The Reality of Antimicrobial Resistance and Antibiotic Usage Data in Asia: The CAPTURA Experience.

Antimicrobial resistance (AMR), particularly in low- and middle-income countries, is threatening to undermine advances in health and development. Scarce technical and human resources in these countries limit the collection of quality AMR data for evidence-based decision-making. The CAPTURA consortium, funded by the Fleming Fund, was implemented across 7 countries in the South and Southeast Asian region. The program focused on collating historical bacteriological data for qualitative and quantitative analyses. The team gathered standard data on the quality of laboratories and clinics and the quality and quantity of retrospective historical AMR data. In addition, retrospective data on antimicrobial use and consumption were analyzed. While standard protocols guided the project, a tailored approach for stakeholder engagement was implemented to work with countries and secure data-sharing agreements. The program also had to navigate the challenges of the COVID-19 pandemic, making some innovative adaptations to overcome logistical barriers. From 2018 through 2022, a large body of data was collected that was used to base a series of recommended key measures for strengthening the development of standardized national surveillance programs and to support alignment with international efforts.

Adjunctive rifampicin for Staphylococcus aureus bacteraemia (ARREST): a multicentre, randomised, double-blind, placebo-controlled trial.

BACKGROUND: Staphylococcus aureus bacteraemia is a common cause of severe community-acquired and hospital-acquired infection worldwide. We tested the hypothesis that adjunctive rifampicin would reduce bacteriologically confirmed treatment failure or disease recurrence, or death, by enhancing early S aureus killing, sterilising infected foci and blood faster, and reducing risks of dissemination and metastatic infection. METHODS: In this multicentre, randomised, double-blind, placebo-controlled trial, adults (≥18 years) with S aureus bacteraemia who had received ≤96 h of active antibiotic therapy were recruited from 29 UK hospitals. Patients were randomly assigned (1:1) via a computer-generated sequential randomisation list to receive 2 weeks of adjunctive rifampicin (600 mg or 900 mg per day according to weight, oral or intravenous) versus identical placebo, together with standard antibiotic therapy. Randomisation was stratified by centre. Patients, investigators, and those caring for the patients were masked to group allocation. The primary outcome was time to bacteriologically confirmed treatment failure or disease recurrence, or death (all-cause), from randomisation to 12 weeks, adjudicated by an independent review committee masked to the treatment. Analysis was intention to treat. This trial was registered, number ISRCTN37666216, and is closed to new participants. FINDINGS: Between Dec 10, 2012, and Oct 25, 2016, 758 eligible participants were randomly assigned: 370 to rifampicin and 388 to placebo. 485 (64%) participants had community-acquired S aureus infections, and 132 (17%) had nosocomial S aureus infections. 47 (6%) had meticillin-resistant infections. 301 (40%) participants had an initial deep infection focus. Standard antibiotics were given for 29 (IQR 18-45) days; 619 (82%) participants received flucloxacillin. By week 12, 62 (17%) of participants who received rifampicin versus 71 (18%) who received placebo experienced treatment failure or disease recurrence, or died (absolute risk difference -1·4%, 95% CI -7·0 to 4·3; hazard ratio 0·96, 0·68-1·35, p=0·81). From randomisation to 12 weeks, no evidence of differences in serious (p=0·17) or grade 3-4 (p=0·36) adverse events were observed; however, 63 (17%) participants in the rifampicin group versus 39 (10%) in the placebo group had antibiotic or trial drug-modifying adverse events (p=0·004), and 24 (6%) versus six (2%) had drug interactions (p=0·0005). INTERPRETATION: Adjunctive rifampicin provided no overall benefit over standard antibiotic therapy in adults with S aureus bacteraemia. FUNDING: UK National Institute for Health Research Health Technology Assessment.

Accuracy of Different Bioinformatics Methods in Detecting Antibiotic Resistance and Virulence Factors from Staphylococcus aureus Whole-Genome Sequences.

In principle, whole-genome sequencing (WGS) can predict phenotypic resistance directly from a genotype, replacing laboratory-based tests. However, the contribution of different bioinformatics methods to genotype-phenotype discrepancies has not been systematically explored to date. We compared three WGS-based bioinformatics methods (Genefinder [read based], Mykrobe [de Bruijn graph based], and Typewriter [BLAST based]) for predicting the presence/absence of 83 different resistance determinants and virulence genes and overall antimicrobial susceptibility in 1,379 Staphylococcus aureus isolates previously characterized by standard laboratory methods (disc diffusion, broth and/or agar dilution, and PCR). In total, 99.5% (113,830/114,457) of individual resistance-determinant/virulence gene predictions were identical between all three methods, with only 627 (0.5%) discordant predictions, demonstrating high overall agreement (Fleiss' kappa = 0.98, P < 0.0001). Discrepancies when identified were in only one of the three methods for all genes except the cassette recombinase, ccrC(b). The genotypic antimicrobial susceptibility prediction matched the laboratory phenotype in 98.3% (14,224/14,464) of cases (2,720 [18.8%] resistant, 11,504 [79.5%] susceptible). There was greater disagreement between the laboratory phenotypes and the combined genotypic predictions (97 [0.7%] phenotypically susceptible, but all bioinformatic methods reported resistance; 89 [0.6%] phenotypically resistant, but all bioinformatics methods reported susceptible) than within the three bioinformatics methods (54 [0.4%] cases, 16 phenotypically resistant, 38 phenotypically susceptible). However, in 36/54 (67%) cases, the consensus genotype matched the laboratory phenotype. In this study, the choice between these three specific bioinformatic methods to identify resistance determinants or other genes in S. aureus did not prove critical, with all demonstrating high concordance with each other and phenotypic/molecular methods. However, each has some limitations; therefore, consensus methods provide some assurance.

Monkeypox encephalitis with transverse myelitis in a female patient.

The 2022 monkeypox outbreak has affected 110 countries worldwide, outside of classic endemic areas (ie, west Africa and central Africa). On July 23, 2022, the outbreak was classified by WHO as a public health emergency of international concern. Clinical presentation varies from mild to life-changing symptoms; neurological complications are relatively uncommon and there are few therapeutic interventions for monkeypox disease. In this Grand Round, we present a case of monkeypox with encephalitis complicated by transverse myelitis in a previously healthy woman aged 35 years who made an almost complete recovery from her neurological symptoms after treatment with tecovirimat, cidofovir, steroids, and plasma exchange. We describe neurological complications associated with orthopoxvirus infections and laboratory diagnosis, the radiological features in this case, and discuss treatment options.

Antimicrobial resistance determinants are associated with Staphylococcus aureus bacteraemia and adaptation to the healthcare environment: a bacterial genome-wide association study.

Staphylococcus aureus is a major bacterial pathogen in humans, and a dominant cause of severe bloodstream infections. Globally, antimicrobial resistance (AMR) in S. aureus remains challenging. While human risk factors for infection have been defined, contradictory evidence exists for the role of bacterial genomic variation in S. aureus disease. To investigate the contribution of bacterial lineage and genomic variation to the development of bloodstream infection, we undertook a genome-wide association study comparing bacteria from 1017 individuals with bacteraemia to 984 adults with asymptomatic S. aureus nasal carriage. Within 984 carriage isolates, we also compared healthcare-associated (HA) carriage with community-associated (CA) carriage. All major global lineages were represented in both bacteraemia and carriage, with no evidence for different infection rates. However, kmers tagging trimethoprim resistance-conferring mutation F99Y in dfrB were significantly associated with bacteraemia-vs-carriage (P=10-8.9-10-9.3). Pooling variation within genes, bacteraemia-vs-carriage was associated with the presence of mecA (HMP=10-5.3) as well as the presence of SCCmec (HMP=10-4.4). Among S. aureus carriers, no lineages were associated with HA-vs-CA carriage. However, we found a novel signal of HA-vs-CA carriage in the foldase protein prsA, where kmers representing conserved sequence allele were associated with CA carriage (P=10-7.1-10-19.4), while in gyrA, a ciprofloxacin resistance-conferring mutation, L84S, was associated with HA carriage (P=10-7.2). In an extensive study of S. aureus bacteraemia and nasal carriage in the UK, we found strong evidence that all S. aureus lineages are equally capable of causing bloodstream infection, and of being carried in the healthcare environment. Genomic variation in the foldase protein prsA is a novel genomic marker of healthcare origin in S. aureus but was not associated with bacteraemia. AMR determinants were associated with both bacteraemia and healthcare-associated carriage, suggesting that AMR increases the propensity not only to survive in healthcare environments, but also to cause invasive disease.

The potential of fosfomycin for multi-drug resistant sepsis: an analysis of in vitro activity against invasive paediatric Gram-negative bacteria.

PURPOSE: Antimicrobial resistance (AMR) is of increasing global concern, threatening to undermine recent progress in reducing child and neonatal mortality. Repurposing older antimicrobials is a prominent strategy to combat multidrug-resistant sepsis. A potential agent is fosfomycin, however, there is scarce data regarding its in vitro activity and pharmacokinetics in the paediatric population. METHODOLOGY: We analysed a contemporary, systematically collected archive of community-acquired (CA) and hospital-acquired (HA) paediatric Gram-negative bacteraemia isolates for their susceptibility to fosfomcyin. MICs were determined using agar serial dilution methods and validated by disk diffusion testing where breakpoints are available. Disk diffusion antimicrobial susceptibility testing was also conducted for current empirical therapies (ampicillin, gentamicin, ceftriaxone) and amikacin (proposed in the literature as a new combination empirical therapeutic option). RESULTS: Fosfomycin was highly active against invasive Gram-negative isolates, including 90  % (202/224) of Enterobacteriaceae and 96  % (22/23) of Pseudomonas spp. Fosfomycin showed high sensitivity against both CA isolates (94 %, 142/151) and HA isolates (81 %, 78/96; P =0.0015). CA isolates were significantly more likely to be susceptible to fosfomycin than the current first-line empirical therapy (96  % vs 59  %, P <0.0001). Extended spectrum ß-lactamases (ESBL) production was detected in 34  % (85/247) of isolates with no significant difference in fosfomycin susceptibility between ESBL-positive or -negative isolates [73/85 (86  %) vs 147/162 (91  %) respectively, P =0.245]. All isolates were susceptible to a fosfomycin-amikacin combination. CONCLUSION: Gram-negative paediatric bacteraemia isolates are highly susceptible to fosfomycin, which could be combined with aminoglycosides as a new, carbapenem-sparing regimen to achieve excellent coverage to treat antimicrobial-resistant neonatal and paediatric sepsis.

Robust Prediction of Resistance to Trimethoprim in Staphylococcus aureus.

The rise of antibiotic resistance threatens modern medicine; to combat it new diagnostic methods are required. Sequencing the whole genome of a pathogen offers the potential to accurately determine which antibiotics will be effective to treat a patient. A key limitation of this approach is that it cannot classify rare or previously unseen mutations. Here we demonstrate that alchemical free energy methods, a well-established class of methods from computational chemistry, can successfully predict whether mutations in Staphylococcus aureus dihydrofolate reductase confer resistance to trimethoprim. We also show that the method is quantitatively accurate by calculating how much the most common resistance-conferring mutation, F99Y, reduces the binding free energy of trimethoprim and comparing predicted and experimentally measured minimum inhibitory concentrations for seven different mutations. Finally, by considering up to 32 free energy calculations for each mutation, we estimate its specificity and sensitivity.

Survival following Staphylococcus aureus bloodstream infection: A prospective multinational cohort study assessing the impact of place of care.

BACKGROUND: Staphylococcus aureus bloodstream infection (SAB) is a common, life-threatening infection with a high mortality. Survival can be improved by implementing quality of care bundles in hospitals. We previously observed marked differences in mortality between hospitals and now assessed whether mortality could serve as a valid and easy to implement quality of care outcome measure. METHODS: We conducted a prospective observational study between January 2013 and April 2015 on consecutive, adult patients with SAB from 11 tertiary care centers in Germany, South Korea, Spain, Taiwan, and the United Kingdom. Factors associated with mortality at 90 days were analyzed by Cox proportional hazards regression and flexible parametric models. RESULTS: 1851 patients with a median age of 66 years (64% male) were analyzed. Crude 90-day mortality differed significantly between hospitals (range 23-39%). Significant variation between centers was observed for methicillin-resistant S. aureus, community-acquisition, infective foci, as well as measures of comorbidities, and severity of disease. In multivariable analysis, factors independently associated with mortality at 90 days were age, nosocomial acquisition, unknown infective focus, pneumonia, Charlson comorbidity index, SOFA score, and study center. The risk of death varied over time differently for each infective focus. Crude mortality differed markedly from adjusted mortality. DISCUSSION: We observed significant differences in adjusted mortality between hospitals, suggesting differences in quality of care. However, mortality is strongly influenced by patient mix and thus, crude mortality is not a suitable quality indicator.

Adjunctive rifampicin to reduce early mortality from Staphylococcus aureus bacteraemia: the ARREST RCT.

BACKGROUND: Staphylococcus aureus bacteraemia is a common and frequently fatal infection. Adjunctive rifampicin may enhance early S. aureus killing, sterilise infected foci and blood faster, and thereby reduce the risk of dissemination, metastatic infection and death. OBJECTIVES: To determine whether or not adjunctive rifampicin reduces bacteriological (microbiologically confirmed) failure/recurrence or death through 12 weeks from randomisation. Secondary objectives included evaluating the impact of rifampicin on all-cause mortality, clinically defined failure/recurrence or death, toxicity, resistance emergence, and duration of bacteraemia; and assessing the cost-effectiveness of rifampicin. DESIGN: Parallel-group, randomised (1 : 1), blinded, placebo-controlled multicentre trial. SETTING: UK NHS trust hospitals. PARTICIPANTS: Adult inpatients (≥ 18 years) with meticillin-resistant or susceptible S. aureus grown from one or more blood cultures, who had received < 96 hours of antibiotic therapy for the current infection, and without contraindications to rifampicin. INTERVENTIONS: Adjunctive rifampicin (600-900 mg/day, oral or intravenous) or placebo for 14 days in addition to standard antibiotic therapy. Investigators and patients were blinded to trial treatment. Follow-up was for 12 weeks (assessments at 3, 7, 10 and 14 days, weekly until discharge and final assessment at 12 weeks post randomisation). MAIN OUTCOME MEASURES: The primary outcome was all-cause bacteriological (microbiologically confirmed) failure/recurrence or death through 12 weeks from randomisation. RESULTS: Between December 2012 and October 2016, 758 eligible participants from 29 UK hospitals were randomised: 370 to rifampicin and 388 to placebo. The median age was 65 years [interquartile range (IQR) 50-76 years]. A total of 485 (64.0%) infections were community acquired and 132 (17.4%) were nosocomial; 47 (6.2%) were caused by meticillin-resistant S. aureus. A total of 301 (39.7%) participants had an initial deep infection focus. Standard antibiotics were given for a median of 29 days (IQR 18-45 days) and 619 (81.7%) participants received flucloxacillin. By 12 weeks, 62 out of 370 (16.8%) patients taking rifampicin versus 71 out of 388 (18.3%) participants taking the placebo experienced bacteriological (microbiologically confirmed) failure/recurrence or died [absolute risk difference -1.4%, 95% confidence interval (CI) -7.0% to 4.3%; hazard ratio 0.96, 95% CI 0.68 to 1.35; p = 0.81]. There were 4 (1.1%) and 5 (1.3%) bacteriological failures (p = 0.82) in the rifampicin and placebo groups, respectively. There were 3 (0.8%) versus 16 (4.1%) bacteriological recurrences (p = 0.01), and 55 (14.9%) versus 50 (12.9%) deaths without bacteriological failure/recurrence (p = 0.30) in the rifampicin and placebo groups, respectively. Over 12 weeks, there was no evidence of differences in clinically defined failure/recurrence/death (p = 0.84), all-cause mortality (p = 0.60), serious (p = 0.17) or grade 3/4 (p = 0.36) adverse events (AEs). However, 63 (17.0%) participants in the rifampicin group versus 39 (10.1%) participants in the placebo group experienced antibiotic or trial drug-modifying AEs (p = 0.004), and 24 (6.5%) participants in the rifampicin group versus 6 (1.5%) participants in the placebo group experienced drug-interactions (p = 0.0005). Evaluation of the costs and health-related quality-of-life impacts revealed that an episode of S. aureus bacteraemia costs an average of £12,197 over 12 weeks. Rifampicin was estimated to save 10% of episode costs (p = 0.14). After adjustment, the effect of rifampicin on total quality-adjusted life-years (QALYs) was positive (0.004 QALYs), but not statistically significant (standard error 0.004 QALYs). CONCLUSIONS: Adjunctive rifampicin provided no overall benefit over standard antibiotic therapy in adults with S. aureus bacteraemia. FUTURE WORK: Given the substantial mortality, other antibiotic combinations or improved source management should be investigated. TRIAL REGISTRATIONS: Current Controlled Trials ISRCTN37666216, EudraCT 2012-000344-10 and Clinical Trials Authorisation 00316/0243/001. FUNDING: This project was funded by the National Institute for Health Research (NIHR) Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 22, No. 59. See the NIHR Journals Library website for further project information.

AMR Surveillance in low and middle-income settings - A roadmap for participation in the Global Antimicrobial Surveillance System (GLASS).

Drug-resistant infections caused by bacteria with increasing antimicrobial resistance (AMR) threaten our ability to treat life-threatening conditions. Tackling AMR requires international collaboration and partnership. An early and leading priority to do this is to strengthen AMR surveillance, particularly in low-income countries where the burden of infectious diseases is highest and where data are most limited. The World Health Organization (WHO) has developed the Global AMR Surveillance System (GLASS) as one of a number of measures designed to tackle the problem of AMR, and WHO member states have been encouraged to produce National Action Plans for AMR by 2017. However, low-income countries are unlikely to have the resources or capacity to implement all the components in the GLASS manual. To facilitate their efforts, we developed a guideline that is aligned to the GLASS procedures, but written specifically for implementation in low-income countries. The guideline allows for flexibility across different systems, but has sufficient standardisation of core protocols to ensure that, if followed, data will be valid and comparable. This will ensure that the surveillance programme can provide health intelligence data to inform evidence-based interventions at local, national and international levels.

Severe infections emerge from commensal bacteria by adaptive evolution.

Bacteria responsible for the greatest global mortality colonize the human microbiota far more frequently than they cause severe infections. Whether mutation and selection among commensal bacteria are associated with infection is unknown. We investigated de novo mutation in 1163 Staphylococcus aureus genomes from 105 infected patients with nose colonization. We report that 72% of infections emerged from the nose, with infecting and nose-colonizing bacteria showing parallel adaptive differences. We found 2.8-to-3.6-fold adaptive enrichments of protein-altering variants in genes responding to rsp, which regulates surface antigens and toxin production; agr, which regulates quorum-sensing, toxin production and abscess formation; and host-derived antimicrobial peptides. Adaptive mutations in pathogenesis-associated genes were 3.1-fold enriched in infecting but not nose-colonizing bacteria. None of these signatures were observed in healthy carriers nor at the species-level, suggesting infection-associated, short-term, within-host selection pressures. Our results show that signatures of spontaneous adaptive evolution are specifically associated with infection, raising new possibilities for diagnosis and treatment.

Identifying lineage effects when controlling for population structure improves power in bacterial association studies.

Bacteria pose unique challenges for genome-wide association studies because of strong structuring into distinct strains and substantial linkage disequilibrium across the genome(1,2). Although methods developed for human studies can correct for strain structure(3,4), this risks considerable loss-of-power because genetic differences between strains often contribute substantial phenotypic variability(5). Here, we propose a new method that captures lineage-level associations even when locus-specific associations cannot be fine-mapped. We demonstrate its ability to detect genes and genetic variants underlying resistance to 17 antimicrobials in 3,144 isolates from four taxonomically diverse clonal and recombining bacteria: Mycobacterium tuberculosis, Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae. Strong selection, recombination and penetrance confer high power to recover known antimicrobial resistance mechanisms and reveal a candidate association between the outer membrane porin nmpC and cefazolin resistance in E. coli. Hence, our method pinpoints locus-specific effects where possible and boosts power by detecting lineage-level differences when fine-mapping is intractable.

Rapid antibiotic-resistance predictions from genome sequence data for Staphylococcus aureus and Mycobacterium tuberculosis.

The rise of antibiotic-resistant bacteria has led to an urgent need for rapid detection of drug resistance in clinical samples, and improvements in global surveillance. Here we show how de Bruijn graph representation of bacterial diversity can be used to identify species and resistance profiles of clinical isolates. We implement this method for Staphylococcus aureus and Mycobacterium tuberculosis in a software package ('Mykrobe predictor') that takes raw sequence data as input, and generates a clinician-friendly report within 3 minutes on a laptop. For S. aureus, the error rates of our method are comparable to gold-standard phenotypic methods, with sensitivity/specificity of 99.1%/99.6% across 12 antibiotics (using an independent validation set, n=470). For M. tuberculosis, our method predicts resistance with sensitivity/specificity of 82.6%/98.5% (independent validation set, n=1,609); sensitivity is lower here, probably because of limited understanding of the underlying genetic mechanisms. We give evidence that minor alleles improve detection of extremely drug-resistant strains, and demonstrate feasibility of the use of emerging single-molecule nanopore sequencing techniques for these purposes.

The usefulness of whole genome sequencing in the management of Staphylococcus aureus infections.

Staphylococcus aureus remains a leading cause of hospital-acquired and community-associated infection worldwide. The burden of disease is exacerbated by the emergence of virulent strains with reduced susceptibility to commonly used antibiotics and their dissemination in healthcare settings and in the community. Whole genome sequencing (WGS) has the potential to revolutionize our understanding and management of S. aureus infection. As a research tool, WGS has provided insights into the origins of antibiotic-resistant strains, the genetic basis of virulence, the emergence and spread of lineages, and the population structure of S. aureus. As a frontline tool, WGS offers the prospect of a method that could be used to predict resistance, assess virulence, and type isolates at the highest possible resolution. The results generated could be used to guide clinical management and infection control practice. Studies using bench-top sequencing machines have already demonstrated the feasibility of such approaches. Infection control management is compromised by our incomplete understanding of transmission, which in turn reflects the suboptimal resolution offered by conventional typing methods. As the costs of sequencing begin to approach those of conventional methods, high-resolution typing with WGS could realistically be implemented for hospital infection control, as well as for local and national surveillance practice. Translation into routine practice will require the development of a knowledge base, reliable automated bioinformatic tools, the capacity to store, exchange and interrogate large volumes of genomic data, and an acceptance of WGS by clinicians, infection control specialists, and laboratory staff.

A pilot study of rapid benchtop sequencing of Staphylococcus aureus and Clostridium difficile for outbreak detection and surveillance.

OBJECTIVES: To investigate the prospects of newly available benchtop sequencers to provide rapid whole-genome data in routine clinical practice. Next-generation sequencing has the potential to resolve uncertainties surrounding the route and timing of person-to-person transmission of healthcare-associated infection, which has been a major impediment to optimal management. DESIGN: The authors used Illumina MiSeq benchtop sequencing to undertake case studies investigating potential outbreaks of methicillin-resistant Staphylococcus aureus (MRSA) and Clostridium difficile. SETTING: Isolates were obtained from potential outbreaks associated with three UK hospitals. PARTICIPANTS: Isolates were sequenced from a cluster of eight MRSA carriers and an associated bacteraemia case in an intensive care unit, another MRSA cluster of six cases and two clusters of C difficile. Additionally, all C difficile isolates from cases over 6 weeks in a single hospital were rapidly sequenced and compared with local strain sequences obtained in the preceding 3 years. MAIN OUTCOME MEASURE: Whole-genome genetic relatedness of the isolates within each epidemiological cluster. RESULTS: Twenty-six MRSA and 15 C difficile isolates were successfully sequenced and analysed within 5 days of culture. Both MRSA clusters were identified as outbreaks, with most sequences in each cluster indistinguishable and all within three single nucleotide variants (SNVs). Epidemiologically unrelated isolates of the same spa-type were genetically distinct (≥21 SNVs). In both C difficile clusters, closely epidemiologically linked cases (in one case sharing the same strain type) were shown to be genetically distinct (≥144 SNVs). A reconstruction applying rapid sequencing in C difficile surveillance provided early outbreak detection and identified previously undetected probable community transmission. CONCLUSIONS: This benchtop sequencing technology is widely generalisable to human bacterial pathogens. The findings provide several good examples of how rapid and precise sequencing could transform identification of transmission of healthcare-associated infection and therefore improve hospital infection control and patient outcomes in routine clinical practice.