 23.

Department of Pathology, Stanford University School of Medicine, Stanford, California, USA

Pathogen cell-free DNA (pcfDNA) in blood and urine is an attractive biomarker; however, the impact of preanalytical factors is not well understood. Blood and urine samples from healthy donors spiked with cfDNA from , , , and Epstein-Barr virus (EBV) and samples from tuberculosis patients were used to evaluate the impact of blood collection tube, urine preservative, processing delay, processing method, freezing and thawing, and sample volume on pcfDNA. The PCR cycle threshold ( ) was used to measure amplifiable cfDNA. In spiked samples, the median values for , , and EBV cfDNA were significantly lower in blood collected in KEDTA tubes than those in Streck and PAXgene blood collection tubes, and they were was significantly lower in urine preserved with EDTA (EDTA-urine) than in urine preserved with Streck reagent (Streck-urine). Blood and urine samples from TB patients preserved with KEDTA and Tris-EDTA, respectively, showed significantly lower median values than with the Streck blood collection tube and Streck urine preservative. Processing delay increased the median pathogen values for Streck and PAXgene but not KEDTA blood samples and for urine preserved with Streck reagent but not EDTA. Double-spin compared with single-spin plasma separation increased the median pathogen regardless of blood collection tube. No differences were observed between whole urine and supernatant and between fresh and thawed plasma and urine after 24 weeks at -80°C. Larger plasma and urine volumes in contrived and patient samples showed a significantly lower median These findings suggest that large-volume single-spin KEDTA-plasma and EDTA-whole urine with up to a 24-h processing delay may optimize pcfDNA detection.
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http://dx.doi.org/10.1128/JCM.00782-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6813001PMC
November 2019

Point of care Xpert MTB/RIF versus smear microscopy for tuberculosis diagnosis in southern African primary care clinics: a multicentre economic evaluation.

Lancet Glob Health 2019 06;7(6):e798-e807

Centre for Lung Infection and Immunity, Division of Pulmonology, Department of Medicine and University of Cape Town (UCT) Lung Institute and South African MRC/UCT Centre for the Study of Antimicrobial Resistance, UCT, Cape Town, South Africa; Faculty of Infectious and Tropical Diseases, Department of Immunology and Infection, London School of Hygiene & Tropical Medicine, London, UK. Electronic address:

Background: Rapid on-site diagnosis facilitates tuberculosis control. Performing Xpert MTB/RIF (Xpert) at point of care is feasible, even when performed by minimally trained health-care workers, and when compared with point-of-care smear microscopy, reduces time to diagnosis and pretreatment loss to follow-up. However, whether Xpert is cost-effective at point of care remains unclear.

Methods: We empirically collected cost (US$, 2014) and clinical outcome data from participants presenting to primary health-care facilities in four African countries (South Africa, Zambia, Zimbabwe, and Tanzania) during the TB-NEAT trial. Costs were determined using an bottom-up ingredients approach. Effectiveness measures from the trial included number of cases diagnosed, initiated on treatment, and completing treatment. The primary outcome was the incremental cost-effectiveness of point-of-care Xpert relative to smear microscopy. The study was performed from the perspective of the health-care provider.

Findings: Using data from 1502 patients, we calculated that the mean Xpert unit cost was lower when performed at a centralised laboratory (Lab Xpert) rather than at point of care ($23·00 [95% CI 22·12-23·88] vs $28·03 [26·19-29·87]). Per 1000 patients screened, and relative to smear microscopy, point-of-care Xpert cost an additional $35 529 (27 054-40 025) and was associated with an additional 24·3 treatment initiations ([-20·0 to 68·5]; $1464 per treatment), 63·4 same-day treatment initiations ([27·3-99·4]; $511 per same-day treatment), and 29·4 treatment completions ([-6·9 to 65·6]; $1211 per completion). Xpert costs were most sensitive to test volume, whereas incremental outcomes were most sensitive to the number of patients initiating and completing treatment. The probability of point-of-care Xpert being cost-effective was 90% at a willingness to pay of $3820 per treatment completion.

Interpretation: In southern Africa, although point-of-care Xpert unit cost is higher than Lab Xpert, it is likely to offer good value for money relative to smear microscopy. With the current availability of point-of-care nucleic acid amplification platforms (eg, Xpert Edge), these data inform much needed investment and resource allocation strategies in tuberculosis endemic settings.

Funding: European Union European and Developing Countries Clinical Trials Partnership.
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http://dx.doi.org/10.1016/S2214-109X(19)30164-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7197817PMC
June 2019

The microbiome and tuberculosis: state of the art, potential applications, and defining the clinical research agenda.

Lancet Respir Med 2019 10 22;7(10):892-906. Epub 2019 Mar 22.

Department of Science and Technology-National Research Foundation (DST-NRF) Centre of Excellence for Biomedical Tuberculosis Research, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa; South African Medical Research Council Centre for Tuberculosis Research, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa; Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa; African Microbiome Institute, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa. Electronic address:

The diverse microbial communities within our bodies produce metabolites that modulate host immune responses. Even the microbiome at distal sites has an important function in respiratory health. However, the clinical importance of the microbiome in tuberculosis, the biggest infectious cause of death worldwide, is only starting to be understood. Here, we critically review research on the microbiome's association with pulmonary tuberculosis. The research indicates five main points: (1) susceptibility to infection and progression to active tuberculosis is altered by gut Helicobacter co-infection, (2) aerosol Mycobacterium tuberculosis infection changes the gut microbiota, (3) oral anaerobes in the lung make metabolites that decrease pulmonary immunity and predict progression, (4) the increased susceptibility to reinfection of patients who have previously been treated for tuberculosis is likely due to the depletion of T-cell epitopes on commensal gut non-tuberculosis mycobacteria, and (5) the prolonged antibiotic treatment required for cure of tuberculosis has long-term detrimental effects on the microbiome. We highlight knowledge gaps, considerations for addressing these knowledge gaps, and describe potential targets for modifying the microbiome to control tuberculosis.
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http://dx.doi.org/10.1016/S2213-2600(18)30501-0DOI Listing
October 2019

Prevalence, Predictors, and Successful Treatment Outcomes of Xpert MTB/RIF-identified Rifampicin-resistant Tuberculosis in Post-conflict Eastern Democratic Republic of the Congo, 2012-2017: A Retrospective Province-Wide Cohort Study.

Clin Infect Dis 2019 09;69(8):1278-1287

Department of Epidemiology, University of Pittsburgh Graduate School of Public Health, Pennsylvania.

Background: Multidrug-resistant tuberculosis (MDR-TB) jeopardizes global TB control. The prevalence and predictors of Rifampicin-resistant (RR) TB, a proxy for MDR-TB, and the treatment outcomes with standard and shortened regimens have not been assessed in post-conflict regions, such as the South Kivu province in the eastern Democratic Republic of the Congo (DRC). We aimed to fill this knowledge gap and to inform the DRC National TB Program.

Methods: of adults and children evaluated for pulmonary TB by sputum smear microscopy and Xpert MTB/RIF (Xpert) from February 2012 to June 2017. Multivariable logistic regression, Kaplan-Meier estimates, and multivariable Cox regression were used to assess independent predictors of RR-TB and treatment failure/death.

Results: Of 1535 patients Xpert-positive for TB, 11% had RR-TB. Independent predictors of RR-TB were a positive sputum smear (adjusted odds ratio [aOR] 2.42, 95% confidence interval [CI] 1.63-3.59), retreatment of TB (aOR 4.92, 95% CI 2.31-10.45), and one or more prior TB episodes (aOR 1.77 per episode, 95% CI 1.01-3.10). Over 45% of RR-TB patients had no prior TB history or treatment. The median time from Xpert diagnosis to RR-TB treatment initiation was 12 days (interquartile range 3-60.2). Cures were achieved in 30/36 (83%) and 84/114 (74%) of patients on 9- vs 20/24-month MDR-TB regimens, respectively (P = .06). Predictors of treatment failure/death were the absence of directly observed therapy (DOT; adjusted hazard ratio [aHR] 2.77, 95% CI 1.2-6.66) and any serious adverse drug event (aHR 4.28, 95% CI 1.88-9.71).

Conclusions: Favorable RR-TB cure rates are achievable in this post-conflict setting with a high RR-TB prevalence. An expanded Xpert scale-up; the prompt initiation of shorter, safer, highly effective MDR-TB regimens; and treatment adherence support are critically needed to optimize outcomes.
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http://dx.doi.org/10.1093/cid/ciy1105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6763636PMC
September 2019

Effect of Xpert MTB/RIF on clinical outcomes in routine care settings: individual patient data meta-analysis.

Lancet Glob Health 2019 02;7(2):e191-e199

Division of Pulmonary and Critical Care Medicine, Zuckerberg San Francisco General Hospital, University of California, San Francisco, CA, USA. Electronic address:

Background: Xpert MTB/RIF, the most widely used automated nucleic acid amplification test for tuberculosis, is available in more than 130 countries. Although diagnostic accuracy is well documented, anticipated improvements in patient outcomes have not been clearly identified. We performed an individual patient data meta-analysis to examine improvements in patient outcomes associated with Xpert MTB/RIF.

Methods: We searched PubMed, Embase, ClinicalTrials.gov, and the Pan African Clinical Trials Registry from inception to Feb 1, 2018, for randomised controlled trials (RCTs) comparing the use of Xpert MTB/RIF with sputum smear microscopy as tests for tuberculosis diagnosis in adults (aged 18 years or older). We excluded studies of patients with extrapulmonary tuberculosis, and studies in which mortality was not assessed. We used a two-stage approach for our primary analysis and a one-stage approach for the sensitivity analysis. To assess the primary outcome of cumulative 6-month all-cause mortality, we first performed logistic regression models (random effects for cluster randomised trials, with robust SEs for multicentre studies) for each trial, and then pooled the odds ratio (OR) estimates by a fixed-effects (inverse variance) or random-effects (Der Simonian Laird) meta-analysis. We adjusted for age and gender, and stratified by HIV status and previous tuberculosis-treatment history. The study protocol has been registered with PROSPERO, number CRD42014013394.

Findings: Our search identified 387 studies, of which five RCTs were eligible for analysis. 8567 adult clinic attendees (4490 [63·5%] of 7074 participants for whom data were available were HIV-positive) were tested for tuberculosis with Xpert MTB/RIF (Xpert group) versus sputum smear microscopy (sputum smear group), across five low-income and middle-income countries (South Africa, Brazil, Zimbabwe, Zambia, and Tanzania). The primary outcome (reported in three studies) occurred in 182 (4·5%) of 4050 patients in the Xpert group and 217 (5·3%) of 4093 patients in the smear group (pooled adjusted OR 0·88, 95% CI 0·68-1·14 [p=0·34]; for HIV-positive individuals OR 0·83, 0·65-1·05 [p=0·12]). Kaplan-Meier estimates showed a lower rate of death (12·73 per 100 person-years in the Xpert group vs 16·38 per 100 person-years in the sputum smear group) for HIV-positive patients (hazard ratio 0·76, 95% CI 0·60-0·97; p=0·03). The risk of bias was assessed as reasonable and the statistical heterogeneity across studies was low (I<20% for the primary outcome).

Interpretation: Despite individual patient data analysis from five RCTs, we were unable to confidently rule in nor rule out an Xpert MTB/RIF-associated reduction in mortality among outpatients tested for tuberculosis. Reduction in mortality among HIV-positive patients in a secondary analysis suggests the possibility of population-level impact.

Funding: US National Institutes of Health.
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http://dx.doi.org/10.1016/S2214-109X(18)30458-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6366854PMC
February 2019

The Injectable Contraceptive Medroxyprogesterone Acetate Attenuates Mycobacterium tuberculosis-Specific Host Immunity Through the Glucocorticoid Receptor.

J Infect Dis 2019 04;219(8):1329-1337

Centre for Lung Infection and Immunity, Division of Pulmonology and UCT Lung Institute, Department of Medicine, University of Cape Town.

Background: The effects of the widely used progestin-only injectable contraceptives, medroxyprogesterone acetate (MPA) and norethisterone acetate (NET-A), on host susceptibility to Mycobacterium tuberculosis (Mtb) are unknown.

Methods: We recruited human immunodeficiency virus-uninfected females, not taking any contraceptives, from Cape Town, South Africa, to evaluate the effect of MPA, NET-A, and dexamethasone on Mtb containment in monocyte-derived macrophages co-incubated with purified protein derivative (PPD)-driven peripheral blood-derived effector cells.

Results: MPA (P < .005) and dexamethasone (P < .01), but not NET-A, significantly attenuated Mtb containment in Mtb-infected macrophages co-cultured with PPD-driven effector cells at physiologically relevant concentrations and in a dose-dependent manner. Antagonizing the glucocorticoid receptor with mifepristone (RU486) abrogated the reduction in Mtb containment. In PPD-stimulated peripheral blood mononuclear cells, MPA and dexamethasone, but not NET-A, upregulated (median [interquartile range]) regulatory T cells (5.3% [3.1%-18.2%]; P < .05), reduced CD4+ T-cell interferon-γ (21% [0.5%-28%]; P < .05) and granzyme B production (12.6% [7%-13.5%]; P < .05), and reduced CD8+ perforin activity (2.2% [0.1%-7%]; P < .05). RU486 reversed regulatory T-cell up-regulation and the inhibitory effect on Th1 and granzyme/perforin-related pathways.

Conclusions: MPA, but not NET-A, subverts mycobacterial containment in vitro and downregulates pathways associated with protective CD8+- and CD4+-related host immunity via the glucocorticoid receptor. These data potentially inform the selection and use of injectable contraceptives in tuberculosis-endemic countries.
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http://dx.doi.org/10.1093/infdis/jiy657DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6452311PMC
April 2019

Evaluation of the airway microbiome in nontuberculous mycobacteria disease.

Eur Respir J 2018 10 25;52(4). Epub 2018 Oct 25.

Division of Pulmonary, Critical Care, and Sleep Medicine, New York University School of Medicine, New York, NY, USA.

Aspiration is associated with nontuberculous mycobacterial (NTM) pulmonary disease and airway dysbiosis is associated with increased inflammation. We examined whether NTM disease was associated with a distinct airway microbiota and immune profile.297 oral wash and induced sputum samples were collected from 106 participants with respiratory symptoms and imaging abnormalities compatible with NTM. Lower airway samples were obtained in 20 participants undergoing bronchoscopy. 16S rRNA gene and nested mycobacteriome sequencing approaches characterised microbiota composition. In addition, inflammatory profiles of lower airway samples were examined.The prevalence of NTM cultures was 58%. Few changes were noted in microbiota characteristics or composition in oral wash and sputum samples among groups. Among NTM samples, 27% of the lower airway samples were enriched with A mycobacteriome approach identified in a greater percentage of samples, including some nonpathogenic strains. In NTM lower airway samples, taxa identified as oral commensals were associated with increased inflammatory biomarkers.The 16S rRNA gene sequencing approach is not sensitive in identifying NTM among airway samples that are culture-positive. However, associations between lower airway inflammation and microbiota signatures suggest a potential role for these microbes in the inflammatory process in NTM disease.
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http://dx.doi.org/10.1183/13993003.00810-2018DOI Listing
October 2018

Direct genotyping of Mycobacterium tuberculosis from Xpert MTB/RIF remnants.

Tuberculosis (Edinb) 2018 07 15;111:202-206. Epub 2018 May 15.

Servicio Microbiología Clínica y Enfermedades Infecciosas, Hospital General Universitario Gregorio Marañón, Madrid, Spain; Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain; CIBER Enfermedades respiratorias, CIBERES, Spain. Electronic address:

Genotyping of Mycobacterium tuberculosis (MTB) isolates has markedly improved our knowledge of its transmission dynamics. MIRU-VNTR is considered the reference molecular tool for MTB fingerprinting. However, the dependence of this technique on cultured isolates means that we lack molecular epidemiology data from many settings where culture facilities have not been implemented. Efforts have been made to adapt the MIRU-VNTR procedure to direct analysis of clinical specimens, although implementation of these efforts has not proven successful. The large-scale roll-out of Xpert MTB/RIF (Xpert) technology, which is now in almost every TB-endemic country, including many where MTB is not cultured, provides us with a new opportunity to explore whether MTB genotyping could be performed from the remnants of the Xpert cartridge. We ran a pilot study in Mozambique in which the remnants of 24 positive Xpert assays for detection of MTB were used as template material for the 15-locus or the more discriminatory 24-locus MIRU-VNTR technique. MTB fingerprinting was possible in specimens with a high bacterial burden, according to the Xpert load categories, and within the first week after Xpert was performed. Given the wide availability, simple processing, and rapid reporting of results with Xpert, our findings suggest that MIRU-VNTR-based fingerprinting from remnants of Xpert could play a major role in extending MTB molecular epidemiology studies to settings where information on the transmission dynamics of this pathogen is lacking.
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http://dx.doi.org/10.1016/j.tube.2018.05.008DOI Listing
July 2018

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

Correlation of Xpert MTB/RIF with measures to assess Mycobacterium tuberculosis bacillary burden in high HIV burden areas of Southern Africa.

Sci Rep 2018 03 26;8(1):5201. Epub 2018 Mar 26.

ISGlobal, Hospital Clínic - Universitat de Barcelona, Barcelona, Spain.

Traditionally, smear microscopy has been used as a point-of-care measure of bacillary burden in tuberculosis patients to inform infection control and contact tracing. Xpert MTB/RIF has the potential to replace smear. However, data to support the use of its quantitative output [cycle threshold (C)] as an alternate point-of-care measure of bacillary burden are limited. This study assessed the correlation (Spearman's) between C, smear, culture time-to-positivity (TTP), and clinical factors in patients with Xpert-positive sputum from Mozambique (n = 238) and South Africa (n = 462). Mean CT and smear grade correlated well (ρ0.72); compared to TTP and smear (ρ0.61); and mean C and TTP (ρ0.50). In multivariate analyses, lower C (higher bacillary load) was associated with negative HIV serostatus and low BMI. A smear positivity rule-out (95% sensitivity) C cut-off of 28.0 was identified, with 54.1% specificity, 2.07 positive likelihood ratio, 0.09 negative likelihood ratio and 79.0% correctly classified. Cut-offs were higher for HIV positive compared to HIV negative individuals for any set sensitivity level. This study suggests Xpert C values correlate well with smear, both in HIV positive and negative individuals, and that C cut-offs might be broadly applicable to multiple settings. Studies to directly assess the association of C with infectiousness are needed.
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http://dx.doi.org/10.1038/s41598-018-23066-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5980110PMC
March 2018

Of Testing and Treatment: Implications of Implementing New Regimens for Multidrug-Resistant Tuberculosis.

Clin Infect Dis 2017 10;65(7):1206-1211

Division of Infectious Diseases, Johns Hopkins University School of Medicine, Baltimore, Maryland.

A novel, shorter-course regimen for treating multidrug-resistant (MDR) tuberculosis was recently recommended by the World Health Organization. However, the most appropriate use of drug susceptibility testing (DST) to support this regimen is less clear. Implementing countries must therefore often choose between using a standardized regimen despite high levels of underlying drug resistance or require more stringent DST prior to treatment initiation. The former carries a high likelihood of exposing patients to de facto monotherapy with a critical drug class (fluoroquinolones), whereas the latter could exclude large groups of patients from their most effective treatment option. We discuss the implications of this dilemma and argue for an approach that will integrate DST into the delivery of any novel antimicrobial regimen, without excessively stringent requirements. Such guidance could make the novel MDR tuberculosis regimen available to most patients while reducing the risk of generating additional drug resistance.
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http://dx.doi.org/10.1093/cid/cix486DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5850434PMC
October 2017