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Evolutionary history of the global emergence of the Escherichia coli epidemic clone ST131
ABSTRACT Background Escherichia coli sequence type 131 (ST131) has emerged globally as the most predominant lineage within this clinically important species, and its association with fluoroquinolone and extended-spectrum cephalosporin resistance impacts significantly on treatment. The evolutionary histories of this lineage, and of important antimicrobial resistance elements within it, remain unclearly defined. Results This study of the largest worldwide collection (n = 215) of sequenced ST131 E. coli isolates to date demonstrates that clonal expansion of two previously recognized antimicrobial-resistant clades, C1/H30R and C2/H30Rx, started around 25 years ago, consistent with the widespread introduction of fluoroquinolones and extended-spectrum cephalosporins in clinical medicine. These two clades appear to have emerged in the United States, with the expansion of the C2/H30Rx clade driven by the acquisition of a blaCTX-M-15-containing IncFII-like plasmid that has subsequently undergone extensive rearrangement. Several other evolutionary processes influencing the trajectory of this drug-resistant lineage are described, including sporadic acquisitions of CTX-M resistance plasmids, and chromosomal integration of blaCTX-M within sub-clusters followed by vertical evolution. These processes are also occurring for another family of CTX-M gene variants more recently observed amongst ST131, the blaCTX-M-14/14-like group. Conclusions The complexity of the evolutionary history of ST131 has important implications for antimicrobial resistance surveillance, epidemiological analysis, and control of emerging clinical lineages of E. coli. These data also highlight the global imperative to reduce specific antibiotic selection pressures, and demonstrate the important and varied roles played by plasmids and other mobile genetic elements in the perpetuation of antimicrobial resistance within lineages.
Insights into platypus population structure and history from whole-genome sequencing
AbstractThe platypus is an egg-laying mammal which, alongside the echidna, occupies a unique place in the mammalian phylogenetic tree. Despite widespread interest in its unusual biology, little is known about its population structure or recent evolutionary history. To provide new insights into the dispersal and demographic history of this iconic species, we sequenced the genomes of 57 platypuses from across the whole species range in eastern mainland Australia and Tasmania. Using a highly-improved reference genome, we called over 6.7M SNPs, providing an informative genetic data set for population analyses. Our results show very strong population structure in the platypus, with our sampling locations corresponding to discrete groupings between which there is no evidence for recent gene flow. Genome-wide data allowed us to establish that 28 of the 57 sampled individuals had at least a third-degree relative amongst other samples from the same river, often taken at different times. Taking advantage of a sampled family quartet, we estimated thede novomutation rate in the platypus at 7.0×10−9/bp/generation (95% CI 4.1×10−9− 1.2×10−8/bp/generation). We estimated effective population sizes of ancestral populations and haplotype sharing between current groupings, and found evidence for bottlenecks and long-term population decline in multiple regions, and early divergence between populations in different regions. This study demonstrates the power of whole-genome sequencing for studying natural populations of an evolutionarily important species.
Evaluation of an electronic clinical decision support algorithm to improve primary care management of acute febrile illness in rural Cambodia: protocol for a cluster-randomised trial
IntroductionAcute febrile illness (AFI), traditionally attributed to malaria, is a common reason for seeking primary healthcare in rural South and Southeast Asia. However, malaria transmission has declined while health workers are often poorly equipped to manage non-malarial AFIs. This results in indiscriminate antibiotic prescribing and care escalation, which promotes antibiotic resistance and may increase healthcare costs. To address this problem, an electronic clinical decision support algorithm (eCDSA) called ‘Electronic clinical Decision support for Acute fever Management (EDAM)’ has been developed for primary health workers which integrates clinical, epidemiological and vital sign data with simple point-of-care tests to produce a diagnosis and management plan.Methods and analysisThis is a pragmatic cluster-randomised trial aiming to assess the effect of EDAM and related training on antibiotic prescribing rates in rural Cambodian primary health centres (PHCs) as the primary outcome, along with a range of secondary outcomes including safety. Patients with AFI are eligible for recruitment if they are aged ≥1 year. A cluster is defined as a PHC and PHCs will be randomised to control (standard of care) and intervention (EDAM and associated training) arms, with 15 PHCs per arm. Patients will be followed up after 7 days to ascertain the safety profile of EDAM. Each PHC will recruit 152 patients (total 4560), based on a baseline antibiotic prescription rate of 25% and expected reduction to 17.5% with EDAM.Ethics and disseminationResults will be published in international peer-reviewed journals to inform the design of future versions of EDAM and of future trials of similar eCDSAs and other digital health interventions targeted towards rural populations. This study was approved by the Oxford University Tropical Research Ethics Committee (550-23) and the Cambodian National Ethics Committee for Health Research (395-NECHR).Trial registration numberInternational Standard Randomized Controlled Trial Number Registry (ISRCTN15157105).
Targeted capture and sequencing of Orientia tsutsugamushi genomes from chiggers and humans.
Scrub typhus is a febrile disease caused by Orientia tsutsugamushi, transmitted by larval stage Trombiculid mites (chiggers), whose primary hosts are small mammals. The phylogenomics of O. tsutsugamushi in chiggers, small mammals and humans remains poorly understood. To combat the limitations imposed by the low relative quantities of pathogen DNA in typical O. tsutsugamushi clinical and ecological samples, along with the technical, safety and cost limitations of cell culture, a novel probe-based target enrichment sequencing protocol was developed. The method was designed to capture variation among conserved genes and facilitate phylogenomic analysis at the scale of population samples. A whole-genome amplification step was incorporated to enhance the efficiency of sequencing by reducing duplication rates. This resulted in on-target capture rates of up to 93% for a diverse set of human, chigger, and rodent samples, with the greatest success rate in samples with real-time PCR Ct values below 35. Analysis of the best-performing samples revealed phylogeographic clustering at local, provincial and international scales. Applying the methodology to a comprehensive set of samples could yield a more complete understanding of the ecology, genomic evolution and population structure of O. tsutsugamushi and other similarly challenging organisms, with potential benefits in the development of diagnostic tests and vaccines.
Oxford Nanopore MinION sequencing enables rapid whole-genome assembly ofRickettsia typhiin a resource-limited setting
AbstractThe infrastructure challenges and costs of next-generation sequencing have been largely overcome, for many sequencing applications, by Oxford Nanopore Technologies’ portable MinION sequencer. However the question remains open whether MinION-based bacterial whole-genome sequencing (WGS) is by itself sufficient for the accurate assessment of phylogenetic and epidemiological relationships between isolates and whether such tasks can be undertaken in resource-limited settings. To investigate this question, we sequenced the genome of an isolate ofRickettsia typhi, an important and neglected cause of fever across much of the tropics and subtropics, for which only three genomic sequences previously existed. We prepared and sequenced libraries on a MinION in Vientiane, Lao PDR using v9.5 chemistry and in parallel we sequenced the same isolate on the Illumina platform in a genomics laboratory in the UK. The MinION sequence reads yielded a single contiguous assembly, in which the addition of Illumina data revealed 226 base-substitution and 5,856 in/del errors. The combined assembly represents the first complete genome sequence of a humanR. typhiisolate collected in the last 50 years and differed from the genomes of existing strains collected over a 90-year time period at very few sites, and with no re-arrangements. Filtering based on the known error profile of MinION data improved the accuracy of the Nanopore-only assembly. However, the frequency of false-positive errors remained greater than true sequence divergence from recorded sequences. While Nanopore-only sequencing cannot yet recover phylogenetic signal inR. typhi, such an approach may be applicable for more diverse organisms.
Organization and client perspectives on homelessness in Boston, MA during the COVID-19 Pandemic: A descriptive qualitative study
The Coronavirus Disease 2019 (COVID-19) pandemic has placed a magnifying glass on what we have been seeing for a long time. With large-scale impacts across the globe, an increased burden has been placed on people experiencing homelessness who already face barriers to necessities such as housing, food, and quality health care. This qualitative descriptive study explored the experiences of people experiencing homelessness during the COVID-19 pandemic and of those working for supporting organizations in the Greater Boston Area between July-November 2020. Additionally, the study identified key recommendations for policymakers or service providers to use in creating inclusive policies and emergency response plans that consider the needs of the homeless community. The study comprised individual interviews and focus group discussions from two groups: 1) employees from supporting organizations (medical organizations, shelters, universities, and housing organizations) and 2) people experiencing homelessness. An inductive content analysis of interview transcripts was conducted to identify emerging themes and recommendations. Community dialogues were held with participants to confirm results and explore any new potential topics or recommendations. Four key themes were identified from interviews: 1) Social inclusion, 2) Services and resources, 3) Community support and collaboration, and 4) Government response and policy. Overall, the COVID-19 pandemic has highlighted major gaps in existing support for people experiencing homelessness on the state and federal levels while also emphasizing the importance of community support and collaboration for this vulnerable population. Further work must be conducted to develop and implement more inclusive policies and regulations to support this community.
Experiences, perceptions and ethical considerations of the malaria infection study in Thailand.
BackgroundThailand has made significant progress in malaria control efforts in the past decade, with a decline in the number of reported cases. However, due to cross-border movements over the past 5 years, reported malaria cases in Thailand have risen. The Malaria Infection Study in Thailand (MIST) involves deliberate infection of healthy volunteers with Plasmodium vivax malaria parasites, and the assessment of the efficacy of potential vaccine and drug candidates in order to understand acquired protection against malaria parasites.MethodsThis paper drew from ethics and social science qualitative study called MIST-ETHICS embedded within the MIST studies. MIST-ETHICS aimed to describe and understand the experiences, perceptions and ethical considerations of the MIST studies. Data were obtained from semi-structured interviews and a focus group discussion. A total of 46 participants participated in MIST-ETHICS .ResultsThree major themes emerged: experiences and perceptions of MIST, reasons for joining MIST, and ethical considerations. We found that although compensation was a motivation for participation, this was secondary to it being beneficial to self (health checks; link to health networks; building merit) and others (medical research contribution; altruism). Participants expressed varied opinions regarding the requirement of a university degree as one of the inclusion criteria for MIST.ConclusionsOur study revealed widespread concerns about long-term health effects and safety. Ethical considerations, including obtaining valid informed consent and ensuring participant inclusivitiy, were deem essential. Despite some debate regarding eligibility criteria, most participants agreed that the informed consent process was robust, accompanied by a strong sense of responsibility to contribute to the greater good. We emphasize the importance of continuously gathering participants' feedback for quality control, such as improving information materials to clarify the purpose of initial phases, their contributing to later phases, and the rationale behind each selection criterion.Trial registrationThis manuscript is part of the clinical trials registered under ClinicalTrials.gov IDs NCT04083508 (MIST1) registered on 5 Sep 2019 and NCT05071079 (MIST2) registered on 28 July 2021. However, the manuscript pertains to a qualitative study that does not require trial registration.
Cross-sectional evaluation of host biomarkers for guiding antibiotic use in bacterial and non-bacterial acute febrile illness in low- and middle-income tropical settings.
OBJECTIVES: To evaluate the effectiveness of 18 different host biomarkers in differentiating bacterial from non-bacterial acute febrile illness (AFI) in resource-limited settings, specifically in Brazil, Malawi and Gabon. DESIGN: Multinational, cross-sectional study. SETTING: The study was carried out across multiple primary healthcare facilities, including urban and rural settings, with a total of three participating centres. Recruitment took place from October 2018 to July 2019 in Brazil, May to November 2019 in Gabon and April 2017 to April 2018 in Malawi. PARTICIPANTS: A total of 1915 participants, including children and adults aged 21-65 years with a fever of≤7 days, were recruited through convenience sampling from outpatient clinics in Brazil, Gabon and Malawi. Individuals with signs of severe illness were excluded. Written consent was obtained from all participants or their guardians. INTERVENTION: This is not applicable as the study primarily focused on biomarker evaluation without specific therapeutic interventions. PRIMARY AND SECONDARY OUTCOME MEASURES: The primary outcome measure was the ability of each host biomarker to differentiate between bacterial and non-bacterial AFI, as evaluated by area under the receiver operating characteristic (AUROC) curves. Secondary outcomes included the performance of individual biomarkers across the different study sites and in a multivariable setting. RESULTS: A Kruskal-Wallis test, adjusted by Benjamini-Hochberg, was performed for each biomarker to identify covariates with a significant difference in the distribution of biomarker values. The analysis revealed that country of origin (Brazil, Gabon, Malawi), age, sex and malaria status significantly impacted biomarker distribution (p≤0.001). The most widely known biomarkers, such as white blood cell (WBC) count and C-reactive protein (CRP), demonstrated the best performance in distinguishing between bacterial and non-bacterial infections, with AUROCs reaching up to 0.83 (0.77-0.88) for WBC count and 0.71 (0.59-0.82) for CRP. However, none of the evaluated novel host biomarkers exhibited high performance (AUROC<0.70 in most cases) and variations in biomarker performance were observed across the three settings. Multivariable analyses demonstrated that while the best combination of biomarkers achieved higher AUROCs, the increase was modest (1-13%), suggesting that the interaction of biomarkers contributed minimally to predictive accuracy. CONCLUSIONS: There is a continued need for innovation in the host-biomarker space as the available markers do not meet the needs of diverse populations around the globe. This highlights the importance of targeted evaluations in non-severe patients in multiple settings to understand the true potential for real-life use. The findings highlight that not one-marker fits all settings and novel innovations remain urgently needed. TRIAL REGISTRATION NUMBER: Clinical trial number: NCT03047642.
COVID-19 Transmission among Healthcare Workers at a Quarantine Facility in Thailand: Genomic and Outbreak Investigations.
During the COVID-19 pandemic, Thailand implemented a quarantine program at approved quarantine facilities for every international traveler. Here, we report an epidemiological and genomic investigation of a COVID-19 cluster consisting of seven healthcare workers (HCWs) at a quarantine facility and its partnered hospital in Thailand. Outbreak investigations were implemented to obtain contact tracing data and to establish chains of transmission. Genomic sequencing of SARS-CoV-2 with samples within the cohort was performed. Investigations of 951 HCWs and staff with quarantined travelers were implemented to determine the chain of transmission. Genomic and outbreak investigations identified the international travelers infected with the B.1.1.31 SARS-CoV-2 lineage as the source of this outbreak. The genomic data and the investigated timeline revealed a putative transmission chain among HCWs, pointing toward the transmission via the use of common living quarters at the investigated quarantine site. The evaluation of this cohort has led to a policy recommendation on quarantine facility management. International travel quarantine is an important strategy to contain importation of COVID-19 cases. However, a quarantine facility is likely to become a potential hotspot, requiring thorough preventive measures. Reducing the exposure risk by providing private living quarters and scheduling clinical duties at a quarantine site separated from the conventional healthcare workforce have been implemented.
Genomic surveillance of SARS-CoV-2 in Thailand reveals mixed imported populations, a local lineage expansion and a virus with truncated ORF7a.
Coronavirus Disease 2019 (COVID-19) is a global public health threat. Genomic surveillance of SARS-CoV-2 was implemented in March of 2020 at a major diagnostic hub in Bangkok, Thailand. Several virus lineages supposedly originated in many countries were found, and a Thai-specific lineage, designated A/Thai-1, has expanded to be predominant in Thailand. A virus sample in the SARS-CoV-2 A/Thai-1 lineage contains a frame-shift deletion at ORF7a, encoding a putative host antagonizing factor of the virus.
Comparative analysis of targeted next-generation sequencing for Plasmodium falciparum drug resistance markers.
Well-defined molecular resistance markers are available for a range of antimalarial drugs, and molecular surveillance is increasingly important for monitoring antimalarial drug resistance. Different genotyping platforms are available, but these have not been compared in detail. We compared Targeted Amplicon Deep sequencing (TADs) using Ion Torrent PGM with Illumina MiSeq for the typing of antimalarial drug resistance genes. We developed and validated protocols to type the molecular resistance markers pfcrt, pfdhfr, pfdhps, pfmdr1, pfkelch, and pfcytochrome b, in Plasmodium falciparum for the Ion Torrent PGM and Illumina MiSeq sequencing platforms. With P. falciparum 3D7 and K1 as reference strains, whole blood samples (N = 20) and blood spots from Rapid Diagnostic Test (RDT) samples (N = 5) from patients with uncomplicated falciparum malaria from Ubon Ratchathani were assessed on both platforms and compared for coverage (average reads per amplicon), sequencing accuracy, variant accuracy, false positive rate, false negative rate, and alternative allele detection, with conventional Sanger sequencing as the reference method for SNP calling. Both whole blood and RDT samples could be successfully sequenced using the Ion Torrent PGM and Illumina MiSeq platforms. Coverage of reads per amplicon was higher with Illumina MiSeq (28,886 reads) than with Ion Torrent PGM (1754 reads). In laboratory generated artificial mixed infections, the two platforms could detect the minor allele down to 1% density at 500X coverage. SNPs calls from both platforms were in complete agreement with conventional Sanger sequencing. The methods can be multiplexed with up to 96 samples per run, which reduces cost by 86% compared to conventional Sanger sequencing. Both platforms, using the developed TAD protocols, provide an accurate method for molecular surveillance of drug resistance markers in P. falciparum, but Illumina MiSeq provides higher coverage than Ion Torrent PGM.
AmpC β-lactamases detected in Southeast Asian Escherichia coli and Klebsiella pneumoniae
Abstract Objectives AmpC β-lactamases are neglected compared with ESBL as a cause of third-generation cephalosporin (3GC) resistance in Enterobacterales in low- and middle-income countries and the burden is unknown. The aim of this study was to investigate the presence of AmpC β-lactamase-producing Escherichia coli and Klebsiella pneumoniae in clinical specimens from three clinical research laboratories in Southeast Asia. Methods Stored clinical isolates of E. coli and K. pneumoniae resistant to ceftriaxone or ceftazidime or cefpodoxime and ESBL confirmation test negative were screened using MASTDISCS AmpC, ESBL and Carbapenemase Detection Set—D72C. Short-read WGS was performed to identify ampC genes. Results Of 126 isolates collected between 2010 and 2020, 31 (24.6%) and 16 (12.7%) were phenotypically AmpC and inducible AmpC positive by MASTDISCS testing, respectively. All inducible AmpC isolates were ceftriaxone susceptible and 97.7% of AmpC/inducible AmpC isolates tested against cefoxitin were resistant. Through WGS, 17 and eight different STs were detected for the AmpC/inducible AmpC E. coli and K. pneumoniae isolates, respectively. Twelve different β-lactamase resistance genes were detected, with blaCMY-2 most commonly in AmpC-positive isolates (20/31; 64.5%; 15 chromosomal, five plasmid). All inducible AmpC-positive isolates had the blaDHA-1 gene (seven chromosomal, nine plasmid). Conclusions Though uncommon, AmpC and inducible AmpC β-lactamases in E. coli and K. pneumoniae are an important cause of infection in Southeast Asia. With current testing methods, these infections may be going undetected, resulting in patients receiving suboptimal treatment.
Molecular diagnosis and genotyping of Orientia tsutsugamushi in Maesot and Chiangrai, Thailand
IntroductionScrub typhus is a neglected tropical disease with an estimated 1 million cases annually. The Asia-Pacific region is an endemic area for scrub typhus, especially in Thailand.MethodsBetween June 2018 and December 2019, 31 patients with acute undifferentiated febrile illness (AUFI) were recruited for clinical trials and tested positive by a scrub typhus IgM RDT.ResultsOf the 17 buffy coat patient samples tested by 47kDa real-time PCR and 56kDa type-specific antigen (TSA) nested PCR, 94% (16/17) were positive, and of the 11 patients that presented with eschar lesions, 100% (11/11) of the eschar samples were confirmed positive. Genetic analysis of the 560 bp partial 56-kDa TSA gene demonstrated that most Orientia tsutsugamushi (Ot) infections were with Karp, Gilliam, Taiwan, P23, and CM606-like strains.DiscussionThis is the second occasion that the CM606-like and P23-like strains were reported in northern Thailand (first reported in 2011 and 2013, respectively). This study demonstrates that 1) the eschar remains the most reliable biological sample for PCR diagnosis of scrub typhus and 2) Northwestern Thailand has significant diversity of Ot strains, which underlines the requirement for ongoing surveillance to increase our understanding of Ot diversity to ensure accurate diagnostics and treatment.
Detection of maternal transmission of resistant Gram-negative bacteria in a Cambodian hospital setting
Infection with Extended-spectrum beta-lactamase -producing Enterobacterales (ESBL-E) is common in infants and leads to increased intensive care unit admission and mortality, but the role of maternal transmission in colonization of infants is unclear. Using paired isolates from 50 pairs of mothers and neonates admitted to a Cambodian hospital, we investigated antimicrobial resistance in Escherichia coli and Klebsiella pneumoniae using whole genome sequencing. We detected a wide variety of ESBL-E genes present in this population along with high levels of multidrug resistance. From 21 pairs where the same organism was present in both mother and neonate, we identified eight pairs with identical or near-identical isolates from both individuals suggestive of transmission at or around birth, including a pair with transmission of multiple strains. We found no evidence for transmission of plasmids only from mother to infant. This suggests vertical transmission outside hospitals as a common cause of ESBL-E colonization in neonates.
Clinical Antiviral Efficacy of Remdesivir in Coronavirus Disease 2019: An Open-Label, Randomized Controlled Adaptive Platform Trial (PLATCOV)
Abstract Background Uncertainty over the therapeutic benefit of parenteral remdesivir in coronavirus disease 2019 (COVID-19) has resulted in varying treatment guidelines. Methods In a multicenter open-label, controlled, adaptive, pharmacometric platform trial, low-risk adult patients with early symptomatic COVID-19 were randomized to 1 of 8 treatment arms including intravenous remdesivir (200 mg followed by 100 mg daily for 5 days) or no study drug. The primary outcome was the rate of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) clearance (estimated under a linear model fit to the daily log10 viral densities, days 0–7) in standardized duplicate oropharyngeal swab eluates, in a modified intention-to-treat population. This ongoing adaptive trial is registered at ClinicalTrials.gov (NCT05041907). Results The 2 study arms enrolled 131 patients (remdesivir n = 67, no study drug n = 64) and estimated viral clearance rates from a median of 18 swab samples per patient (a total of 2356 quantitative polymerase chain reactions). Under the linear model, compared with the contemporaneous control arm (no study drug), remdesivir accelerated mean estimated viral clearance by 42% (95% credible interval, 18%–73%). Conclusions Parenteral remdesivir accelerates viral clearance in early symptomatic COVID-19. Pharmacometric assessment of therapeutics using the method described can determine in vivo clinical antiviral efficacy rapidly and efficiently.
Geographical mapping and seroprevalence of Burkholderia pseudomallei amongst livestock species in Lao People's Democratic Republic.
The Gram-negative bacterium Burkholderia pseudomallei causes a severe infectious disease known as melioidosis in humans and animals. It is considered endemic in tropical countries, including Thailand, Lao PDR (Laos), and Northern Australia. B. pseudomallei is a saprophyte found in contaminated soil and surface water. Humans and animals can become infected via direct exposure to contaminated water or soil and inhalation of dust or water droplets. Despite the high morbidity and mortality rates of melioidosis, there is a lack of knowledge of its geographical distribution and seroprevalence, even within endemic countries, raising a significant public health concern. For a better understanding of melioidosis in livestock in Laos, both as an animal health concern and as an indicator of human risk, we collected serum samples from an abattoir monitoring program for B. pseudomallei antibody testing using the Indirect Haemagglutination Assay (IHA). Out of the 917 sera collected, major findings included the identification of a significant cluster (p = 0.041) in the southwest border region adjoining northeastern Thailand, in the province of Savannakhet in Laos. Sera collected in January 2020 had the highest B. pseudomallei seroprevalence (17.0%), and cattle had the highest seroprevalence (22.8%), followed by buffalo (19.7%) and swine (4.0%). The B. pseudomallei seroprevalence results among the common livestock species and the maps generated can assist with future monitoring, prevention, and detection of melioidosis in Laos.