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Antibiotic tolerance in Mycobacterium tuberculosis reduces bacterial killing, worsens treatment outcomes, and contributes to resistance. We studied rifampicin tolerance in isolates with or without isoniazid resistance (IR). Using a minimum duration of killing assay, we measured rifampicin survival in isoniazid-susceptible (IS, n=119) and resistant (IR, n=84) isolates, correlating tolerance with bacterial growth, rifampicin minimum inhibitory concentrations (MICs), and isoniazid-resistant mutations. Longitudinal IR isolates were analyzed for changes in rifampicin tolerance and genetic variant emergence. The median time for rifampicin to reduce the bacterial population by 90% (MDK90) increased from 1.23 days (IS) and 1.31 days (IR) to 2.55 days (IS) and 1.98 days (IR) over 15–60 days of incubation, indicating fast and slow-growing tolerant sub-populations. A 6 log10-fold survival fraction classified tolerance as low, medium, or high, showing that IR is linked to increased tolerance and faster growth (OR = 2.68 for low vs. medium, OR = 4.42 for low vs. high, p-trend = 0.0003). High tolerance in IR isolates was associated with rifampicin treatment in patients and genetic microvariants. These findings suggest that IR tuberculosis should be assessed for high rifampicin tolerance to optimize treatment and prevent the development of multi-drug-resistant tuberculosis.

ABSTRACT A previous investigation has elucidated the landscape of Mtb genomic diversity and transmission dynamics in Ho Chi Minh City, Vietnam. Here, we expand the scope of this survey by adding a substantial number of additional genomes (total sample size: 2,542) and phenotypic drug susceptibility data for the majority of isolates. We aim to explore the prevalence and evolutionary dynamics of drug resistance and our ability to predict drug resistance from sequencing data. Among isolates tested phenotypically against first-line drugs, we observed high rates of streptomycin [STR, 37.7% (N = 573/1,520)] and isoniazid resistance [INH, 25.7% (N = 459/1,786)] and lower rates of resistance to rifampicin [RIF, 4.9% (N = 87/1,786)] and ethambutol [EMB, 4.2% (N = 75/1,785)]. Relative to global benchmarks, resistance to STR and INH was predicted accurately when applying the TB-Profiler algorithm to whole genome sequencing data (sensitivities of 0.81 and 0.87, respectively), while resistance to RIF and EMB was predicted relatively poorly (sensitivities of 0.70 and 0.44, respectively). Exploring the evolution of drug resistance revealed the main phylogenetic lineages to display differing dynamics and tendencies to evolve resistance via mutations in certain genes. The Beijing sublineage L2.2.1 was found to acquire de novo resistance mutations more frequently than isolates from other lineages and to suffer no apparent fitness cost acting to impede the transmission of resistance. Mutations conferring resistance to INH and STR arose earlier, on average, than those conferring resistance to RIF and are now more widespread across the phylogeny. The high prevalence of “background” INH resistance, combined with high rates of RIF mono-resistance (20.7%, N = 18/87), suggests that rapid assays for INH resistance will be valuable in this setting. These tests will allow the detection of INH mono-resistance and will allow multi-drug-resistant isolates to be distinguished from isolates with RIF mono-resistance. IMPORTANCE Drug-resistant tuberculosis (TB) infection is a growing and potent concern, and combating it will be necessary to achieve the WHO’s goal of a 95% reduction in TB deaths by 2035. While prior studies have explored the evolution and spread of drug resistance, we still lack a clear understanding of the fitness costs (if any) imposed by resistance-conferring mutations and the role that Mtb genetic lineage plays in determining the likelihood of resistance evolution. This study offers insight into these questions by assessing the dynamics of resistance evolution in a high-burden Southeast Asian setting with a diverse lineage composition. It demonstrates that there are clear lineage-specific differences in the dynamics of resistance acquisition and transmission and shows that different lineages evolve resistance via characteristic mutational pathways.

Bacterial populations in the in vitro laboratory cultures, environment, and patients contain metabolically different subpopulations that respond differently to stress agents, including antibiotics, and emerge as stress tolerant or resistant strains. To contain the emergence of such strains, it is important to study the features of the metabolic status and response of the subpopulations to stress agents. For this purpose, an efficient method is required for the fractionation and isolation of the subpopulations from the cultures. Here we describe in detail the manual setting up of a simple, easy-to-do, reproducibly robust Percoll discontinuous density gradient centrifugation for the fractionation of subpopulations of short-sized cells (SCs) and normal/long-sized cells (NCs) from Mycobacterium smegmatis and Mycobacterium tuberculosis cultures, which we had reported earlier. About 90-98% enrichment was obtained respectively for SCs and NCs for M. smegmatis and 69-67% enrichment was obtained respectively for the SCs and NCs for M. tuberculosis. • The Percoll discontinuous density gradient centrifugation helps the fractionation and isolation of mycobacterial subpopulations that differ in density. • The method offers a consistently reproducible high enrichment of the subpopulations of SCs and NCs from the in vitro cultures of M. smegmatis and M. tuberculosis. • Our earlier reports on the consistency in the differential response of the subpopulations, enriched using the method, to oxidative, nitrite, and antibiotic stress proves its validity.

Summary: Background: Molecular diagnostics are considered the most promising route to achievement of rapid, universal drug susceptibility testing for Mycobacterium tuberculosis complex (MTBC). We aimed to generate a WHO-endorsed catalogue of mutations to serve as a global standard for interpreting molecular information for drug resistance prediction. Methods: In this systematic analysis, we used a candidate gene approach to identify mutations associated with resistance or consistent with susceptibility for 13 WHO-endorsed antituberculosis drugs. We collected existing worldwide MTBC whole-genome sequencing data and phenotypic data from academic groups and consortia, reference laboratories, public health organisations, and published literature. We categorised phenotypes as follows: methods and critical concentrations currently endorsed by WHO (category 1); critical concentrations previously endorsed by WHO for those methods (category 2); methods or critical concentrations not currently endorsed by WHO (category 3). For each mutation, we used a contingency table of binary phenotypes and presence or absence of the mutation to compute positive predictive value, and we used Fisher's exact tests to generate odds ratios and Benjamini-Hochberg corrected p values. Mutations were graded as associated with resistance if present in at least five isolates, if the odds ratio was more than 1 with a statistically significant corrected p value, and if the lower bound of the 95% CI on the positive predictive value for phenotypic resistance was greater than 25%. A series of expert rules were applied for final confidence grading of each mutation. Findings: We analysed 41 137 MTBC isolates with phenotypic and whole-genome sequencing data from 45 countries. 38 215 MTBC isolates passed quality control steps and were included in the final analysis. 15 667 associations were computed for 13 211 unique mutations linked to one or more drugs. 1149 (7·3%) of 15 667 mutations were classified as associated with phenotypic resistance and 107 (0·7%) were deemed consistent with susceptibility. For rifampicin, isoniazid, ethambutol, fluoroquinolones, and streptomycin, the mutations' pooled sensitivity was more than 80%. Specificity was over 95% for all drugs except ethionamide (91·4%), moxifloxacin (91·6%) and ethambutol (93·3%). Only two resistance mutations were identified for bedaquiline, delamanid, clofazimine, and linezolid as prevalence of phenotypic resistance was low for these drugs. Interpretation: We present the first WHO-endorsed catalogue of molecular targets for MTBC drug susceptibility testing, which is intended to provide a global standard for resistance interpretation. The existence of this catalogue should encourage the implementation of molecular diagnostics by national tuberculosis programmes. Funding: Unitaid, Wellcome Trust, UK Medical Research Council, and Bill and Melinda Gates Foundation.

It is uncertain whether differences in Mycobacterium tuberculosis (Mtb) virulence defined in vitro influence clinical tuberculosis pathogenesis, transmission, and mortality. We primarily used a macrophage lysis model to characterize the virulence of Mtb isolates collected from 153 Vietnamese adults with pulmonary tuberculosis. The virulence phenotypes were then investigated for their relationship with sputum bacterial load, bacterial lineages, bacterial growth, and cytokine responses in macrophages. Over 6 days of infection, 34 isolates (22.2%) showed low virulence (< 5% macrophages lysed), 46 isolates (30.1%) showed high virulence (≥90% lysis of macrophages), and 73 isolates (47.7%) were of intermediate virulence (5–90% macrophages lysed). Highly virulent isolates were associated with an increased bacterial load in patients' sputum before anti-tuberculosis therapy (P = 0.02). Isolate-dependent virulence phenotype was consistent in both THP-1 and human monocyte-derived macrophages. High virulence isolates survived better and replicated in macrophages one hundred fold faster than those with low virulence. Macrophages infected with high virulence isolates produced lower concentrations of TNF-α and IL-6 (P = 0.002 and 0.0005, respectively), but higher concentration of IL-1β (P = 5.1 × 10−5) compared to those infected with low virulence isolates. High virulence was strongly associated with East Asian/Beijing lineage [P = 0.002, Odd ratio (OR) = 4.32, 95% confident intervals (CI) 1.68–11.13]. The association between virulence phenotypes, bacterial growth, and proinflammatory cytokines in macrophages suggest the suppression of certain proinflammatory cytokines (TNF-α and IL-6) but not IL-1β allows better intracellular survival of highly virulent Mtb. This could result in rapid macrophage lysis and higher bacterial load in sputum of patients infected with high virulence isolates, which may contribute to the pathogenesis and success of the Beijing lineage.

Introduction: Mycobacteria have several unique cellular characteristics, such as multiple cell envelope layers, elongation at cell poles, asymmetric cell division, and accumulation of intracytoplasmic lipid inclusions, which contributes to their survival under stress conditions. However, the understanding of these characteristics in clinical Mycobacterium tuberculosis (M. tuberculosis) isolates and under host stress is limited. We previously reported the influence of host stress on the cell length distribution in a large set of clinical M. tuberculosis isolates (n = 158). Here, we investigate the influence of host stress on the cellular ultrastructure of few clinical M. tuberculosis isolates (n = 8) from that study. The purpose of this study is to further understand the influence of host stress on the cellular adaptations of clinical M. tuberculosis isolates.Methods: We selected few M. tuberculosis isolates (n = 8) for analyzing the cellular ultrastructure ex vivo in sputum and under in vitro stress conditions by transmission electron microscopy. The cellular adaptations of M. tuberculosis in sputum were correlated with the ultrastructure of antibiotic sensitive and resistant isolates in liquid culture, under oxidative stress, iron deficiency, and exposure to isoniazid.Results: In sputum, M. tuberculosis accumulated intracytoplasmic lipid inclusions. In liquid culture, clinical M. tuberculosis revealed isolate to isolate variation in the extent of intracytoplasmic lipid inclusions, which were absent in the laboratory strain H37Rv. Oxidative stress, iron deficiency, and exposure to isoniazid increased the accumulation of lipid inclusions and decreased the thickness of the cell envelope electron transparent layer in M. tuberculosis cells. Furthermore, intracytoplasmic compartments were observed in iron deficient cells.Conclusion: Our ultrastructural analysis has revealed significant influence of host stress on the cellular adaptations in clinical M. tuberculosis isolates. These adaptations may contribute to the survival of M. tuberculosis under host and antibiotic stress conditions. Variation in the cellular adaptations among clinical M. tuberculosis isolates may correlate with their ability to persist in tuberculosis patients during antibiotic treatment. These observations indicate the need for further analyzing these cellular adaptations in a large set of clinical M. tuberculosis isolates. This will help to determine the significance of these cellular adaptations in the tuberculosis treatment.

Mycobacterial cellular variations in growth and division increase heterogeneity in cell length, possibly contributing to cell-to-cell variation in host and antibiotic stress tolerance. This may be one of the factors influencing Mycobacterium tuberculosis persistence to antibiotics. Tuberculosis (TB) is a major public health problem in developing countries, antibiotic persistence, and emergence of antibiotic resistance further complicates this problem. We wanted to investigate the factors influencing cell-length distribution in clinical M. tuberculosis strains. In parallel we examined M. tuberculosis cell-length distribution in a large set of clinical strains (n = 158) from ex vivo sputum samples, in vitro macrophage models, and in vitro cultures. Our aim was to understand the influence of clinically relevant factors such as host stresses, M. tuberculosis lineages, antibiotic resistance, antibiotic concentrations, and disease severity on the cell size distribution in clinical M. tuberculosis strains. Increased cell size and cell-to-cell variation in cell length were associated with bacteria in sputum and infected macrophages rather than liquid culture. Multidrug-resistant (MDR) strains displayed increased cell length heterogeneity compared to sensitive strains in infected macrophages and also during growth under rifampicin (RIF) treatment. Importantly, increased cell length was also associated with pulmonary TB disease severity. Supporting these findings, individual host stresses, such as oxidative stress and iron deficiency, increased cell-length heterogeneity of M. tuberculosis strains. In addition we also observed synergism between host stress and RIF treatment in increasing cell length in MDR-TB strains. This study has identified some clinical factors contributing to cell-length heterogeneity in clinical M. tuberculosis strains. The role of these cellular adaptations to host and antibiotic tolerance needs further investigation.

Prior criteria for organ dysfunction in critically ill children were based mainly on expert opinion. We convened the Pediatric Organ Dysfunction Information Update Mandate (PODIUM) expert panel to summarize data characterizing single and multiple organ dysfunction and to derive contemporary criteria for pediatric organ dysfunction. The panel was composed of 88 members representing 47 institutions and 7 countries. We conducted systematic reviews of the literature to derive evidence-based criteria for single organ dysfunction for neurologic, cardiovascular, respiratory, gastrointestinal, acute liver, renal, hematologic, coagulation, endocrine, endothelial, and immune system dysfunction. We searched PubMed and Embase from January 1992 to January 2020. Study identification was accomplished using a combination of medical subject headings terms and keywords related to concepts of pediatric organ dysfunction. Electronic searches were performed by medical librarians. Studies were eligible for inclusion if the authors reported original data collected in critically ill children; evaluated performance characteristics of scoring tools or clinical assessments for organ dysfunction; and assessed a patient-centered, clinically meaningful outcome. Data were abstracted from each included study into an electronic data extraction form. Risk of bias was assessed using the Quality in Prognosis Studies tool. Consensus was achieved for a final set of 43 criteria for pediatric organ dysfunction through iterative voting and discussion. Although the PODIUM criteria for organ dysfunction were limited by available evidence and will require validation, they provide a contemporary foundation for researchers to identify and study single and multiple organ dysfunction in critically ill children.

Tuberculous meningitis (TBM) is the most severe form of tuberculous with substantial mortality. In May 2015, 54 researchers from 10 countries met in Da Lat, Vietnam, to discuss advances in TBM. Among the attendees were researchers involved in pivotal studies on the use of Xpert MTB/Rif for TBM diagnosis. Attendees discussed the 2014 World Health Organization strong recommendation favoring the use of Xpert "in preference to conventional microscopy and culture as the initial diagnostic test for cerebrospinal fluid (CSF) if the sample volume is low or if additional specimens cannot be obtained to make a quick diagnosis." Attendees were concerned that the limitations of Xpert testing for TBM are not emphasized. Clear guidance is needed for the investigational pathway for TBM, including recommendations on the diagnostic package of investigations, which does not stop with Xpert testing. Second, emphasis on the large CSF volumes (ideally 8-10 mL) needed for Xpert testing is required. Guidelines should also emphasize that TBM is a medical emergency and early treatment reduces mortality.