In HIV hyperendemic sub-Saharan African communities, particularly in southern Africa, the likelih... more In HIV hyperendemic sub-Saharan African communities, particularly in southern Africa, the likelihood of achieving the Sustainable Development Goal of ending the tuberculosis (TB) epidemic by 2030 is low, due to lack of cost-effective and practical interventions in population settings. We used one of Africa's largest population-based prospective cohorts from rural KwaZulu-Natal Province, South Africa, to measure the spatial variations in the prevalence of recently-diagnosed TB disease, and to quantify the impact of community coverage of antiretroviral therapy (ART) on recently-diagnosed TB disease. We collected data on TB disease episodes from a population-based sample of 41,812 adult individuals between 2009 and 2015. Spatial clusters ('hotspots') of recently-diagnosed TB were identified using a space-time scan statistic. Multilevel logistic regression models were fitted to investigate the relationship between community ART coverage and recently-diagnosed TB. Spatial clusters of recentlydiagnosed TB were identified in a region characterized by a high prevalence of HIV and population movement. Every percentage increase in ART coverage was associated with a 2% decrease in the odds of recently-diagnosed tB (aoR = 0.98, 95% CI:0.97-0.99). We identified for the first time the clear occurrence of recently-diagnosed TB hotspots, and quantified potential benefit of increased community ART coverage in lowering tuberculosis, highlighting the need to prioritize the expansion of such effective population interventions targeting high-risk areas. Tuberculosis (TB) is the leading cause of death from infectious disease in the world 1 , with epidemics being spatially heterogeneous, as indicated by evidence of geographic clustering at different resolution levels 2,3. Given the lack of consistent evidence for scalable community-wide approaches to control TB 4 , particularly in resource-limited regions grappling with the realities of an unrelenting HIV endemic, there is considerable interest in developing sustainable, targeted interventions in a precision public health approach 5. TB accounts for the largest percentage of total mortality in 2016 (6.5%) in South Africa 6 and is the fifth leading cause of years of life lost 7 and disability-adjusted life-years in the country 8 , according to the Global Burden of Disease Study 2015. In sub-Saharan Africa (SSA), particularly southern Africa, as HIV is the major driver of the TB epidemic 9-11 , this also raises questions around how geographically targeted HIV interventions could influence TB epidemiology.
Tuberculosis (TB) remains a serious threat in underdeveloped areas. Mycobacterium tuberculosis cu... more Tuberculosis (TB) remains a serious threat in underdeveloped areas. Mycobacterium tuberculosis curli pili (MTP), a virulence factor, is a potential biomarker for a reliable point of care (POC) test and was evaluated for its ability to react with Immunoglobulin G (IgG) in TB patients. An MTP synthetic peptide in a slot blot assay was used to screen serum/plasma samples (n= 65) in 3 separate cohorts, including 40 TB positive (16 HIV co-infected), 20 TB negative/HIV negative patients and 5 healthy volunteers. Forty samples were true positives (HIV positive, n = 16), 23 true negatives (HIV negative) and 2 false positives (HIV negative). The McNemar test demonstrated a 3.08% accuracy estimate (CI: −2.1%-3.08%). This confirms that MTP is expressed during infection, including HIV-TB co-infection, is likely to be suitable for the design of a POC test and supports the validation of MTP for TB detection in larger patient populations.
2019. Erratum for O'Donnell et al., "Early detection of emergent extensively drugresistant tuberc... more 2019. Erratum for O'Donnell et al., "Early detection of emergent extensively drugresistant tuberculosis by flow cytometry-based phenotyping and whole-genome sequencing." Antimicrob Agents Chemother 63:e00764-19.
Effect of Xpert MTB/RIF on clinical outcomes in routine care settings: individual patient data me... more Effect of Xpert MTB/RIF on clinical outcomes in routine care settings: individual patient data meta-analysis. LANCET GLOBAL HEALTH, 7 (2). e191-e199.
Background A critical gap in tuberculosis (TB) treatment is detection of emergent drug resistance... more Background A critical gap in tuberculosis (TB) treatment is detection of emergent drug resistance. We hypothesized that advanced phenotyping with whole genome sequencing (WGS) will detect low frequency Mycobacterium tuberculosis (MTB) drug-resistance. Methods We assessed a reporter mycobacteriophage (φ2GFP10) in vitro to detect drug-resistant subpopulations and predict MTB bactericidal activity in this pilot study. Subsequently, we prospectively studied 20 TB patients with serial φ2GFP10, Xpert MTB/RIF, and MTB culture through end of treatment. WGS was performed and single nucleotide polymorphisms (SNPs) were examined to detect mixed infection in selected MTB isolates. Results Resistant MTB were detected at 1:100,000 and changes in cytometry gated events were predictive of in vitro MTB bactericidal activity using the φ2GFP10 assay. Emergent drug-resistance was detected in one patient by φ2GFP10 at three weeks but not by conventional testing (MTB culture and GeneXpert). WGS revealed ...
Chest radiographs are used for diagnosis and severity assessment in tuberculosis (TB). The extent... more Chest radiographs are used for diagnosis and severity assessment in tuberculosis (TB). The extent of disease as determined by smear grade and cavitation as a binary measure can predict 2-month smear results, but little has been done to determine whether radiological severity reflects the bacterial burden at diagnosis. Pre-treatment chest x-rays from 1837 participants with smear-positive pulmonary TB enrolled into the REMoxTB trial (Gillespie et al., N Engl J Med 371:1577-87, 2014) were retrospectively reviewed. Two clinicians blinded to clinical details using the Ralph scoring system performed separate readings. An independent reader reviewed discrepant results for quality assessment and cavity presence. Cavitation presence was plotted against time to positivity (TTP) of sputum liquid cultures (MGIT 960). The Wilcoxon rank sum test was performed to calculate the difference in average TTP for these groups. The average lung field affected was compared to log TTP by linear regression. ...
The World Health Organization recommends drug-susceptibility testing of Mycobacterium tuberculosi... more The World Health Organization recommends drug-susceptibility testing of Mycobacterium tuberculosis complex for all patients with tuberculosis to guide treatment decisions and improve outcomes. Whether DNA sequencing can be used to accurately predict profiles of susceptibility to first-line antituberculosis drugs has not been clear. We obtained whole-genome sequences and associated phenotypes of resistance or susceptibility to the first-line antituberculosis drugs isoniazid, rifampin, ethambutol, and pyrazinamide for isolates from 16 countries across six continents. For each isolate, mutations associated with drug resistance and drug susceptibility were identified across nine genes, and individual phenotypes were predicted unless mutations of unknown association were also present. To identify how whole-genome sequencing might direct first-line drug therapy, complete susceptibility profiles were predicted. These profiles were predicted to be susceptible to all four drugs (i.e., pansus...
The use of early morning sputum samples (EMS) to diagnose tuberculosis (TB) can result in treatme... more The use of early morning sputum samples (EMS) to diagnose tuberculosis (TB) can result in treatment delay given the need for the patient to return to the clinic with the EMS, increasing the chance of patients being lost during their diagnostic workup. However, there is little evidence to support the superiority of EMS over spot sputum samples. In this new analysis of the REMoxTB study, we compare the diagnostic accuracy of EMS with spot samples for identifying Mycobacterium tuberculosis pre- and post-treatment. Patients who were smear positive at screening were enrolled into the study. Paired sputum samples (one EMS and one spot) were collected at each trial visit pre- and post-treatment. Microscopy and culture on solid LJ and liquid MGIT media were performed on all samples; those missing corresponding paired results were excluded from the analyses. Data from 1115 pre- and 2995 post-treatment paired samples from 1931 patients enrolled in the REMoxTB study were analysed. Patients wer...
Tuberculosis chemotherapy is dependent on the use of the antibiotic pyrazinamide, which is being ... more Tuberculosis chemotherapy is dependent on the use of the antibiotic pyrazinamide, which is being threatened by emerging drug resistance. Resistance is mediated through mutations in the bacterial gene pncA. Methods for testing pyrazinamide susceptibility are difficult and rarely performed, and this means that the full spectrum of pncA alleles that confer clinical resistance to pyrazinamide is unknown. Here, we performed in vitro saturating mutagenesis of pncA to generate a comprehensive library of PncA polymorphisms resultant from a single-nucleotide polymorphism. We then screened it for pyrazinamide resistance both in vitro and in an infected animal model. We identify over 300 resistance-conferring substitutions. Strikingly, these mutations map throughout the PncA structure and result in either loss of enzymatic activity and/or decrease in protein abundance. Our comprehensive mutational and screening approach should stand as a paradigm for determining resistance mutations and their ...
Multidrug-resistant tuberculosis (MDR-TB), caused by drug-resistant strains of Mycobacterium tube... more Multidrug-resistant tuberculosis (MDR-TB), caused by drug-resistant strains of Mycobacterium tuberculosis, is an increasingly serious problem worldwide. Here we examined a data set of whole-genome sequences from 5,310 M. tuberculosis isolates from five continents. Despite the great diversity of these isolates with respect to geographical point of isolation, genetic background and drug resistance, the patterns for the emergence of drug resistance were conserved globally. We have identified harbinger mutations that often precede multidrug resistance. In particular, the katG mutation encoding p.Ser315Thr, which confers resistance to isoniazid, overwhelmingly arose before mutations that conferred rifampicin resistance across all of the lineages, geographical regions and time periods. Therefore, molecular diagnostics that include markers for rifampicin resistance alone will be insufficient to identify pre-MDR strains. Incorporating knowledge of polymorphisms that occur before the emergen...
A hallmark of pulmonary tuberculosis is the formation of macrophage-rich granulomas. These may re... more A hallmark of pulmonary tuberculosis is the formation of macrophage-rich granulomas. These may restrict Mycobacterium tuberculosis (Mtb) growth, or progress to central necrosis and cavitation, facilitating pathogen growth. To determine factors leading to Mtb proliferation and host cell death, we used live cell imaging to track Mtb infection outcomes in individual primary human macrophages. Internalization of Mtb aggregates caused macrophage death, and phagocytosis of large aggregates was more cytotoxic than multiple small aggregates containing similar numbers of bacilli. Macrophage death did not result in clearance of Mtb. Rather, it led to accelerated intracellular Mtb growth regardless of prior activation or macrophage type. In contrast, bacillary replication was controlled in live phagocytes. Mtb grew as a clump in dead cells, and macrophages which internalized dead infected cells were very likely to die themselves, leading to a cell death cascade. This demonstrates how pathogen ...
Persisters are the minor subpopulation of bacterial cells that lack alleles conferring resistance... more Persisters are the minor subpopulation of bacterial cells that lack alleles conferring resistance to a specific bactericidal antibiotic but can survive otherwise lethal concentrations of that antibiotic. In infections with Mycobacterium tuberculosis , such persisters underlie the need for long-term antibiotic therapy and contribute to treatment failure in tuberculosis cases. Here, we demonstrate the value of d ual- r eporter m ycobacteriophages (Φ 2 DRMs) for characterizing M. tuberculosis persisters. The addition of isoniazid (INH) to exponentially growing M. tuberculosis cells consistently resulted in a 2- to 3-log decrease in CFU within 4 days, and the remaining ≤1% of cells, which survived despite being INH sensitive, were INH-tolerant persisters with a distinct transcriptional profile. We fused the promoters of several genes upregulated in persisters to the red fluorescent protein tdTomato gene in Φ 2 GFP10, a mycobacteriophage constitutively expressing green fluorescent protei...
Background KwaZulu-Natal (KZN) has the highest burden of notified multidrug-resistant tuberculosi... more Background KwaZulu-Natal (KZN) has the highest burden of notified multidrug-resistant tuberculosis (MDR TB) and extensively drug-resistant (XDR) TB cases in South Africa. A better understanding of spatial heterogeneity in the risk of drug-resistance may help to prioritize local responses. Methods Between July 2012 and June 2013, we conducted a two-way Lot Quality Assurance Sampling (LQAS) study to classify the burden of rifampicin (RIF)-resistant TB among incident TB cases notified within the catchment areas of seven laboratories in two northern and one southern district of KZN. Decision rules for classification of areas as having either a high-or low-risk of RIF resistant TB (based on proportion of RIF resistance among all TB cases) were based on consultation with local policy makers. Results We classified five areas as high-risk and two as low-risk. High-risk areas were identified in both Southern and Northern districts, with the greatest proportion of RIF resistance observed in the northernmost area, the Manguzi community situated on the Mozambique border. Conclusion Our study revealed heterogeneity in the risk of RIF resistant disease among incident TB cases in KZN. This study demonstrates the potential for LQAS to detect geographic heterogeneity in areas where access to drug susceptibility testing is limited.
A more complete understanding of the genetic basis of drug resistance in Mycobacterium tuberculos... more A more complete understanding of the genetic basis of drug resistance in Mycobacterium tuberculosis is critical for prompt diagnosis and optimal treatment, particularly for toxic secondline drugs like D-cycloserine. Here, we used whole-genome sequences from 498 strains of M. tuberculosis to identify novel resistance-conferring genotypes. By combining association and Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
Many hospital inpatients in South Africa have undiagnosed active and drug-resistant tuberculosis ... more Many hospital inpatients in South Africa have undiagnosed active and drug-resistant tuberculosis (TB). Early detection of TB is essential to inform immediate infection control actions to minimize transmission risk. We assessed the utility of Xpert ® MTB/RIF (GeneXpert) as a screening tool for medical admissions at a large public hospital in South Africa. Consecutive adult patients admitted to medical wards between March-June 2013 were enrolled; sputum specimens were collected and tested by GeneXpert, smear microscopy, and culture. Chest X-rays (CXRs) were conducted as standard care for all patients admitted. We evaluated the proportion of patients identified with TB disease through each diagnostic method. Among enrolled patients whose medical charts were available for review postdischarge, 61 (27%) were diagnosed with TB; 34 (56% of diagnosed TB cases) were GeneXpert positive. When patients in whom TB was identified by other means were excluded, GeneXpert yielded only four additional TB cases. However, GeneXpert identified rifampicin-resistant TB in one patient, who was initially diagnosed based on CXR. The utility of GeneXpert for TB screening was limited in an institution where CXR is conducted routinely and which serves a population in which TB and TB/HIV co-infection are highly prevalent, but it allowed for rapid detection of rifampicin resistance. South Africa's tuberculosis (TB) burden is among the highest in the world 1 , and it is the leading cause of death for South Africans between the ages of 15 and 64 2. Approximately 61% of TB cases are co-infected with HIV 1 and in 2014 there were close to 19,000 cases of laboratory-confirmed multidrug-resistant (MDR) TB 1. The burden of TB and MDR TB among medical inpatients in South Africa is particularly high 3,4 , and recent evidence suggests that many hospital patients have unsuspected TB 5. This high prevalence, coupled with the open-ward design of South Africa's public hospitals, creates conditions favouring nosocomial infection among inpatients and hospital staff 4,6-11 , representing an urgent need to improve screening of patients for TB and MDR TB upon admission. Timely case identification and initiation of appropriate treatment are critical for TB control 12,13 but diagnostic tools have been inadequate. Sputum smear microscopy is insensitive, particularly among HIV-infected individuals 14 , and cannot differentiate between drug-susceptible and drug-resistant strains of Mycobacterium tuberculosis (MTB). Sputum culture, the gold standard for detecting MTB, takes weeks to months to yield results, and relies on sophisticated laboratory facilities and skilled technicians. Xpert ® MTB/RIF (GeneXpert) is a newly-developed World Health Organization (WHO)-endorsed nucleic acid amplification test which detects both MTB and rifampicin (RIF)-resistance-a surrogate marker for MDR TB-and produces results in approximately two hours 15. The cartridge-based system requires minimal training and biosafety measures, and can be implemented closer to the point of care than other tests 16,17. Clinical validation trials 17-19 and operational studies 20-23 have demonstrated high sensitivity and specificity of the test for both
In HIV hyperendemic sub-Saharan African communities, particularly in southern Africa, the likelih... more In HIV hyperendemic sub-Saharan African communities, particularly in southern Africa, the likelihood of achieving the Sustainable Development Goal of ending the tuberculosis (TB) epidemic by 2030 is low, due to lack of cost-effective and practical interventions in population settings. We used one of Africa's largest population-based prospective cohorts from rural KwaZulu-Natal Province, South Africa, to measure the spatial variations in the prevalence of recently-diagnosed TB disease, and to quantify the impact of community coverage of antiretroviral therapy (ART) on recently-diagnosed TB disease. We collected data on TB disease episodes from a population-based sample of 41,812 adult individuals between 2009 and 2015. Spatial clusters ('hotspots') of recently-diagnosed TB were identified using a space-time scan statistic. Multilevel logistic regression models were fitted to investigate the relationship between community ART coverage and recently-diagnosed TB. Spatial clusters of recentlydiagnosed TB were identified in a region characterized by a high prevalence of HIV and population movement. Every percentage increase in ART coverage was associated with a 2% decrease in the odds of recently-diagnosed tB (aoR = 0.98, 95% CI:0.97-0.99). We identified for the first time the clear occurrence of recently-diagnosed TB hotspots, and quantified potential benefit of increased community ART coverage in lowering tuberculosis, highlighting the need to prioritize the expansion of such effective population interventions targeting high-risk areas. Tuberculosis (TB) is the leading cause of death from infectious disease in the world 1 , with epidemics being spatially heterogeneous, as indicated by evidence of geographic clustering at different resolution levels 2,3. Given the lack of consistent evidence for scalable community-wide approaches to control TB 4 , particularly in resource-limited regions grappling with the realities of an unrelenting HIV endemic, there is considerable interest in developing sustainable, targeted interventions in a precision public health approach 5. TB accounts for the largest percentage of total mortality in 2016 (6.5%) in South Africa 6 and is the fifth leading cause of years of life lost 7 and disability-adjusted life-years in the country 8 , according to the Global Burden of Disease Study 2015. In sub-Saharan Africa (SSA), particularly southern Africa, as HIV is the major driver of the TB epidemic 9-11 , this also raises questions around how geographically targeted HIV interventions could influence TB epidemiology.
Tuberculosis (TB) remains a serious threat in underdeveloped areas. Mycobacterium tuberculosis cu... more Tuberculosis (TB) remains a serious threat in underdeveloped areas. Mycobacterium tuberculosis curli pili (MTP), a virulence factor, is a potential biomarker for a reliable point of care (POC) test and was evaluated for its ability to react with Immunoglobulin G (IgG) in TB patients. An MTP synthetic peptide in a slot blot assay was used to screen serum/plasma samples (n= 65) in 3 separate cohorts, including 40 TB positive (16 HIV co-infected), 20 TB negative/HIV negative patients and 5 healthy volunteers. Forty samples were true positives (HIV positive, n = 16), 23 true negatives (HIV negative) and 2 false positives (HIV negative). The McNemar test demonstrated a 3.08% accuracy estimate (CI: −2.1%-3.08%). This confirms that MTP is expressed during infection, including HIV-TB co-infection, is likely to be suitable for the design of a POC test and supports the validation of MTP for TB detection in larger patient populations.
2019. Erratum for O'Donnell et al., "Early detection of emergent extensively drugresistant tuberc... more 2019. Erratum for O'Donnell et al., "Early detection of emergent extensively drugresistant tuberculosis by flow cytometry-based phenotyping and whole-genome sequencing." Antimicrob Agents Chemother 63:e00764-19.
Effect of Xpert MTB/RIF on clinical outcomes in routine care settings: individual patient data me... more Effect of Xpert MTB/RIF on clinical outcomes in routine care settings: individual patient data meta-analysis. LANCET GLOBAL HEALTH, 7 (2). e191-e199.
Background A critical gap in tuberculosis (TB) treatment is detection of emergent drug resistance... more Background A critical gap in tuberculosis (TB) treatment is detection of emergent drug resistance. We hypothesized that advanced phenotyping with whole genome sequencing (WGS) will detect low frequency Mycobacterium tuberculosis (MTB) drug-resistance. Methods We assessed a reporter mycobacteriophage (φ2GFP10) in vitro to detect drug-resistant subpopulations and predict MTB bactericidal activity in this pilot study. Subsequently, we prospectively studied 20 TB patients with serial φ2GFP10, Xpert MTB/RIF, and MTB culture through end of treatment. WGS was performed and single nucleotide polymorphisms (SNPs) were examined to detect mixed infection in selected MTB isolates. Results Resistant MTB were detected at 1:100,000 and changes in cytometry gated events were predictive of in vitro MTB bactericidal activity using the φ2GFP10 assay. Emergent drug-resistance was detected in one patient by φ2GFP10 at three weeks but not by conventional testing (MTB culture and GeneXpert). WGS revealed ...
Chest radiographs are used for diagnosis and severity assessment in tuberculosis (TB). The extent... more Chest radiographs are used for diagnosis and severity assessment in tuberculosis (TB). The extent of disease as determined by smear grade and cavitation as a binary measure can predict 2-month smear results, but little has been done to determine whether radiological severity reflects the bacterial burden at diagnosis. Pre-treatment chest x-rays from 1837 participants with smear-positive pulmonary TB enrolled into the REMoxTB trial (Gillespie et al., N Engl J Med 371:1577-87, 2014) were retrospectively reviewed. Two clinicians blinded to clinical details using the Ralph scoring system performed separate readings. An independent reader reviewed discrepant results for quality assessment and cavity presence. Cavitation presence was plotted against time to positivity (TTP) of sputum liquid cultures (MGIT 960). The Wilcoxon rank sum test was performed to calculate the difference in average TTP for these groups. The average lung field affected was compared to log TTP by linear regression. ...
The World Health Organization recommends drug-susceptibility testing of Mycobacterium tuberculosi... more The World Health Organization recommends drug-susceptibility testing of Mycobacterium tuberculosis complex for all patients with tuberculosis to guide treatment decisions and improve outcomes. Whether DNA sequencing can be used to accurately predict profiles of susceptibility to first-line antituberculosis drugs has not been clear. We obtained whole-genome sequences and associated phenotypes of resistance or susceptibility to the first-line antituberculosis drugs isoniazid, rifampin, ethambutol, and pyrazinamide for isolates from 16 countries across six continents. For each isolate, mutations associated with drug resistance and drug susceptibility were identified across nine genes, and individual phenotypes were predicted unless mutations of unknown association were also present. To identify how whole-genome sequencing might direct first-line drug therapy, complete susceptibility profiles were predicted. These profiles were predicted to be susceptible to all four drugs (i.e., pansus...
The use of early morning sputum samples (EMS) to diagnose tuberculosis (TB) can result in treatme... more The use of early morning sputum samples (EMS) to diagnose tuberculosis (TB) can result in treatment delay given the need for the patient to return to the clinic with the EMS, increasing the chance of patients being lost during their diagnostic workup. However, there is little evidence to support the superiority of EMS over spot sputum samples. In this new analysis of the REMoxTB study, we compare the diagnostic accuracy of EMS with spot samples for identifying Mycobacterium tuberculosis pre- and post-treatment. Patients who were smear positive at screening were enrolled into the study. Paired sputum samples (one EMS and one spot) were collected at each trial visit pre- and post-treatment. Microscopy and culture on solid LJ and liquid MGIT media were performed on all samples; those missing corresponding paired results were excluded from the analyses. Data from 1115 pre- and 2995 post-treatment paired samples from 1931 patients enrolled in the REMoxTB study were analysed. Patients wer...
Tuberculosis chemotherapy is dependent on the use of the antibiotic pyrazinamide, which is being ... more Tuberculosis chemotherapy is dependent on the use of the antibiotic pyrazinamide, which is being threatened by emerging drug resistance. Resistance is mediated through mutations in the bacterial gene pncA. Methods for testing pyrazinamide susceptibility are difficult and rarely performed, and this means that the full spectrum of pncA alleles that confer clinical resistance to pyrazinamide is unknown. Here, we performed in vitro saturating mutagenesis of pncA to generate a comprehensive library of PncA polymorphisms resultant from a single-nucleotide polymorphism. We then screened it for pyrazinamide resistance both in vitro and in an infected animal model. We identify over 300 resistance-conferring substitutions. Strikingly, these mutations map throughout the PncA structure and result in either loss of enzymatic activity and/or decrease in protein abundance. Our comprehensive mutational and screening approach should stand as a paradigm for determining resistance mutations and their ...
Multidrug-resistant tuberculosis (MDR-TB), caused by drug-resistant strains of Mycobacterium tube... more Multidrug-resistant tuberculosis (MDR-TB), caused by drug-resistant strains of Mycobacterium tuberculosis, is an increasingly serious problem worldwide. Here we examined a data set of whole-genome sequences from 5,310 M. tuberculosis isolates from five continents. Despite the great diversity of these isolates with respect to geographical point of isolation, genetic background and drug resistance, the patterns for the emergence of drug resistance were conserved globally. We have identified harbinger mutations that often precede multidrug resistance. In particular, the katG mutation encoding p.Ser315Thr, which confers resistance to isoniazid, overwhelmingly arose before mutations that conferred rifampicin resistance across all of the lineages, geographical regions and time periods. Therefore, molecular diagnostics that include markers for rifampicin resistance alone will be insufficient to identify pre-MDR strains. Incorporating knowledge of polymorphisms that occur before the emergen...
A hallmark of pulmonary tuberculosis is the formation of macrophage-rich granulomas. These may re... more A hallmark of pulmonary tuberculosis is the formation of macrophage-rich granulomas. These may restrict Mycobacterium tuberculosis (Mtb) growth, or progress to central necrosis and cavitation, facilitating pathogen growth. To determine factors leading to Mtb proliferation and host cell death, we used live cell imaging to track Mtb infection outcomes in individual primary human macrophages. Internalization of Mtb aggregates caused macrophage death, and phagocytosis of large aggregates was more cytotoxic than multiple small aggregates containing similar numbers of bacilli. Macrophage death did not result in clearance of Mtb. Rather, it led to accelerated intracellular Mtb growth regardless of prior activation or macrophage type. In contrast, bacillary replication was controlled in live phagocytes. Mtb grew as a clump in dead cells, and macrophages which internalized dead infected cells were very likely to die themselves, leading to a cell death cascade. This demonstrates how pathogen ...
Persisters are the minor subpopulation of bacterial cells that lack alleles conferring resistance... more Persisters are the minor subpopulation of bacterial cells that lack alleles conferring resistance to a specific bactericidal antibiotic but can survive otherwise lethal concentrations of that antibiotic. In infections with Mycobacterium tuberculosis , such persisters underlie the need for long-term antibiotic therapy and contribute to treatment failure in tuberculosis cases. Here, we demonstrate the value of d ual- r eporter m ycobacteriophages (Φ 2 DRMs) for characterizing M. tuberculosis persisters. The addition of isoniazid (INH) to exponentially growing M. tuberculosis cells consistently resulted in a 2- to 3-log decrease in CFU within 4 days, and the remaining ≤1% of cells, which survived despite being INH sensitive, were INH-tolerant persisters with a distinct transcriptional profile. We fused the promoters of several genes upregulated in persisters to the red fluorescent protein tdTomato gene in Φ 2 GFP10, a mycobacteriophage constitutively expressing green fluorescent protei...
Background KwaZulu-Natal (KZN) has the highest burden of notified multidrug-resistant tuberculosi... more Background KwaZulu-Natal (KZN) has the highest burden of notified multidrug-resistant tuberculosis (MDR TB) and extensively drug-resistant (XDR) TB cases in South Africa. A better understanding of spatial heterogeneity in the risk of drug-resistance may help to prioritize local responses. Methods Between July 2012 and June 2013, we conducted a two-way Lot Quality Assurance Sampling (LQAS) study to classify the burden of rifampicin (RIF)-resistant TB among incident TB cases notified within the catchment areas of seven laboratories in two northern and one southern district of KZN. Decision rules for classification of areas as having either a high-or low-risk of RIF resistant TB (based on proportion of RIF resistance among all TB cases) were based on consultation with local policy makers. Results We classified five areas as high-risk and two as low-risk. High-risk areas were identified in both Southern and Northern districts, with the greatest proportion of RIF resistance observed in the northernmost area, the Manguzi community situated on the Mozambique border. Conclusion Our study revealed heterogeneity in the risk of RIF resistant disease among incident TB cases in KZN. This study demonstrates the potential for LQAS to detect geographic heterogeneity in areas where access to drug susceptibility testing is limited.
A more complete understanding of the genetic basis of drug resistance in Mycobacterium tuberculos... more A more complete understanding of the genetic basis of drug resistance in Mycobacterium tuberculosis is critical for prompt diagnosis and optimal treatment, particularly for toxic secondline drugs like D-cycloserine. Here, we used whole-genome sequences from 498 strains of M. tuberculosis to identify novel resistance-conferring genotypes. By combining association and Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
Many hospital inpatients in South Africa have undiagnosed active and drug-resistant tuberculosis ... more Many hospital inpatients in South Africa have undiagnosed active and drug-resistant tuberculosis (TB). Early detection of TB is essential to inform immediate infection control actions to minimize transmission risk. We assessed the utility of Xpert ® MTB/RIF (GeneXpert) as a screening tool for medical admissions at a large public hospital in South Africa. Consecutive adult patients admitted to medical wards between March-June 2013 were enrolled; sputum specimens were collected and tested by GeneXpert, smear microscopy, and culture. Chest X-rays (CXRs) were conducted as standard care for all patients admitted. We evaluated the proportion of patients identified with TB disease through each diagnostic method. Among enrolled patients whose medical charts were available for review postdischarge, 61 (27%) were diagnosed with TB; 34 (56% of diagnosed TB cases) were GeneXpert positive. When patients in whom TB was identified by other means were excluded, GeneXpert yielded only four additional TB cases. However, GeneXpert identified rifampicin-resistant TB in one patient, who was initially diagnosed based on CXR. The utility of GeneXpert for TB screening was limited in an institution where CXR is conducted routinely and which serves a population in which TB and TB/HIV co-infection are highly prevalent, but it allowed for rapid detection of rifampicin resistance. South Africa's tuberculosis (TB) burden is among the highest in the world 1 , and it is the leading cause of death for South Africans between the ages of 15 and 64 2. Approximately 61% of TB cases are co-infected with HIV 1 and in 2014 there were close to 19,000 cases of laboratory-confirmed multidrug-resistant (MDR) TB 1. The burden of TB and MDR TB among medical inpatients in South Africa is particularly high 3,4 , and recent evidence suggests that many hospital patients have unsuspected TB 5. This high prevalence, coupled with the open-ward design of South Africa's public hospitals, creates conditions favouring nosocomial infection among inpatients and hospital staff 4,6-11 , representing an urgent need to improve screening of patients for TB and MDR TB upon admission. Timely case identification and initiation of appropriate treatment are critical for TB control 12,13 but diagnostic tools have been inadequate. Sputum smear microscopy is insensitive, particularly among HIV-infected individuals 14 , and cannot differentiate between drug-susceptible and drug-resistant strains of Mycobacterium tuberculosis (MTB). Sputum culture, the gold standard for detecting MTB, takes weeks to months to yield results, and relies on sophisticated laboratory facilities and skilled technicians. Xpert ® MTB/RIF (GeneXpert) is a newly-developed World Health Organization (WHO)-endorsed nucleic acid amplification test which detects both MTB and rifampicin (RIF)-resistance-a surrogate marker for MDR TB-and produces results in approximately two hours 15. The cartridge-based system requires minimal training and biosafety measures, and can be implemented closer to the point of care than other tests 16,17. Clinical validation trials 17-19 and operational studies 20-23 have demonstrated high sensitivity and specificity of the test for both
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