'Those who cannot remember the past are condemned to repeat it': Drug-susceptibility testing for bedaquiline and delamanid.

Despite being fundamental to all treatment decisions, the breakpoints that define susceptibility and resistance to conventional anti-tuberculosis (TB) drugs were traditionally defined based on expert opinion as opposed to modern microbiological principles. As a result, the breakpoints for several key drugs (i.e. amikacin, levofloxacin, and moxifloxacin) were too high, resulting in the systematic misclassification of a proportion of resistant strains as susceptible. Moreover, a recent systematic review of clinical outcome data prompted the World Health Organization (WHO) to make significant changes to its treatment guidelines. For example, capreomycin and kanamycin are no longer recommended for TB treatment because their use correlates with worse clinical outcomes. This history notwithstanding, robust breakpoints still do not exist for bedaquiline and delamanid six years after their approval. This was compounded by the fact that access to both agents for drug-susceptibility testing had initially been restricted. It is incumbent upon the European Medicines Agency, the United States Food and Drug Administration, and WHO to ensure that drug developers generate the necessary data to set breakpoints as a prerequisite for the approval of new agents

Wild-Type and Non-Wild-Type Mycobacterium tuberculosis MIC Distributions for the Novel Fluoroquinolone Antofloxacin Compared with Those for Ofloxacin, Levofloxacin, and Moxifloxacin.

Antofloxacin (AFX) is a novel fluoroquinolone that has been approved in China for the treatment of infections caused by a variety of bacterial species. We investigated whether it could be repurposed for the treatment of tuberculosis by studying its in vitro activity. We determined the wild-type and non-wild-type MIC ranges for AFX as well as ofloxacin (OFX), levofloxacin (LFX), and moxifloxacin (MFX), using the microplate alamarBlue assay, of 126 clinical Mycobacterium tuberculosis strains from Beijing, China, of which 48 were OFX resistant on the basis of drug susceptibility testing on Löwenstein-Jensen medium. The MIC distributions were correlated with mutations in the quinolone resistance-determining regions of gyrA (Rv0006) and gyrB (Rv0005). Pharmacokinetic/pharmacodynamic (PK/PD) data for AFX were retrieved from the literature. AFX showed lower MIC levels than OFX but higher MIC levels than LFX and MFX on the basis of the tentative epidemiological cutoff values (ECOFFs) determined in this study. All strains with non-wild-type MICs for AFX harbored known resistance mutations that also resulted in non-wild-type MICs for LFX and MFX. Moreover, our data suggested that the current critical concentration of OFX for Löwenstein-Jensen medium that was recently revised by the World Health Organization might be too high, resulting in the misclassification of phenotypically non-wild-type strains with known resistance mutations as wild type. On the basis of our exploratory PK/PD calculations, the current dose of AFX is unlikely to be optimal for the treatment of tuberculosis, but higher doses could be effective.The work was supported by the research funding from Infectious Diseases Special Project, Minister of Health of China (2016ZX10003001-12) and Beijing Municipal Administration of Hospitals Clinical Medicine Development of Special Funding Support (ZYLX201304). The strains used in this project were obtained from the ‘Beijing Bio-Bank of clinical resources on Tuberculosis’ (D09050704640000), Beijing Chest Hospital. In addition, this study was supported by the Health Innovation Challenge Fund (HICF-T5-342 and WT098600), a parallel funding partnership between the UK Department of Health and Wellcome Trust. T. S. was supported by grants from the Swedish Heart and Lung Foundation and Marianne and Marcus Wallenberg Foundation. The views expressed in this publication are those of the authors and not necessarily those of the Department of Health, Public Health England, or the Wellcome Trust. C. U. K. is a Junior Research Fellow at Wolfson College, Cambridge.This is the author accepted manuscript. The final version is available from American Society for Microbiology at http://dx.doi.org/10.1128/AAC.00393-16

Drug-resistance mechanisms and tuberculosis drugs.

This publication presents independent research supported by the Health Innovation Challenge Fund (HICF-T5-342 and WT098600), a parallel funding partnership between the UK Department of Health and Wellcome Trust.This is the final version of the article. It first appeared at http://dx.doi.org/10.1016/S0140-6736(14)62450-8

Whole-genome sequencing of multidrug-resistant Mycobacterium tuberculosis isolates from Myanmar.

Drug-resistant tuberculosis (TB) is a major health threat in Myanmar. An initial study was conducted to explore the potential utility of whole-genome sequencing (WGS) for the diagnosis and management of drug-resistant TB in Myanmar. Fourteen multidrug-resistant Mycobacterium tuberculosis isolates were sequenced. Known resistance genes for a total of nine antibiotics commonly used in the treatment of drug-susceptible and multidrug-resistant TB (MDR-TB) in Myanmar were interrogated through WGS. All 14 isolates were MDR-TB, consistent with the results of phenotypic drug susceptibility testing (DST), and the Beijing lineage predominated. Based on the results of WGS, 9 of the 14 isolates were potentially resistant to at least one of the drugs used in the standard MDR-TB regimen but for which phenotypic DST is not conducted in Myanmar. This study highlights a need for the introduction of second-line DST as part of routine TB diagnosis in Myanmar as well as new classes of TB drugs to construct effective regimens.Professor Sandy Smith Memorial ScholarshipThis is the final version of the article. It first appeared from Elsevier via https://doi.org/10.1016/j.jgar.2016.04.00

Genome sequencing defines phylogeny and spread of methicillin-resistant Staphylococcus aureus in a high transmission setting.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of nosocomial infection. Whole-genome sequencing of MRSA has been used to define phylogeny and transmission in well-resourced healthcare settings, yet the greatest burden of nosocomial infection occurs in resource-restricted settings where barriers to transmission are lower. Here, we study the flux and genetic diversity of MRSA on ward and individual patient levels in a hospital where transmission was common. We repeatedly screened all patients on two intensive care units for MRSA carriage over a 3-mo period. All MRSA belonged to multilocus sequence type 239 (ST 239). We defined the population structure and charted the spread of MRSA by sequencing 79 isolates from 46 patients and five members of staff, including the first MRSA-positive screen isolates and up to two repeat isolates where available. Phylogenetic analysis identified a flux of distinct ST 239 clades over time in each intensive care unit. In total, five main clades were identified, which varied in the carriage of plasmids encoding antiseptic and antimicrobial resistance determinants. Sequence data confirmed intra- and interwards transmission events and identified individual patients who were colonized by more than one clade. One patient on each unit was the source of numerous transmission events, and deep sampling of one of these cases demonstrated colonization with a "cloud" of related MRSA variants. The application of whole-genome sequencing and analysis provides novel insights into the transmission of MRSA in under-resourced healthcare settings and has relevance to wider global health.The authors acknowledge financial support from the UKCRC Translational Infection Research (TIR) Initiative and the Medical Research Council (Grant number G1000803), with contributions to the grant from the Biotechnology and Biological Sciences Research Council, the National Institute for Health Research on behalf of the Department of Health, and the Chief Scientist Office of the Scottish Government Health Directorate (to Professor Peacock); from Wellcome Trust grant number 098051 awarded to the Wellcome Trust Sanger Institute; and the NIHR Cambridge Biomedical Research Centre (to Professor Peacock). S.Y.C.T. is an Australian National Health and Medical Research Council Career Development Fellow (1065736)This is the final version of the article. It first appeared at http://www.genome.org/cgi/doi/10.1101/gr.174730.114

Prevalence and genetic profiles of isoniazid resistance in tuberculosis patients: A multicountry analysis of cross-sectional data.

BACKGROUND: The surveillance of drug resistance among tuberculosis (TB) patients is central to combatting the global TB epidemic and preventing the spread of antimicrobial resistance. Isoniazid and rifampicin are two of the most powerful first-line anti-TB medicines, and resistance to either of them increases the risk of treatment failure, relapse, or acquisition of resistance to other drugs. The global prevalence of rifampicin resistance is well documented, occurring in 3.4% (95% CI 2.5%-4.4%) of new TB patients and 18% (95% CI 7.6%-31%) of previously treated TB patients in 2018, whereas the prevalence of isoniazid resistance at global and regional levels is less understood. In 2018, the World Health Organization (WHO) recommended a modified 6-month treatment regimen for people with isoniazid-resistant, rifampicin-susceptible TB (Hr-TB), which includes rifampicin, pyrazinamide, ethambutol, and levofloxacin. We estimated the global prevalence of Hr-TB among TB patients and investigated associated phenotypic and genotypic drug resistance patterns. METHODS AND FINDINGS: Aggregated drug resistance data reported to WHO from either routine continuous surveillance or nationally representative periodic surveys of TB patients for the period 2003-2017 were reviewed. Isoniazid data were available from 156 countries or territories for 211,753 patients. Among these, the global prevalence of Hr-TB was 7.4% (95% CI 6.5%-8.4%) among new TB patients and 11.4% (95% CI 9.4%-13.4%) among previously treated TB patients. Additional data on pyrazinamide and levofloxacin resistance were available from 6 countries (Azerbaijan, Bangladesh, Belarus, Pakistan, the Philippines, and South Africa). There were no cases of resistance to both pyrazinamide and levofloxacin among Hr-TB patients, except for the Philippines (1.8%, 95% CI 0.2-6.4) and Belarus (5.3%, 95% CI 0.1-26.0). Sequencing data for all genomic regions involved in isoniazid resistance were available for 4,563 patients. Among the 1,174 isolates that were resistant by either phenotypic testing or sequencing, 78.6% (95% CI 76.1%-80.9%) had resistance-conferring mutations in the katG gene and 14.6% (95% CI 12.7%-16.8%) in both katG and the inhA promoter region. For 6.8% (95% CI 5.4%-8.4%) of patients, mutations occurred in the inhA promoter alone, for whom an increased dose of isoniazid may be considered. The main limitations of this study are that most analyses were performed at the national rather than individual patient level and that the quality of laboratory testing may vary between countries. CONCLUSIONS: In this study, the prevalence of Hr-TB among TB patients was higher than the prevalence of rifampicin resistance globally. Many patients with Hr-TB would be missed by current diagnostic algorithms driven by rifampicin testing, highlighting the need for new rapid molecular technologies to ensure access to appropriate treatment and care. The low prevalence of resistance to pyrazinamide and fluoroquinolones among patients with Hr-TB provides further justification for the recommended modified treatment regimen

Genomic Diversity among Drug Sensitive and Multidrug Resistant Isolates of Mycobacterium tuberculosis with Identical DNA Fingerprints

complex (MTBC), the causative agent of tuberculosis (TB), is characterized by low sequence diversity making this bacterium one of the classical examples of a genetically monomorphic pathogen. Because of this limited DNA sequence variation, routine genotyping of clinical MTBC isolates for epidemiological purposes relies on highly discriminatory DNA fingerprinting methods based on mobile and repetitive genetic elements. According to the standard view, isolates exhibiting the same fingerprinting pattern are considered direct progeny of the same bacterial clone, and most likely reflect ongoing transmission or disease relapse within individual patients.We generated 23.9 million (K-1) and 33.0 million (K-2) paired 50 bp purity filtered reads corresponding to a mean coverage of 483.5 fold and 656.1 fold respectively. Compared with the laboratory strain H37Rv both Beijing isolates shared 1,209 SNPs. The two Beijing isolates differed by 130 SNPs and one large deletion. The susceptible isolate had 55 specific SNPs, while the MDR variant had 75 specific SNPs, including the five known resistance-conferring mutations. isolates exhibiting identical DNA fingerprinting patterns can harbour substantial genomic diversity. Because this heterogeneity is not captured by traditional genotyping of MTBC, some aspects of the transmission dynamics of tuberculosis could be missed or misinterpreted. Furthermore, a valid differentiation between disease relapse and exogenous reinfection might be impossible using standard genotyping tools if the overall diversity of circulating clones is limited. These findings have important implications for clinical trials of new anti-tuberculosis drugs

dfrA thyA Double Deletion in para-Aminosalicylic Acid-Resistant Mycobacterium tuberculosis Beijing Strains.

This is the final version of the article. It first appeared from the American Society for Microbiology via http://dx.doi.org/10.1128/AAC.00253-16Para-aminosalicylic acid (PAS) is a group 4 anti-tuberculosis agent (1). It targets folate metabolism as shown in Fig. S1, which also summarises the known resistance mechanisms to this pro-drug (2). Recently, we reported a multidrug-resistant (MDR) Mycobacterium tuberculosis Beijing strain harbouring a deletion of both dfrA and thyA from Australia (Fig. 1A and Table S1) (3). Since then, we have found deletions affecting both genes in five further MDR Beijing strains (two isolated in Australia and three from Peru) and one extensively drug-resistant (XDR) Beijing strain from China. The Australian MDR strains were recovered from three patients with no apparent epidemiological links and were likely infected in their country of origin (Table S1). The three Peruvian isolates were closely related and consequently shared the same deletion, whereas the remaining strains were distantly related and had deletions that differed in size (Fig. 1A). Consequently, these five distinct deletions were acquired independently, which can be a signal for positive selection of resistance mechanisms. In line with this hypothesis, the strains from Australia and China were PAS resistant when tested with the BACTEC MGIT 960 system and on L?wenstein-Jensen medium, respectively (Supplementary Methods). Two out of the three Peruvian deletion mutants were also PAS resistant on 7H10 medium at 8 ?g/mL, whereas the two closely related ancestral wild-type strains were susceptible (Fig. 1B). We were unable to retest the strains at 2 ?g/mL, the recommended critical concentration by the Clinical and Laboratory Standards Institute and World Health Organization, which would have clarified whether the susceptible result for the third deletion mutant was an artefact (1, 4).This publication presents independent research supported by the Health Innovation Challenge Fund (HICF-T5-342 and WT098600), a parallel funding partnership between the UK Department of Health and Wellcome Trust, and grant SRG2015-00006-FHS from the University of Macau. The views expressed in this publication are those of the authors and not necessarily those of the Department of Health, Public Health England, or the Wellcome Trust. M. E. T. is a Clinician Scientist Fellow funded by the Academy of Medical Sciences and the Health Foundation and the NIHR Cambridge Biomedical Research Centre. E.M. was supported by the Australian National Health and Medical Research Council?s Centre for Research Excellence in Tuberculosis. C. U. K. is a Junior Research Fellow at Wolfson College, Cambridge

Revised Interpretation of the Hain Lifescience GenoType MTBC To Differentiate Mycobacterium canettii and Members of the Mycobacterium tuberculosis Complex.

Using 894 phylogenetically diverse genomes of the Mycobacterium tuberculosis complex (MTBC), we simulated in silico the ability of the Hain Lifescience GenoType MTBC assay to differentiate the causative agents of tuberculosis. Here, we propose a revised interpretation of this assay to reflect its strengths (e.g., it can distinguish some strains of Mycobacterium canettii and variants of Mycobacterium bovis that are not intrinsically resistant to pyrazinamide) and limitations (e.g., Mycobacterium orygis cannot be differentiated from Mycobacterium africanum)