Evolution and transmission of antibiotic resistance is driven by Beijing lineage Mycobacterium tuberculosis in Vietnam.

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.

Whole genome sequencing of multidrug resistant Enterobacterales identified in children and their household members within Siem Reap, Cambodia.

Objectives: To explore the association of recent hospitalization and asymptomatic carriage of multidrug-resistant Enterobacterales (MDRE) and determine the prevailing strains and antibiotic resistance genes in Siem Reap, Cambodia using WGS. Methods: In this cross-sectional study, faecal samples were collected from two arms: a hospital-associated arm consisted of recently hospitalized children (2-14 years), with their family members; and a community-associated arm comprising children in the matching age group and their family members with no recent hospitalization. Forty-two families in each study arm were recruited, with 376 enrolled participants (169 adults and 207 children) and 290 stool specimens collected from participants. The DNA of ESBL- and carbapenemase-producing Enterobacterales cultured from the faecal samples was subject to WGS on the Illumina NovaSeq platform. Results: Of the 290 stool specimens, 277 Escherichia coli isolates and 130 Klebsiella spp. were identified on CHROMagar ESBL and KPC plates. The DNA of 276 E. coli (one isolate failed quality control test), 89 Klebsiella pneumoniae, 40 Klebsiella quasipneumoniae and 1 Klebsiella variicola was sequenced. CTX-M-15 was the most common ESBL gene found in E. coli (n = 104, 38%), K. pneumoniae (n = 50, 56%) and K. quasipneumoniae (n = 16, 40%). The prevalence of bacterial lineages and ESBL genes was not associated with any specific arm. Conclusions: Our results demonstrate that MDRE is likely to be endemic within the Siem Reap community. ESBL genes, specifically blaCTX-M, can be found in almost all E. coli commensals, indicating that these genes are continuously propagated in the community through various unknown channels at present.

Metabolomic analysis of Mycobacterium tuberculosis reveals metabolic profiles for identification of drug-resistant tuberculosis.

The detection of pre-extensively (pre-XDR) and extensively drug-resistant tuberculosis (XDR-TB) is challenging. Drug-susceptibility tests for some anti-TB drugs, especially ethambutol (ETH) and ethionamide (ETO), are problematic due to overlapping thresholds to differentiate between susceptible and resistant phenotypes. We aimed to identify possible metabolomic markers to detect Mycobacterium tuberculosis (Mtb) strains causing pre-XDR and XDR-TB. The metabolic patterns of ETH- and ETO-resistant Mtb isolates were also investigated. Metabolomics of 150 Mtb isolates (54 pre-XDR, 63 XDR-TB and 33 pan-susceptible; pan-S) were investigated. Metabolomics of ETH and ETO phenotypically resistant subgroups were analyzed using UHPLC-ESI-QTOF-MS/MS. Orthogonal partial least-squares discriminant analysis revealed distinct separation in all pairwise comparisons among groups. Two metabolites (meso-hydroxyheme and itaconic anhydride) were able to differentiate the pre-XDR and XDR-TB groups from the pan-S group with 100% sensitivity and 100% specificity. In comparisons of the ETH and ETO phenotypically resistant subsets, sets of increased (ETH = 15, ETO = 7) and decreased (ETH = 1, ETO = 6) metabolites specific for the resistance phenotype of each drug were found. We demonstrated the potential for metabolomics of Mtb to differentiate among types of DR-TB as well as between isolates that were phenotypically resistant to ETO and ETH. Thus, metabolomics might be further applied for DR-TB diagnosis and patient management.

Comparative Activities of Novel Therapeutic Agents against Molecularly Characterized Clinical Carbapenem-Resistant Enterobacterales Isolates.

Limited treatment options exist for the treatment of carbapenem-resistant Enterobacterales (CRE) bacteria. Fortunately, there are several recently approved antibiotics indicated for CRE infections. Here, we examine the in vitro activity of various novel agents (eravacycline, plazomicin, ceftazidime-avibactam, imipenem-relebactam, and meropenem-vaborbactam) and comparators (tigecycline, amikacin, levofloxacin, fosfomycin, polymyxin B) against 365 well-characterized CRE clinical isolates with various genotypes. Nonduplicate isolates collected from the largest public health hospital in Singapore between 2007 and 2020 were subjected to antimicrobial susceptibility testing (broth microdilution or antibiotic gradient test strips). Susceptibilities were defined using Clinical and Laboratory Standards Institute (CLSI) or Food and Drug Administration (FDA) interpretative criteria. Sequence types and resistance mechanisms were characterized using short-read whole-genome sequencing. Overall, tigecycline and plazomicin exhibited the highest susceptibility rates (89.6% and 80.8%, respectively). However, the tigecycline susceptibility breakpoint utilized here may be outdated in view of prevailing pharmacokinetic-pharmacodynamic (PK/PD) data. Susceptibility varied by carbapenemase genotype; the ß-lactam/ß-lactamase inhibitor combinations were equally active (92.3 to 99.2% susceptible) against KPC producers, but only ceftazidime-avibactam retained high susceptibility (98.7%) against OXA-48-like producers. Against metallo-ß-lactamase producers, only plazomicin exhibited moderate activity (77.0% susceptible). Aminoglycoside activity was also influenced by carbapenemase genotypes. This work provides an insight into the comparative activity and presumptive utility of novel agents in this geographic region. IMPORTANCE This study determined the susceptibilities of carbapenem-resistant Enterobacterales isolates to various novel antimicrobial agents (ceftazidime-avibactam, imipenem-relebactam, meropenem-vaborbactam, eravacycline, and plazomicin). Whole-genome sequencing was performed for all strains. Our study findings provide insights into the comparative activities of novel agents in this geographic region. Plazomicin and ceftazidime-avibactam exhibited the lowest nonsusceptibility rates and may be considered promising agents in the management of carbapenem-resistant Enterobacterales infections. We note also that antibiotic activity is influenced by genotypes and that understanding the geographic region's molecular epidemiology could aid in the definition of the presumptive utility of novel agents and contribute to antibiotic decision-making.

Genomic Surveillance of Carbapenem-Resistant Klebsiella pneumoniae from a Major Public Health Hospital in Singapore.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a global public health threat. In this study, we employed whole-genome sequencing (WGS) to determine the genomic epidemiology of a longitudinal collection of clinical CRKP isolates recovered from a large public acute care hospital in Singapore. Phylogenetic analyses, a characterization of resistance and virulence determinants, and plasmid profiling were performed for 575 unique CRKP isolates collected between 2009 and 2020. The phylogenetic analyses identified the presence of global high-risk clones among the CRKP population (clonal group [CG] 14/15, CG17/20, CG147, CG258, and sequence type [ST] 231), and these clones constituted 50% of the isolates. Carbapenemase production was common (n = 497, 86.4%), and KPC was the predominant carbapenemase (n = 235, 40.9%), followed by OXA-48-like (n = 128, 22.3%) and NDM (n = 93, 16.2%). Hypervirulence was detected in 59 (10.3%) isolates and was most common in the ST231 carbapenemase-producing isolates (21/59, 35.6%). Carbapenemase genes were associated with diverse plasmid replicons; however, there was an association of blaOXA-181/232 with ColKP3 plasmids. This study presents the complex and diverse epidemiology of the CRKP strains circulating in Singapore. Our study highlights the utility of WGS-based genomic surveillance in tracking the population dynamics of CRKP. IMPORTANCE In this study, we characterized carbapenem-resistant Klebsiella pneumoniae clinical isolates collected over a 12-year period in the largest public acute-care hospital in Singapore using whole-genome sequencing. The results of this study demonstrate significant genomic diversity with the presence of well-known epidemic, multidrug-resistant clones amid a diverse pool of nonepidemic lineages. Genomic surveillance involving comprehensive resistance, virulence, and plasmid gene content profiling provided critical information for antimicrobial resistance monitoring and highlighted future surveillance priorities, such as the emergence of ST231 K. pneumoniae strains bearing multidrug resistance, virulence elements, and the potential plasmid-mediated transmission of the blaOXA-48-like gene. The findings here also reinforce the necessity of unique infection control and prevention strategies that take the genomic diversity of local circulating strains into consideration.

Whole-Genome Sequencing and Drug-Susceptibility Analysis of Serial Mycobacterium abscessus Isolates from Thai Patients.

Mycobacterium abscessus is an important pathogen that can cause serious human diseases and is difficult to treat due to antibiotic resistance. In this study, we analyzed, using whole-genome sequence (WGS) data, M. abscessus strains serially isolated from patients at various time intervals. We undertook genetic diversity analysis between subspecies, mutation-rate estimation and identification of drug-resistant mutations with minimum inhibitory concentration (MIC) analysis. Clonal isolates of M. abscessus:­subsp. abscessus (MAB) and subsp. massiliense (MMAS)­causing persistent infection through time, differed by 0−7 and 0−14 SNPs, respectively, despite being isolated 1 to 659 days apart. Two cases caused by MMAS differed by ≥102 SNPs at 350 days apart and were regarded as examples of reinfection. Isolates collected ≤7 days apart exhibited a high mutation rate (133.83 ± 0.00 SNPs/genome (5 Mb)/year for MMAS and 127.75 SNPs/genome (5 Mb)/year for MAB). Mutation rates declined in a time-dependent manner in both subspecies. Based on isolates collected > 180 days apart, MMAS had a significantly higher average mutation rate than MAB (2.89 ± 1.02 versus 0.82 ± 0.83 SNPs/genome (5 Mb)/year, (p = 0.01), respectively). All well-known drug-resistance mutations were found to be strongly associated with high MIC levels for clarithromycin and ciprofloxacin. No known mutations were identified for strains resistant to linezolid and amikacin. MAB strains in the study were susceptible to amikacin, while most MMAS strains were susceptible to clarithromycin, amikacin and linezolid. No hetero-resistance was found in the strains analyzed. Our study reports the genetic diversity and mutation rate of M. abscessus between the two major subspecies and confirms the drug resistance-associated mutations. Information about drug-resistance and associated mutations can be applied in diagnosis and patient management.

Whole-genome sequencing of Mycobacterium tuberculosis from Cambodia.

Cambodia has one of the highest tuberculosis (TB) incidence rates in the WHO Western Pacific region. Remarkably though, the prevalence of multidrug-resistant TB (MDR-TB) remains low. We explored the genetic diversity of Mycobacterium tuberculosis (MTB) circulating in this unique setting using whole-genome sequencing (WGS). From October 2017 until January 2018, we collected one hundred sputum specimens from consenting adults older than 21 years of age, newly diagnosed with bacteriologically confirmed TB in 3 districts of Phnom Penh and Takeo provinces of Cambodia before they commence on their TB treatment, where eighty MTB isolates were successfully cultured and sequenced. Majority of the isolates belonged to Lineage 1 (Indo-Oceanic) (69/80, 86.25%), followed by Lineage 2 (East Asian) (10/80, 12.5%) and Lineage 4 (Euro-American) (1/80, 1.25%). Phenotypic resistance to both streptomycin and isoniazid was found in 3 isolates (3/80, 3.75%), while mono-resistance to streptomycin and isoniazid was identical at 2.5% (N = 2 each). None of the isolates tested was resistant to either rifampicin or ethambutol. The specificities of genotypic prediction for resistance to all drugs tested were 100%, while the sensitivities of genotypic resistance predictions to isoniazid and streptomycin were lower at 40% (2/5) and 80% (4/5) respectively. We identified 8 clusters each comprising of two to five individuals all residing in the Takeo province, making up half (28/56, 50%) of all individuals sampled in the province, indicating the presence of multiple ongoing transmission events. All clustered isolates were of Lineage 1 and none are resistant to any of the drugs tested. This study while demonstrating the relevance and utility of WGS in predicting drug resistance and inference of disease transmission, highlights the need to increase the representation of genotype-phenotype TB data from low and middle income countries in Asia and Africa to improve the accuracies for prediction of drug resistance.

Country-wide genotyping of Mycobacterium tuberculosis complex in Singapore, 2011-2017.

OBJECTIVE: To describe the molecular epidemiology of Mycobacterium tuberculosis complex (MTBC) and factors associated with its transmission in Singapore. METHODS: Spoligotyping, 24-loci mycobacterial interspersed repetitive units - variable number of tandem repeats (MIRU-VNTR) typing and demographic data from the national TB notification registry of MTBC culture-positive cases notified from January 2011 to December 2017 were analysed. RESULTS: Of the 12,046 culture-positive cases notified, complete spoligotyping and MIRU-VNTR typing results were available for 8690 (72.1%) belonging to 4950 (57.0%) local-born and 3740 (43.0%) foreign-born persons. From these, 4810 (55.3%) were identified in 883 clusters. The proportion of recent transmission was 45.2%. The East-Asian Lineage 2 accounted for 4045 (47.1%) of isolates, and disproportionately accounted for large clusters. Clustered cases were more likely to be older than 50 years, male, Malay, local-born, Singapore citizens or Permanent Residents, of lower socioeconomic status, imprisoned; to harbour East-Asian Lineage 2 strain; to have cavitary pulmonary TB, positive sputum smear or be recalcitrant treatment defaulters. They were less likely to have multidrug-resistant, or isoniazid or rifampicin mono-resistant TB. CONCLUSION: We demonstrated the diversity of MTBC strains and, notwithstanding the likely over-estimation of clustering using these genotyping methods, elucidated factors associated with TB transmission in Singapore.

Clustering biological sequences with dynamic sequence similarity threshold.

BACKGROUND: Biological sequence clustering is a complicated data clustering problem owing to the high computation costs incurred for pairwise sequence distance calculations through sequence alignments, as well as difficulties in determining parameters for deriving robust clusters. While current approaches are successful in reducing the number of sequence alignments performed, the generated clusters are based on a single sequence identity threshold applied to every cluster. Poor choices of this identity threshold would thus lead to low quality clusters. There is however little support provided to users in selecting thresholds that are well matched with the input sequences. RESULTS: We present a novel sequence clustering approach called ALFATClust that exploits rapid pairwise alignment-free sequence distance calculations and community detection in graph for clusters generation. Instead of a single threshold applied to every generated cluster, ALFATClust is capable of dynamically determining the cut-off threshold for each individual cluster by considering both cluster separation and intra-cluster sequence similarity. Benchmarking analysis shows that ALFATClust generally outperforms existing approaches by simultaneously maintaining cluster robustness and substantial cluster separation for the benchmark datasets. The software also provides an evaluation report for verifying the quality of the non-singleton clusters obtained. CONCLUSIONS: ALFATClust is able to generate sequence clusters having high intra-cluster sequence similarity and substantial separation between clusters without having users to decide precise similarity cut-off thresholds.

Optimized analysis parameters of variant calling for whole genome-based phylogeny of Mycobacteroides abscessus.

Whole-genome sequence (WGS) analysis provides the best resolution for reconstructing bacterial phylogeny. However, the resulting tree could vary according to parameters used in the WGS pipeline, making it difficult to compare results across multiple studies. This study compares effects on phylogenies when applying different parameter stringencies. We used as the study model to optimize parameters strains of Mycobacteroides abscessus serially isolated at various intervals, isolates known to represent persistent infection (PI) cases or re-infection (RI) cases and isolates from different subspecies. Un-optimized parameters with low stringency provided an excessive number of SNPs (823) compared to the optimized setting (3 SNPs) between paired strains isolated 1 day apart from PI cases, discordant tree topology and misclassification of subspecies and of instances of RI. We demonstrated that using high-quality variants provides more accuracy for recognizing serial isolates of the same clone versus different clones and for phylogenetic analysis of M. abscessus. Our approach might be used as a model for analyses requiring phylogenetic reconstruction of other bacteria.

Epidemiology of Extended-Spectrum Beta-Lactamase and Carbapenemase-Producing Enterobacterales in the Greater Mekong Subregion: A Systematic-Review and Meta-Analysis of Risk Factors Associated With Extended-Spectrum Beta-Lactamase and Carbapenemase Isolation.

Background: Despite the rapid spread of extended-spectrum beta-lactamase (ESBL) producing-Enterobacterales (ESBL-E) and carbapenemase-producing Enterobacterales (CPE), little is known about the extent of their prevalence in the Greater Mekong Subregion (GMS). In this systematic review, we aimed to determine the epidemiology of ESBL-E and CPE in clinically significant Enterobacterales: Escherichia coli and Klebsiella pneumoniae from the GMS (comprising of Cambodia, Laos, Myanmar, Thailand, Vietnam and Yunnan province and Guangxi Zhuang region of China). Methods: Following a list of search terms adapted to subject headings, we systematically searched databases: Medline, EMBASE, Scopus and Web of Science for articles published on and before October 20th, 2020. The search string consisted of the bacterial names, methods involved in detecting drug-resistance phenotype and genotype, GMS countries, and ESBL and carbapenemase detection as the outcomes. Meta-analyses of the association between the isolation of ESBL from human clinical and non-clinical specimens were performed using the "METAN" function in STATA 14. Results: One hundred and thirty-nine studies were included from a total of 1,513 identified studies. Despite the heterogeneity in study methods, analyzing the prevalence proportions on log-linear model scale for ESBL producing-E. coli showed a trend that increased by 13.2% (95%CI: 6.1-20.2) in clinical blood specimens, 8.1% (95%CI: 1.7-14.4) in all clinical specimens and 17.7% (95%CI: 4.9-30.4) increase in carriage specimens. Under the log-linear model assumption, no significant trend over time was found for ESBL producing K. pneumoniae and ESBL-E specimens. CPE was reported in clinical studies and carriage studies past 2010, however a trend could not be determined because of the small dataset. Twelve studies were included in the meta-analysis of risk factors associated with isolation of ESBL. Recent antibiotic exposure was the most studied variable and showed a significant positive association with ESBL-E isolation (pooled OR: 2.9, 95%CI: 2.3-3.8) followed by chronic kidney disease (pooled OR: 4.7, 95%CI: 1.8-11.9), and other co-morbidities (pooled OR: 1.6, 95%CI: 1.2-2.9). Conclusion: Data from GMS is heterogeneous with significant data-gaps, especially in community settings from Laos, Myanmar, Cambodia and Yunnan and Guangxi provinces of China. Collaborative work standardizing the methodology of studies will aid in better monitoring, surveillance and evaluation of interventions across the GMS.

In vitro Bactericidal Activities of Combination Antibiotic Therapies Against Carbapenem-Resistant Klebsiella pneumoniae With Different Carbapenemases and Sequence Types.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is becoming increasingly problematic due to the limited effectiveness of new antimicrobials or other factors such as treatment cost. Thus, combination therapy remains a suitable treatment option. We aimed to evaluate the in vitro bactericidal activity of various antibiotic combinations against CRKP with different carbapenemase genotypes and sequence types (STs). Thirty-seven CRKP with various STs and carbapenemases were exposed to 11 antibiotic combinations (polymyxin B or tigecycline in combination with ß-lactams including aztreonam, cefepime, piperacillin/tazobactam, doripenem, meropenem, and polymyxin B with tigecycline) in static time-kill studies (TKS) using clinically achievable concentrations. Out of the 407 isolate-combination pairs, only 146 (35.8%) were bactericidal (≥3 log10CFU/mL decrease from initial inoculum). Polymyxin B in combination with doripenem, meropenem, or cefepime was the most active, each demonstrating bactericidal activity in 27, 24, and 24 out of 37 isolates, respectively. Tigecycline in combination with ß-lactams was rarely bactericidal. Aside from the lower frequency of bactericidal activity in the dual-carbapenemase producers, there was no apparent difference in combination activity among the strains with other carbapenemase types. In addition, bactericidal combinations were varied even in strains with similar STs, carbapenemases, and other genomic characteristics. Our findings demonstrate that the bactericidal activity of antibiotic combinations is highly strain-specific likely owing to the complex interplay of carbapenem-resistance mechanisms, i.e., carbapenemase genotype alone cannot predict in vitro bactericidal activity. The availability of WGS information can help rationalize the activity of certain combinations. Further studies should explore the use of genomic markers with phenotypic information to predict combination activity.

Dynamics of the fecal microbiome and antimicrobial resistome in commercial piglets during the weaning period.

This study aimed to characterize the alteration of the fecal microbiome and antimicrobial resistance (AMR) determinants in 24 piglets at day 3 pre-weaning (D. - 3), weaning day (D.0), days 3 (D.3) and 8 post-weaning (D.8), using whole-genome shotgun sequencing. Distinct clusters of microbiomes and AMR determinants were observed at D.8 when Prevotella (20.9%) was the major genus, whereas at D. - 3-D.3, Alistipes (6.9-12.7%) and Bacteroides (5.2-8.5%) were the major genera. Lactobacillus and Escherichia were notably observed at D. - 3 (1.2%) and D. - 3-D.3 (0.2-0.4%), respectively. For AMR, a distinct cluster of AMR determinants was observed at D.8, mainly conferring resistance to macrolide-lincosamide-streptogramin (mefA), ß-lactam (cfxA6 and aci1) and phenicol (rlmN). In contrast, at D. - 3-D.3, a high abundance of determinants with aminoglycoside (AMG) (sat, aac(6')-aph(2''), aadA and acrF), ß-lactam (fus-1, cepA and mrdA), multidrug resistance (MDR) (gadW, mdtE, emrA, evgS, tolC and mdtB), phenicol (catB4 and cmlA4), and sulfonamide patterns (sul3) was observed. Canonical correlation analysis (CCA) plot associated Escherichia coli with aac(6')-aph(2''), emrA, mdtB, catB4 and cmlA4 at D. - 3, D.0 and/or D.3 whereas at D.8 associations between Prevotella and mefA, cfxA6 and aci1 were identified. The weaning age and diet factor played an important role in the microbial community composition.

Genomic characterization of carbapenem-non-susceptible Pseudomonas aeruginosa in Singapore.

Pseudomonas aeruginosa is a clinically important pathogen implicated in many hospital-acquired infections. Its propensity to acquire broad-spectrum resistance has earned the organism its status as a severe public health threat requiring urgent control measures. While whole-genome sequencing-based genomic surveillance provides a means to track antimicrobial resistance, its use in molecular epidemiological surveys of P. aeruginosa remains limited, especially in the Southeast Asian region. We sequenced the whole genomes of 222 carbapenem-non-susceptible P. aeruginosa (CNPA) isolates collected in 2006-2020 at the largest public acute care hospital in Singapore. Antimicrobial susceptibilities were determined using broth microdilution. Clonal relatedness, multi-locus sequence types, and antimicrobial resistance determinants (acquired and chromosomal) were determined. In this study, CNPA exhibited broad-spectrum resistance (87.8% multi-drug resistance), retaining susceptibility only to polymyxin B (95.0%) and amikacin (55.0%). Carbapenemases were detected in 51.4% of the isolates, where IMP and NDM metallo-ß-lactamases were the most frequent. Carbapenem resistance was also likely associated with OprD alterations or efflux mechanisms (ArmZ/NalD mutations), which occurred in strains with or without carbapenemases. The population of CNPA in the hospital was diverse; the 222 isolates grouped into 68 sequence types (ST), which included various high-risk clones. We detected an emerging clone, the NDM-1-producing ST308, in addition to the global high-risk ST235 clone which was the predominant clone in our population. Our results thus provide a "snapshot" of the circulating lineages of CNPA locally and the prevailing genetic mechanisms contributing to carbapenem resistance. This database also serves as the baseline for future prospective surveillance studies.

Cross-sectional study of prevalence and risk factors, and a cost-effectiveness evaluation of screening and preventive treatment strategies for latent tuberculosis among migrants in Singapore.

OBJECTIVES: WHO recommends that low burden countries consider systematic screening and treatment of latent tuberculosis infection (LTBI) in migrants from high incidence countries. We aimed to determine LTBI prevalence and risk factors and evaluate cost-effectiveness of screening and treating LTBI in migrants to Singapore from a government payer perspective. DESIGN: Cross-sectional study and cost-effectiveness analysis. SETTING: Migrants in Singapore. PARTICIPANTS: 3618 migrants who were between 20 and 50 years old, have not worked in Singapore previously and stayed in Singapore for less than a year were recruited. PRIMARY AND SECONDARY OUTCOME MEASURES: Costs, quality-adjusted life-years (QALYs), threshold length of stay, incremental cost-effectiveness ratios (ICERs), cost per active TB case averted. RESULTS: Of 3584 migrants surveyed, 20.4% had positive interferon-gamma release assay (IGRA) results, with the highest positivity in Filipinos (33.2%). Higher LTBI prevalence was significantly associated with age, marital status and past TB exposure. The cost-effectiveness model projected an ICER of S$57 116 per QALY and S$12 422 per active TB case averted for screening and treating LTBI with 3 months once weekly isoniazid and rifapentine combination regimen treatment compared with no screening over a 50-year time horizon. ICER was most sensitive to the cohort's length of stay in Singapore, yearly disease progression rates from LTBI to active TB, followed by the cost of IGRA testing. CONCLUSIONS: For LTBI screening and treatment of migrants to be cost-effective, migrants from high burden countries would have to stay in Singapore for ~50 years. Risk-stratified approaches based on projected length of stay and country of origin and/or age group can be considered.

Whole-genome analysis of drug-resistant Mycobacterium tuberculosis reveals novel mutations associated with fluoroquinolone resistance.

Multidrug-resistant and extensively drug-resistant tuberculosis (M/XDR-TB) remains a global public-health challenge. Known mutations in quinolone resistance-determination regions cannot fully explain phenotypic fluoroquinolone (FQ) resistance in Mycobacterium tuberculosis (Mtb). The aim of this study was to look for novel mutations in Mtb associated with resistance to FQ drugs using whole-genome sequencing analysis. Whole-genome sequences of 659 Mtb strains, including 214 with phenotypic FQ resistance and 445 pan-susceptible isolates, were explored for mutations associated with FQ resistance overall and with resistance to individual FQ drugs (ofloxacin, levofloxacin, moxifloxacin and gatifloxacin). Three novel genes (recC, Rv2005c and PPE59) associated with FQ resistance were identified (P < 0.00001 based on screening analysis and absence of relevant mutations in a pan-susceptible validation set of 360 strains). Nine novel single nucleotide polymorphisms (SNPs), including in gyrB (G5383A and G6773A), gyrA (G7892A), recC (G725900C and G726857T/C), Rv2005c (C2251373G, G2251420C and C2251725T) and PPE59 (C3847269T), were used for diagnostic performance analysis. Enhancing the known SNP set with five of these novel SNPs, including gyrA [G7892A (Leu247Leu)], recC [G725900C (Leu893Leu) and G726857T/C (Arg484Arg)], Rv2005c [G2251420C (Pro205Arg)] and PPE59 [C3847269T (Asn35Asn)] increased the sensitivity of detection of FQ-resistant Mtb from 83.2% (178/214) to 86.9% (186/214) while maintaining 100% specificity (360/360). No specific mutation associated with resistance to only a single drug (ofloxacin, levofloxacin, moxifloxacin or gatifloxacin) was found. In conclusion, this study reports possible additional mutations associated with FQ resistance in Mtb.

Genomic epidemiology of tuberculosis in eastern Malaysia: insights for strengthening public health responses.

Tuberculosis is a leading public health priority in eastern Malaysia. Knowledge of the genomic epidemiology of tuberculosis can help tailor public health interventions. Our aims were to determine tuberculosis genomic epidemiology and characterize resistance mutations in the ethnically diverse city of Kota Kinabalu, Sabah, located at the nexus of Malaysia, Indonesia, Philippines and Brunei. We used an archive of prospectively collected Mycobacterium tuberculosis samples paired with epidemiological data. We collected sputum and demographic data from consecutive consenting outpatients with pulmonary tuberculosis at the largest tuberculosis clinic from 2012 to 2014, and selected samples from tuberculosis inpatients from the tertiary referral centre during 2012-2014 and 2016-2017. Two hundred and eight M. tuberculosis sequences were available for analysis, representing 8 % of cases notified during the study periods. Whole-genome phylogenetic analysis demonstrated that most strains were lineage 1 (195/208, 93.8 %), with the remainder being lineages 2 (8/208, 3.8 %) or 4 (5/208, 2.4 %). Lineages or sub-lineages were not associated with patient ethnicity. The lineage 1 strains were diverse, with sub-lineage 1.2.1 being dominant (192, 98 %). Lineage 1.2.1.3 isolates were geographically most widely distributed. The greatest diversity occurred in a border town sub-district. The time to the most recent common ancestor for the three major lineage 1.2.1 clades was estimated to be the year 1966 (95 % HPD 1948-1976). An association was found between failure of culture conversion by week 8 of treatment and infection with lineage 2 (4/6, 67 %) compared with lineage 1 strains (4/83, 5 %) (P<0.001), supporting evidence of greater virulence of lineage 2 strains. Eleven potential transmission clusters (SNP difference ≤12) were identified; at least five included people living in different sub-districts. Some linked cases spanned the whole 4-year study period. One cluster involved a multidrug-resistant tuberculosis strain matching a drug-susceptible strain from 3 years earlier. Drug resistance mutations were uncommon, but revealed one phenotype-genotype mismatch in a genotypically multidrug-resistant isolate, and rare nonsense mutations within the katG gene in two isolates. Consistent with the regionally mobile population, M. tuberculosis strains in Kota Kinabalu were diverse, although several lineage 1 strains dominated and were locally well established. Transmission clusters - uncommonly identified, likely attributable to incomplete sampling - showed clustering occurring across the community, not confined to households or sub-districts. The findings indicate that public health priorities should include active case finding and early institution of tuberculosis management in mobile populations, while there is a need to upscale effective contact investigation beyond households to include other contacts within social networks.

Government trust, perceptions of COVID-19 and behaviour change: cohort surveys, Singapore.

OBJECTIVE: To evaluate how public perceptions and trust in government communications affected the adoption of protective behaviour in Singapore during the coronavirus disease 2019 (COVID-19) pandemic. METHODS: We launched our community-based cohort to assess public perceptions of infectious disease outbreaks in mid-2019. After the first case of COVID-19 was reported in Singapore on 23 January, we launched a series of seven COVID-19 surveys to both existing and regularly enrolled new participants every 2 weeks. As well as sociodemographic properties of the participants, we recorded changing responses to judge awareness of the situation, trust in various information sources and perceived risk. We used multivariable logistic regression models to evaluate associations with perceptions of risk and self-reported adopted frequencies of protective behaviour. FINDINGS: Our cohort of 633 participants provided 2857 unique responses during the seven COVID-19 surveys. Most agreed or strongly agreed that information from official government sources (99.1%; 528/533) and Singapore-based news agencies (97.9%; 522/533) was trustworthy. Trust in government communication was significantly associated with higher perceived threat (odds ratio, OR: 2.2; 95% confidence interval, CI: 1.6-3.0), but inversely associated with perceived risk of infection (OR: 0.6; 95% CI: 0.4-0.8) or risk of death if infected (OR: 0.6; 95% CI: 0.4-0.9). Trust in government communication was also associated with a greater likelihood of adopting protective behaviour. CONCLUSION: Our findings show that trust is a vital commodity when managing an evolving outbreak. Our repeated surveys provided real-time feedback, allowing an improved understanding of the interplay between perceptions, trust and behaviour.

Ceftolozane/Tazobactam Resistance and Mechanisms in Carbapenem-Nonsusceptible Pseudomonas aeruginosa.

This study established the in vitro activity of ceftolozane/tazobactam (C/T) and its genotypic resistance mechanisms by whole-genome sequencing (WGS) in 195 carbapenem-nonsusceptible Pseudomonas aeruginosa (CNSPA) clinical isolates recovered from Singapore between 2009 and 2020. C/T susceptibility rates were low, at 37.9%. Cross-resistance to ceftazidime/avibactam was observed, although susceptibility to the agent was slightly higher, at 41.0%. Whole-genome sequencing revealed that C/T resistance was largely mediated by the presence of horizontally acquired ß-lactamases, especially metallo-ß-lactamases. These were primarily disseminated in well-recognized high-risk clones belonging to sequence types (ST) 235, 308, and 179. C/T resistance was also observed in several non-carbapenemase-producing isolates, in which resistance was likely mediated by ß-lactamases and, to a smaller extent, mutations in AmpC-related genes. There was no obvious mechanism of resistance observed in five isolates. The high C/T resistance highlights the limited utility of the agent as an empirical agent in our setting. Knowledge of local molecular epidemiology is crucial in determining the potential of therapy with novel agents.IMPORTANCEPseudomonas aeruginosa infection is one of the most difficult health care-associated infections to treat due to the ability of the organism to acquire a multitude of resistance mechanisms and express the multidrug resistance phenotype. Ceftolozane/tazobactam (C/T), a novel ß-lactam/ß-lactamase inhibitor combination, addresses an unmet medical need in patients with these multidrug-resistant P. aeruginosa infections. Our findings demonstrate geographical variation in C/T susceptibility owing to the distinct local molecular epidemiology. This study adds on to the growing knowledge of C/T resistance, particularly mutational resistance, and will aid in the design of future ß-lactams and ß-lactamase inhibitors. WGS proved to be a useful tool to understand the P. aeruginosa resistome and its contribution to emerging resistance in novel antimicrobial agents.

Paternal gene pool of Malays in Southeast Asia and its applications for the early expansion of Austronesians.

OBJECTIVES: The origin and differentiation of Austronesian populations and their languages have long fascinated linguists, archeologists, and geneticists. However, the founding process of Austronesians and when they separated from their close relatives, such as the Daic and Austro-Asiatic populations in the mainland of Asia, remain unclear. In this study, we explored the paternal origin of Malays in Southeast Asia and the early differentiation of Austronesians. MATERIALS AND METHODS: We generated whole Y-chromosome sequences of 50 Malays and co-analyzed 200 sequences from other Austronesians and related populations. We generated a revised phylogenetic tree with time estimation. RESULTS: We identified six founding paternal lineages among the studied Malays samples. These founding lineages showed a surprisingly coincident expansion age at 5000 to 6000 years ago. We also found numerous mostly close related samples of the founding lineages of Malays among populations from Mainland of Asia. CONCLUSION: Our analyses provided a refined phylogenetic resolution for the dominant paternal lineages of Austronesians found by previous studies. We suggested that the co-expansion of numerous founding paternal lineages corresponds to the initial differentiation of the most recent common ancestor of modern Austronesians. The splitting time and divergence pattern in perspective of paternal Y-chromosome evidence are highly consistent with the previous theories of ethnologists, linguists, and archeologists.