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We have developed an efficient and inexpensive pipeline for streamlining large-scale collection and genome sequencing of bacterial isolates. Evaluation of this method involved a worldwide research collaboration focused on the model organism Salmonella enterica, the 10KSG consortium. By optimising a logistics pipeline that collected isolates as thermolysates, permitting shipment in ambient conditions, the project assembled a diverse collection of 10,419 clinical and environmental isolates from low- and middle-income countries in less than one year. The bacteria were obtained from 51 countries/territories dating from 1949 to 2017, with a focus on Africa and Latin-America. All isolates were collected in barcoded tubes and genome sequenced using an optimised DNA extraction method and the LITE pipeline for library construction. After Illumina sequencing, the total reagent cost was less than USD$10 per genome. Our method can be applied to genome-sequence other large bacterial collections at a relatively low cost, within a limited timeframe, to support global collaborations.

Background Uropathogenic Escherichia coli (UPEC) are amongst the most frequent causes of urinary tract infections. We report a systematic review and meta-analysis of virulence factors and antimicrobial resistance of UPEC isolated from urinary tract infections. Methods A systematic review and meta-analysis were performed using PRISMA guidelines (Research Registry ref. 5874). Data were extracted from PubMed/MEDLINE and ScienceDirect databases for studies published from January 1, 2000 to December 31, 2019. Studies reporting antimicrobial resistance and virulence factors of UPEC isolated in confirmed urinary tract infections (≥105CFU/ml) were eligible. Prevalence of antimicrobial resistance and virulence factors of UPEC were estimated using random-effects meta-analysis model. Estimates with 95% confidence intervals, I-square (I2) statistic, and Cochran’s Q test were computed using the score statistic and the exact binomial method by incorporating the Freeman-Tukey double arcsine transformation of proportions. Results Our search returned 2504 hits, of which 13 studies were included in the meta-analysis, totalling 1888 UPEC isolates. Highest antimicrobial resistance rates were observed among the antibiotic class of tetracycline in 69.1% (498/721), followed by sulphonamides in 59.3% (1119/1888), quinolones in 49.4% (1956/3956), and beta-lactams in 36.9% (4410/11964). Among beta-lactams, high resistance was observed in aminopenicillins in 74.3% (1157/1557) and first generation cephalosporins in 38.8% (370/953). Meanwhile, virulence factors with highest prevalence were immune suppressors (54.1%) followed by adhesins (45.9%). Taken individually, the most observed virulence genes were shiA (92.1%), CSH (80.0%), fimH/MSHA (75.3%), traT (75.1%), sisA (72.2%), iucD (65.7%), iutA (61.8%), kpsMTII (60.6%), and PAI (55.2%). Conclusions The increased antibiotic resistance of UPEC isolates was demonstrated and suggested a need for reassessment of empirical therapies in urinary tract infections treatment caused by this pathogen. In addition, this pathotype exhibited diverse surface and secreted virulence factors.

BackgroundLow- and middle-income countries (LMICs) have high morbidity and mortality from drug-resistant infections, especially from enteric bacteria such as Escherichia coli. LMICs have varying infrastructure and services in the community to separate people from human and animal waste, creating risks for ESBL-Enterobacterales (ESBL-E) transmission. Limited data exist from Southern Africa on the prevalence of ESBL-E the community.Methods and findingsIn this longitudinal cohort study we took a one-health approach to investigating prevalence and distribution of ESBL-E in urban, peri-urban and rural Malawian households between May 2018 and October 2020. We described human health, antibiotic usage (ABU), health seeking behaviour, structural and behavioural environmental health practices, and animal husbandry at these households. In parallel, human and animal stool and diverse environmental samples were collected and cultured to identify presence of ESBL E. coli and ESBL K. pneumoniae. Univariable and multivariable analysis was performed to determine associations with human ESBL-E colonisation.We recruited 300 households, totalling 841 visits, and a paucity of environmental health infrastructure and materials for safe sanitation was noted across all sites. In total, 11,975 samples were cultured and ESBL-E were isolated from 41.8% (n=1190) of human stool and 29.8% (n=290) of animal stool samples. Animal species with particularly high rates of ESBL-E colonisation included pigs (56.8%, n=21) poultry (32.5%, n=148) and dogs (58.8% n= 30). ESBL-E were isolated from 66.2% (n=339) of river water samples and 46.0% (n=138) of drain samples. Urban areas had greater ESBL-E contamination of food, household surfaces, floors and the external environment, alongside the highest rates of ESBL-E colonisation in humans (47.1%, n=384) and animals (55.1%, n=65). Multivariable models illustrated that human ESBL E. coli colonisation was associated with the wet season (aOR = 1.66, 95%CrI: 1.38-2.00), living in urban areas (aOR = 2.01, 95%CrI: 1.26-3.24), advanced age (aOR = 1.14, 95%CrI: 1.05-1.24) and in households where animals were observed interacting with food (aOR = 1.62, 95%CrI: 1.17-2.28) or kept inside (aOR = 1.58, 95%CrI: 1.00-2.43). Human ESBL K. pneumoniae colonisation was also associated with the wet season (aOR = 2.23, 95%CrI: 1.63-2.76.ConclusionWe identified extremely high levels of ESBL-E colonisation in humans and animals and contamination of the environment in Southern Malawi. Urbanisation and season are key risks for ESBL-E colonisation, perhaps reflecting environmental contamination as toilets overflow in high population density areas in heavy rains in the wet season. Without adequate efforts to improve environmental health, ESBL transmission is likely to persist in this setting.

2.AbstractSalmonella Enteritidis is one of the most commonly reported serovars of non-typhoidal Salmonella causing human disease and is responsible for both gastroenteritis and invasive non-typhoidal Salmonella (iNTS) disease worldwide. Whole-genome sequence (WGS) comparison of Salmonella Enteritidis isolates from across the world have identified three distinct clades, named Global Epidemic, Central/East African and West African, all of which have been implicated in epidemics: the Global Epidemic clade was linked to poultry-associated gastroenteritis, while the two African clades were related to iNTS disease. However, the distribution and epidemiology of these clades across Africa is poorly understood because identification of these clades currently requires whole genome sequencing capacity. Here, we report a sensitive, time- and cost-effective real-time PCR assay capable of differentiating between the Salmonella Enteritidis clades to facilitate surveillance and to inform public health responses.3.Impact statementChallenges in the diagnosis and treatment of invasive Salmonella Enteritidis (S. Enteritidis) bloodstream infections in sub–Saharan Africa are responsible for a case fatality rate of approximately 15% (12). It is important to identify distinct clades of S. Enteritidis in diagnostic laboratories in the African setting to determine whether particular outbreaks are associated with different health outcomes. Here, we have described the development of a high-quality molecular classification assay for the clade-typing of S. Enteritidis that is ideal for use in public health laboratories in resource-limited settings.

Background Diverse environmental exposures and risk factors have been implicated in the transmission of Salmonella Typhi, but the dominant transmission pathways through the environment to susceptible humans remain unknown. Here, we use spatial, bacterial genomic, and hydrological data to refine our view of typhoid transmission in an endemic setting. Methods A total of 546 patients presenting to Queen Elizabeth Central Hospital in Blantyre, Malawi, with blood culture–confirmed typhoid fever between April 2015 and January 2017 were recruited to a cohort study. The households of a subset of these patients were geolocated, and 256 S. Typhi isolates were whole-genome sequenced. Pairwise single-nucleotide variant distances were incorporated into a geostatistical modeling framework using multidimensional scaling. Results Typhoid fever was not evenly distributed across Blantyre, with estimated minimum incidence ranging across the city from 100 cases per 100 000 population per year. Pairwise single-nucleotide variant distance and physical household distances were significantly correlated (P = .001). We evaluated the ability of river catchment to explain the spatial patterns of genomics observed, finding that it significantly improved the fit of the model (P = .003). We also found spatial correlation at a smaller spatial scale, of households living Conclusions These findings reinforce the emerging view that hydrological systems play a key role in the transmission of typhoid fever. By combining genomic and spatial data, we show how multifaceted data can be used to identify high incidence areas, explain the connections between them, and inform targeted environmental surveillance, all of which will be critical to shape local and regional typhoid control strategies.

Bloodstream infections caused by nontyphoidal Salmonella are a major public health concern in Africa, causing ~49,600 deaths every year. The most common Salmonella enterica pathovariant associated with invasive nontyphoidal Salmonella disease is Salmonella Typhimurium sequence type (ST)313. It has been proposed that antimicrobial resistance and genome degradation has contributed to the success of ST313 lineages in Africa, but the evolutionary trajectory of such changes was unclear. Here, to define the evolutionary dynamics of ST313, we sub-sampled from two comprehensive collections of Salmonella isolates from African patients with bloodstream infections, spanning 1966 to 2018. The resulting 680 genome sequences led to the discovery of a pan-susceptible ST313 lineage (ST313 L3), which emerged in Malawi in 2016 and is closely related to ST313 variants that cause gastrointestinal disease in the United Kingdom and Brazil. Genomic analysis revealed degradation events in important virulence genes in ST313 L3, which had not occurred in other ST313 lineages. Despite arising only recently in the clinic, ST313 L3 is a phylogenetic intermediate between ST313 L1 and L2, with a characteristic accessory genome. Our in-depth genotypic and phenotypic characterization identifies the crucial loss-of-function genetic events that occurred during the stepwise evolution of invasive S. Typhimurium across Africa., Stepwise evolution of invasive Salmonella Typhimurium in Africa is defined using genotypic and phenotypic analyses of isolates collected over a 50-yr period.

Methods A priority-setting process (PSP) was launched to define priorities for patient-centred antimicrobial resistance (AMR) surveillance and research in low- and middle-income countries (LMICs). A list of uncertainties related to AMR surveillance in human health was generated using an online survey of stakeholders in LMICs which asked for unanswered questions about diagnosis, treatment or prevention of antibiotic resistance. Results A total of 445 respondents generated 1076 questions that were mapped to a final shortlist of 107 questions. The most common theme was the treatment of drug-resistant infections, followed by diagnosis, then prevention and then requests for local AMR data. The most asked question was a request for local AMR data, revealing the lack of basic information in many LMICs to guide actions to tackle AMR. The steering group recommended three research areas to be prioritised for funding in the next five years: infection prevention and control in LMICs, improved electronic patient records, starting with laboratory information management systems, and sustainable behavior change among doctors and other health care professionals with a focus on diagnostic stewardship. Keywords Antimicrobial resistance; surveillance; drug-resistant infections; priority-setting; low- and middle-income countries

Background Typhoid fever remains a major source of morbidity and mortality in low-income settings. Its most feared complication is intestinal perforation. However, due to the paucity of diagnostic facilities in typhoid-endemic settings, including microbiology, histopathology, and radiology, the etiology of intestinal perforation is frequently assumed but rarely confirmed. This poses a challenge for accurately estimating burden of disease. Methods We recruited a prospective cohort of patients with confirmed intestinal perforation in 2016 and performed enhanced microbiological investigations (blood and tissue culture, plus tissue polymerase chain reaction [PCR] for Salmonella Typhi). In addition, we used a Poisson generalized linear model to estimate excess perforations attributed to the typhoid epidemic, using temporal trends in S. Typhi bloodstream infection and perforated abdominal viscus at Queen Elizabeth Central Hospital from 2008–2017. Results We recruited 23 patients with intraoperative findings consistent with intestinal perforation. 50% (11/22) of patients recruited were culture or PCR positive for S. Typhi. Case fatality rate from typhoid-associated intestinal perforation was substantial at 18% (2/11). Our statistical model estimates that culture-confirmed cases of typhoid fever lead to an excess of 0.046 perforations per clinical typhoid fever case (95% CI, .03–.06). We therefore estimate that typhoid fever accounts for 43% of all bowel perforation during the period of enhanced surveillance. Conclusions The morbidity and mortality associated with typhoid abdominal perforations are high. By placing clinical outcome data from a cohort in the context of longitudinal surgical registers and bacteremia data, we describe a valuable approach to adjusting estimates of the burden of typhoid fever.

The burden of antimicrobial resistance is a major threat to global health; however, prospective clinical outcome data from Africa are scarce. In Malawi, third-generation cephalosporins are the antibiotics of choice in patients admitted to hospital despite a rapid proliferation of resistance to these drugs. We aimed to quantify the effect of resistance to third-generation cephalosporins on mortality and length of hospital stay among patients with bloodstream infections.We did a prospective cohort study of patients admitted to Queen Elizabeth Central Hospital in Blantyre, Malawi. Patients of all ages who had positive blood cultures for Enterobacterales were included, with the exception of those from the genus Salmonella, and were followed up for 180 days. We characterised blood culture isolates using whole-genome sequencing and used Cox regression models to estimate the effect of resistance to third-generation cephalosporins on length of hospital stay, in-hospital mortality, and survival.Between Jan 31, 2018, and Jan 13, 2020, we recruited 326 patients, from whom 220 (68%) of 326 isolates were resistant to third-generation cephalosporins. The case fatality proportion was 45% (99 of 220) in patients with bloodstream infections that were resistant to third-generation cephalosporins, and 34% (36 of 106) in patients with bloodstream infections that were sensitive to third-generation cephalosporins. Resistance to third-generation cephalosporins was associated with an increased probability of in-hospital mortality (hazard ratio [HR] 1·44, 95% CI 1·02-2·04), longer hospital stays (1·5 days, 1·0-2·0) and decreased probability of discharge alive (HR 0·31, 0·22-0·45). Whole-genome sequencing showed a high diversity of sequence types of both Escherichia coli and Klebsiella pneumoniae. Although isolates associated with death were distributed across clades, we identified three E coli clades (ST410, ST617, and ST648) that were isolated from 14 patients who all died.Resistance to third-generation cephalosporins is associated with increased mortality and longer hospital stays in patients with bloodstream infections in Malawi. These data show the urgent need for allocation of resources towards antimicrobial resistance mitigation strategies in Africa.Wellcome Trust and Wellcome Asia and Africa Programme.

Members of the Klebsiella pneumoniae species complex, particularly K. pneumoniae subsp. pneumoniae are antimicrobial resistance (AMR) associated pathogens of global importance, and polyvalent vaccines targeting Klebsiella O-antigens are in development. Whole-genome sequencing has provided insight into O-antigen distribution in the K. pneumoniae species complex, as well as population structure and virulence determinants, but genomes from sub-Saharan Africa are underrepresented in global sequencing efforts. We therefore carried out a genomic analysis of extended-spectrum beta-lactamase (ESBL)-producing K. pneumoniae species complex isolates colonizing adults in Blantyre, Malawi. We placed these isolates in a global genomic context, and compared colonizing to invasive isolates from the main public hospital in Blantyre. In total, 203 isolates from stool and rectal swabs from adults were whole-genome sequenced and compared to a publicly available multicounty collection and previously sequenced Malawian and Kenyan isolates from blood or sterile sites. We inferred phylogenetic relationships and analysed the diversity of genetic loci linked to AMR, virulence, capsule and LPS O-antigen (O-types). We find that the diversity of Malawian K. pneumoniae subsp. pneumoniae isolates represents the species’ population structure, but shows distinct local signatures concerning clonal expansions. Siderophore and hypermucoidy genes were more frequent in invasive versus colonizing isolates (present in 13 % vs 1 %) but still generally lacking in most invasive isolates. O-antigen population structure and distribution was similar in invasive and colonizing isolates, with O4 more common (14%) than in previously published studies (2–5 %). We conclude that host factors, pathogen opportunity or alternate virulence loci not linked to invasive disease elsewhere are likely to be the major determinants of invasive disease in Malawi. Distinct ST and O-type distributions in Malawi highlight the need to sample locations where the burden of invasive Klebsiella disease is greatest to robustly define secular trends in Klebsiella diversity to assist in the development of a useful vaccine. Colonizing and invasive isolates in Blantyre are similar, hence O-typing of colonizing Klebsiella isolates may be a rapid and cost-effective approach to describe global diversity and guide vaccine development.

In sub-Saharan Africa (sSA), there is high morbidity and mortality from severe bacterial infection and this is compounded by antimicrobial resistance, in particular, resistance to 3rd-generation cephalosporins. This resistance is typically mediated by extended-spectrum beta lactamases (ESBLs). To interrupt ESBL transmission it will be important to investigate how human behaviour, water, sanitation, and hygiene (WASH) practices, environmental contamination, and antibiotic usage in both urban and rural settings interact to contribute to transmission of ESBL E. coli and ESBL K. pneumoniae between humans, animals, and the environment. Here we present the protocol for the Drivers of Resistance in Uganda and Malawi (DRUM) Consortium, in which we will collect demographic, geospatial, clinical, animal husbandry and WASH data from a total of 400 households in Uganda and Malawi. Longitudinal human, animal and environmental sampling at each household will be used to isolate ESBL E. coli and ESBL K. pneumoniae. This will be complimented by a Risks, Attitudes, Norms, Abilities and Self-Regulation (RANAS) survey and structured observations to understand the contextual and psychosocial drivers of regional WASH practices. Bacterial isolates and plate sweeps will be further characterised using a mixture of short-,long-read and metagenomic whole-genome sequencing. These datasets will be integrated into agent-based models to describe the transmission of EBSL resistance in Uganda and Malawi and allow us to inform the design of interventions for interrupting transmission of ESBL-bacteria.

Background: Neonatal sepsis is responsible for a considerable burden of morbidity and mortality in sub-Saharan African countries. Outcomes from neonatal sepsis are worsening due to increasing rates of antimicrobial resistance. Sub-optimal Infection Prevention and Control (IPC) practices of health care workers and caregivers are important drivers of infection transmission. The Chatinkha Neonatal Unit at Queen Elizabeth Central Hospital, Blantyre, Malawi has experienced multiple outbreaks of neonatal sepsis, associated with drug resistant Klebsiella pneumoniae. We aimed to understand the barriers to implementation of optimal IPC focusing on hand hygiene practice. Methods: We used a qualitative research methodology to meet the study aim. Combining participant observation (PO) over a seven-month period with semi structured interviews (SSI) to provide an in-depth understanding of activities relating to hygiene and IPC existing on the ward. Results: While most staff and some caregivers, had a good understanding of ideal IPC and understood the importance of good handwashing practices, they faced substantial structural limitations, and scarce resources (both material and human) which made implementation challenging. For staff, the overwhelming numbers of patients meant the workload was often unmanageable and practicing optimal IPC was challenging. Caregivers lacked access to basic amenities, including linen and chairs, meaning that it was almost impossible for them to maintain good hand hygiene. Limited access to soap and the erratic water supply for both caregivers and healthcare workers further worsened the situation. Communication challenges between different cadres of staff and with patient caregivers meant that those handling neonates and cleaning the wards were often unaware of outbreaks of drug resistant infection. Conclusion: For IPC to be improved, interventions need to address the chronic shortages of material resources and create an enabling environment for HCWs and patient caregivers.

ObjectivesWe studied neonates with suspected early-onset sepsis (EOS, sepsis developing in the first 72 hours after delivery) in Malawi to (1) describe clinical characteristics and microbiological findings, (2) identify which patient characteristics may be associated with pathogen positivity on blood culture, and (3) describe mortality and its potential determinants.DesignProspective observational study (May 2018–June 2019).SettingNeonatal ward in Queen Elizabeth Central Hospital, the largest government hospital in Malawi.PatientsAll neonates with suspected EOS in whom a blood culture was obtained.ResultsOut of 4308 neonatal admissions, 1244 (28.9%) had suspected EOS. We included 1149 neonates, of which 109 blood cultures had significant growth (9.5%). The most commonly isolated pathogens wereStaphylococcus aureus,Klebsiella pneumoniae,Enterobacter cloacae,Escherichia coliandAcinetobacter baumanii. Many of the Gram negatives were extended-spectrum beta lactamase-producing Enterobacteriaceae, and these were 40–100% resistant to first-line and second-line antimicrobials. Gestational age (GA) of ConclusionIn Malawi, EOS was suspected in nearly a third of neonatal admissions and had a high mortality. Ten per cent were culture-confirmed and predicted by low GA. To reduce the impact of suspected neonatal sepsis in least developed countries, improved maternal and antenatal care and development of rapid point of care methods to more accurately guide antimicrobial use could simultaneously improve outcome and reduce antimicrobial resistance.

Typhoid fever is a major cause of illness and mortality in low- and middle-income settings. We investigated the association of typhoid fever and rainfall in Blantyre, Malawi, where multi-drug-resistant typhoid has been transmitting since 2011. Peak rainfall preceded the peak in typhoid fever by approximately 15 weeks [95% confidence interval (CI) 13.3, 17.7], indicating no direct biological link. A quasi-Poisson generalised linear modelling framework was used to explore the relationship between rainfall and typhoid incidence at biologically plausible lags of 1–4 weeks. We found a protective effect of rainfall anomalies on typhoid fever, at a two-week lag (P = 0.006), where a 10 mm lower-than-expected rainfall anomaly was associated with up to a 16% reduction in cases (95% CI 7.6, 26.5). Extreme flooding events may cleanse the environment of S. Typhi, while unusually low rainfall may reduce exposure from sewage overflow. These results add to evidence that rainfall anomalies may play a role in the transmission of enteric pathogens, and can help direct future water and sanitation intervention strategies for the control of typhoid fever.

A special-care neonatal unit from a large public hospital in Malawi was noted as having more frequent, difficult-to-treat infections, and a suspected outbreak of multi-drug-resistant Klebsiella pneumoniae was investigated using genomic characterisation. All K. pneumoniae bloodstream infections (BSIs) from patients in the neonatal ward (n=62), and a subset of K. pneumoniae BSI isolates (n=38) from other paediatric wards in the hospital, collected over a 4 year period were studied. After whole genome sequencing, the strain sequence types (STs), plasmid types, virulence and resistance genes were identified. One ST340 clone, part of clonal complex 258 (CC258) and an ST that drives hospital outbreaks worldwide, harbouring numerous resistance genes and plasmids, was implicated as the likely cause of the outbreak. This study contributes molecular information necessary for tracking and characterizing this important hospital pathogen in sub-Saharan Africa.

Background: The World Health Organisation recommends prioritised use of recently prequalified typhoid conjugate vaccines in countries with the highest incidence of typhoid fever. However, representative typhoid surveillance data are lacking in many low-income countries because of the costs and challenges of diagnostic clinical microbiology. Environmental surveillance (ES) of Salmonella Typhi in sewage and wastewater using molecular methods may offer a low-cost alternative, but its performance in comparison with clinical surveillance has not been assessed. Methods: We developed a harmonised protocol for typhoid ES and its implementation in communities in India and Malawi where it will be compared with findings from hospital-based surveillance for typhoid fever. The protocol includes methods for ES site selection based on geospatial analysis, grab and trap sample collection at sewage and wastewater sites, and laboratory methods for sample processing, concentration and quantitative polymerase chain reaction (PCR) to detect Salmonella Typhi. The optimal locations for ES sites based on digital elevation models and mapping of sewage and river networks are described for each community and their suitability confirmed through field investigation. We will compare the prevalence and abundance of Salmonella Typhi in ES samples collected each month over a 12-month period to the incidence of blood culture confirmed typhoid cases recorded at referral hospitals serving the study areas. Conclusions: If environmental detection of Salmonella Typhi correlates with the incidence of typhoid fever estimated through clinical surveillance, typhoid ES may be a powerful and low-cost tool to estimate the local burden of typhoid fever and support the introduction of typhoid conjugate vaccines. Typhoid ES could also allow the impact of vaccination to be assessed and rapidly identify circulation of drug resistant strains.

BackgroundInvasiveSalmonellainfections cause significant morbidity and mortality in Sub-Saharan Africa. However, the routes of transmission are uncertain. We conducted a case-control study of index-case and geographically-matched control households in Blantyre, Malawi, samplingSalmonellaisolates from index cases, healthy people, animals, and the household environment.MethodologySixty index cases of human invasiveSalmonellainfection were recruited (March 2015-Oct 2016). Twenty-eight invasive Non-TyphoidalSalmonella(iNTS) disease and 32 typhoid patients consented to household sampling. Each index-case household was geographically matched to a control household. Extensive microbiological sampling included stool sampling from healthy household members, stool or rectal swabs from household-associated animals and boot-sock sampling of the household environment.Findings1203 samples from 120 households, yielded 43 non-TyphoidalSalmonella(NTS) isolates from 25 households (overall sample positivity 3.6%). In the 28 iNTS patients, disease was caused by 3 STs of Salmonella Typhimurium, mainly ST313. In contrast, the isolates from households spanned 15 sequence types (STs). TwoS. Typhimurium isolates from index cases closely matched isolates from their respective asymptomatic household members (2 and 3 SNP differences respectively). Despite the recovery of a diverse range of NTS, there was no overlap between the STs causing iNTS disease with any environmental or animal isolates.ConclusionsThe finding of NTS strains from index cases that matched household members, coupled with lack of related animal or environmental isolates, supports a hypothesis of human to human transmission of iNTS infections in the household. The breadth of NTS strains found in animals and the household environment demonstrated the robustness of NTS sampling and culture methodology, and suggests a diverse ecology ofSalmonellain this setting. Healthy typhoid (S. Typhi) carrier state was not detected. The lack ofS. Typhi isolates from the household environment suggests that further methodological development is needed to cultureS. Typhi from the environment.Author summaryInvasiveSalmonellainfections cause the loss of millions of disability adjusted life years (DALYs) every year globally. The two main types of invasiveSalmonellainfections in Africa are i) typhoid fever, caused bySalmonellaTyphi, and ii) invasive Non-Typhoidal Salmonella (iNTS) disease, primarily caused in our setting bySalmonellaTyphimurium. Despite the high disease burden, and the observed differences between the epidemiology of typhoid and iNTS disease, we lack an understanding of the reservoirs and transmission routes of iNTS. Therefore, we carried out extensive microbiological sampling of the household members, domestic animals, and living environments of patients with invasiveSalmonellainfections, and of geographically-matched control households, and investigated the genetic relationships between householdSalmonellaand index-case blood-stream isolates by whole genome sequencing (WGS). We identified a wide range of NTS serovars / sequence types across all households and sample-types, but only identifiedSalmonellathat matched iNTS that matched invasive cases strains in the stool of healthy people from the same households. Our findings support, but cannot prove, the hypothesis that iNTS-associated strains are transmitted from person-to-person. Boot-sock sampling of the household environment gave the highest yield ofSalmonellaof any of our sampling strategies. None of the 41 environmentalSalmonellaisolates from non-human sources, including 4 domestic animal-associated isolates, matched the disease-causing sequence types. Our findings are consistent with a hypothesis that the reservoir of Typhimurium iNTS infections is the human gastrointestinal tract, and transmission occurs within households. Longitudinal studies are required, however, to confirm this hypothesis.

Shortening courses of antimicrobials has been proposed to reduce risk of antimicrobial resistant (AMR) infections, but acquisition and selection dynamics under antimicrobial pressure at the individual level are poorly understood. We combine multi-state modelling and whole-genome sequencing to understand colonisation dynamics of extended-spectrum beta-lactamase producing Enterobacterales (ESBL-E) in Malawian adults. We demonstrate prolonged post-exposure antibiotic effect, meaning short courses exert similar colonisation pressure to longer ones. Genome data does not identify widespread hospital-associated ESBL-E transmission, hence apparent acquisitions may be selected from the patient microbiota by antimicrobial exposure. Understanding ESBL-E dynamics under antimicrobial pressure is crucial for evidence-based stewardship protocols.

Drug-resistant bacteria of the order Enterobacterales which produce extended-spectrum beta-lactamase enzymes (ESBL-Enterobacterales, ESBL-E) are global priority pathogens. Antimicrobial stewardship interventions proposed to curb their spread include shorter courses of antimicrobials to reduce selection pressure but individual-level acquisition and selection dynamics are poorly understood. We sampled stool of 425 adults (aged 16–76 years) in Blantyre, Malawi, over 6 months and used multistate modelling and whole-genome sequencing to understand colonization dynamics of ESBL-E. Models suggest a prolonged effect of antimicrobials such that truncating an antimicrobial course at 2 days has a limited effect in reducing colonization. Genomic analysis shows largely indistinguishable diversity of healthcare-associated and community-acquired isolates, hence some apparent acquisition of ESBL-E during hospitalization may instead represent selection from a patient’s microbiota by antimicrobial exposure. Our approach could help guide stewardship protocols; interventions that aim to review and truncate courses of unneeded antimicrobials may be of limited use in preventing ESBL-E colonization.

Klebsiella pneumoniae is an antimicrobial resistance (AMR) associated pathogen of global importance, and polyvalent vaccines targeting K. pneumoniae O-antigens are in development. Genomes from sub-Saharan Africa (sSA) are underrepresented in global sequencing efforts. We therefore carried out a genomic analysis of extended-spectrum beta-lactamase (ESBL)-producing K. pneumoniae complex isolates colonising adults in Blantyre, Malawi, placed these isolates in a global genomic context, and compared colonising to invasive isolates from the main public hospital in Blantyre. 203 isolates from stool and rectal swabs from adults were whole-genome sequenced and compared to a publicly available multicountry collection of 484 K. pneumoniae genomes sampled to cover maximum diversity of the species, 150 previously sequenced Malawian and 66 Kenyan isolates from blood or sterile sites. We inferred phylogenetic relationships and analysed the diversity of genetic loci linked to AMR, virulence, capsule (K-) and LPS O-antigen (O-types). We find that the diversity of Malawian Klebsiella isolates is representative of the species’ population structure, but with local success and expansion of sequence types (STs) ST14, ST15, ST340 and ST307. Siderophore and hypermucoidy genes were more frequent in invasive versus carriage isolates (present in 13% vs 1%, p < 0.001) but still generally lacking in most invasive isolates. The population structure and distribution of O-antigen types was similar in Malawian invasive and carriage isolates, with O4 being more common in Malawian isolates (14%) than in previously published studies (2-5%). We conclude that host factors, pathogen opportunity or alternate virulence loci not linked to invasive disease elsewhere are likely to be the major determinants of invasive disease in Malawi. Distinct ST and O-type distributions in Malawi highlights the need for geographically aware sampling to robustly define secular trends in Klebsiella diversity. Colonising and invasive isolates in Blantyre are similar and hence O-typing of colonising Klebsiella isolates may be a rapid and cost-effective approach to describe global diversity and guide vaccine development.Data SummaryAll data and code to replicate this analysis is available as the blantyreESBL v1.0.0 R package (https://doi.org/10.5281/zenodo.5554082) available at https://github.com/joelewis101/blantyreESBL. Reads from all isolates sequenced as part of this study have been deposited in the European Nucleotide Archive, and accession numbers (as well as accession numbers of publicly available genomes used in this analysis) are provided in the R package.

Escherichia coli is a ubiquitous bacterium and one of the most prevalent Gram-negative species associated with drug resistant infections. The large number of sequenced genomes available have provided us with a consistently growing knowledge base to further understand pathogenesis and epidemiology of this organism. However, data from sub-Saharan Africa (sSA) are underrepresented in global sequencing efforts and E. coli genetic diversity from this region is poorly described. To reduce this gap, we investigated extended-spectrum beta-lactamase (ESBL)-producing E. coli colonising adults in Blantyre, Malawi to assess the bacterial diversity and AMR determinants and to place these isolates in the context of the wider population structure. We performed short-read whole-genome sequencing of 473 colonising ESBL E. coli isolated from human stool and contextualised the genomes with a previously curated multi-country species wide collection of 10,146 genomes. The most frequently identified sequence types (STs) in our collection were the globally successful ST131, ST410 and ST167, and the dominant ESBL genes were blaCTX-M, mirroring global trends. However, 37% of Malawian isolates did not cluster with any isolates in the curated multicountry collection and a core gene phylogeny was consistent with locally spreading subclades within globally dominant clones, including in ST410 and ST167. We also found Carbapenemase genes in our collection at low frequency; we used long read sequencing to characterise selected ESBL and carbapenemase-associated plasmids, demonstrating the presence of globally distributed carbapenemase carrying plasmids. Increased genomic surveillance of E. coli from Malawi and sSA is necessary to understand local, regional and global transmission of both E. coli and the AMR genes they commonly carry.Impact StatementDrug-resistant Escherichia coli producing extended-spectrum beta lactamase (ESBL) or carbapenemase enzymes have been identified by the World Health Organisation as priority pathogens of global concern, and whole genome sequencing has provided insight into mechanisms of virulence, antimicrobial resistance, and the spread of high-risk clones. However, studies analysing large numbers of E. coli using whole-genome data often focus on opportunistic use of hospital diagnostic collections in high-income settings. Understanding how the genomic epidemiology of E. coli in low- and middle-income countries (including many of the nations of sub-Saharan Africa) differs is essential to provide insight into local, and global drivers of transmission. We therefore sequenced 473 ESBL-producing E. coli genomes colonising adults in Blantyre, Malawi. We analyse determinants of antimicrobial resistance and virulence and place the isolates in wider context using a previously published global E. coli collection that was generated to represent the whole species diversity of sequences publicly available at the time of generation. We find that there is diversity in Malawian isolates not reflected in the curated global collection: widely successful antimicrobial-resistance associated E. coli sequence types are represented in Blantyre, but locally circulating subclades are apparent. Furthermore, given the high number of ESBL producing pathogens causing infections there is an unmet need for carbapenem antimicrobials which are still active against ESBL-producers but are not yet widely available in our setting. We find that carbapenemases (enzymes that can render bacteria resistant to carbapenems) in our collection are unusual but present and carried on globally disseminated plasmids. So too are globally successful, stably carbapenemase-associated E. coli lineages. Although the Malawian isolates analysed typically lacked carbapenemases, carbapenem use is increasing in Malawi and their unstewarded use will accelerate selection for carbapememases in E. coli in the future. Our study highlights the need for robust stewardship protocols and ongoing genomic surveillance as these agents are introduced.Data SummaryAll data and code to replicate this analysis are available as the blantyreESBL v1.3 R package (https://doi.org/10.5281/zenodo.5554081) available at https://github.com/joelewis101/blantyreESBL. Reads from all isolates sequenced as part of this study have been deposited in the European Nucleotide Archive, under PRJEB26677, PRJEB28522 and PRJEB36486 (short reads) and PRJNA869071 (Nanopore reads and hybrid assemblies). Accession numbers (as well as accession numbers of publicly available genomes used in this analysis) linked to sample metadata are provided in the R package and as supplementary data to this manuscript.

Background\ud There are limited data on healthcare-associated infections (HAI) from African countries like Malawi.\ud \ud Aim\ud We undertook a point prevalence survey of HAI and antimicrobial use in the surgery department of Queen Elizabeth Central Hospital (QECH) in Malawi and ascertained the associated risk factors for HAI.\ud \ud Methods\ud A cross-sectional point prevalence survey (PPS) was carried out in the surgery department of QECH. The European Centre for Disease Prevention and Control PPS protocol version 5.3 was adapted to our setting and used as a data collection tool.\ud \ud Findings\ud 105 patients were included in the analysis; median age was 34 (IQR: 24-47) years and 55.2% patients were male. Point prevalence of HAI was 11.4% (n=12/105) (95% CI: 6.0%-19.1%), including four surgical site infections, four urinary tract infections, three bloodstream infections and one bone/joint infection. We identified the following risk factors for HAI; length-of-stay between 8 and 14 days (OR=14.4, 95% CI: 1.65-124.7, p=0.0143), presence of indwelling urinary catheter (OR=8.3, 95% CI: 2.24-30.70, p=0.003) and history of surgery in the past 30 days (OR=5.11, 95% CI: 1.46-17.83, p=0.011). 29/105 patients (27.6%) were prescribed antimicrobials, most commonly the 3rd-generation cephalosporin, ceftriaxone (n=15).\ud \ud Conclusion\ud The prevalence rates of HAI and antimicrobial use in surgery wards at QECH are relatively high. Hospital infection prevention and control measures need to be strengthened to reduce the burden of HAI at QECH.

Background Sepsis is an important cause of mortality globally, although population incidence estimates from low-income settings, including sub-Saharan Africa, are absent. We aimed to estimate sepsis incidence burden using routinely available data from a large urban hospital in Malawi. Methods We linked routine-care databases at Queen Elizabeth Central Hospital, Blantyre, Malawi, to provide admission and discharge data for 217 149 adults from 2013–2016. Using a definition of sepsis based on systemic inflammatory response syndrome criteria and Blantyre census population data, we calculated population incidence estimates of sepsis and severe sepsis and used negative binomial regression to assess for trends over time. Missing data were multiply imputed with chained equations. Results We estimate that the incidence rate of emergency department–attending sepsis and severe sepsis in adults was 1772 per 100 000 person-years (95% confidence interval [CI], 1754–1789) and 303 per 100 000 person-years (95% CI, 295–310), respectively, between 2013 and 2016, with a year-on-year decrease in incidence. In-hospital mortality for patients admitted to the hospital with sepsis and severe sepsis was 23.7% (95% CI, 22.7–24.7%) and 28.1% (95% CI, 26.1 – 30.0%), respectively, with no clear change over time. Conclusions Sepsis incidence is higher in Blantyre, Malawi, than in high-income settings, from where the majority of sepsis incidence data are derived. Worldwide sepsis burden is likely to be underestimated, and data from low-income countries are needed to inform the public health response., Sepsis epidemiology in sub-Saharan Africa (sSA) is poorly described. Using large databases from a Malawian teaching hospital, we present some of the first estimates of sepsis population incidence from sSA and find it to be high compared with high-income settings.

Aims This study evaluated detection methods for Salmonella Typhi (S. Typhi) in the environment, to establish a novel pathway from field sampling to isolation of viable organisms and molecular confirmation from complex environmental samples, thus enabling environmental surveillance of typhoid. Methods and Results Multiple media were assessed using clinical isolates from the Public Health England’s (PHE) Culture collection. The culture pathway selected consisted of a primary 2% bile broth and secondary Selenite F broth, followed by modified Chromogenic Agar for Salmonella Esterase (mCASE). A qPCR assay was adapted from a validated S. Typhi PCR panel for confirmation of isolates, with comparison to biochemical and serological tests showing good specificity. Sampling locations in Blantyre, Malawi were used to compare sampling methods. Viable S. Typhi were isolated from a mixture of trap and grab river water samples on six occasions. Conclusions Culture of viable S. Typhi from environmental samples was possible using effective capture and culture techniques. Significance and impact of study Whilst several studies have attempted to detect S. Typhi from the environment, this is the first successful attempt to isolate the organism from river water since the 1980’s. Supplementing clinical data with environmental screening offers the potential for enhanced surveillance, which might inform interventions and assess vaccination programmes.

Background The World Health Organisation recommends prioritised use of recently prequalified typhoid conjugate vaccines in countries with the highest incidence of typhoid fever. However, representative typhoid surveillance data are lacking in many low-income countries because of the costs and challenges of diagnostic clinical microbiology. Environmental surveillance (ES) of Salmonella Typhi in sewage and wastewater using molecular methods may offer a low-cost alternative, but its performance in comparison with clinical surveillance has not been assessed. Methodology/Principal Findings We developed a harmonised protocol for typhoid ES and its implementation in communities in India and Malawi where it will be compared with findings from hospital-based surveillance for typhoid fever. The protocol includes methods for ES site selection based on geospatial analysis, grab and trap sample collection at sewage and wastewater sites, and laboratory methods for sample processing, concentration and quantitative PCR to detect Salmonella Typhi. The optimal locations for ES sites based on digital elevation models and mapping of sewage and river networks are described for each community and their suitability confirmed through field investigation. We will compare the prevalence and abundance of Salmonella Typhi in ES samples collected each month over a 12-month period to the incidence of blood culture confirmed typhoid estimated from cases recorded at referral hospitals serving the study areas and community surveys of healthcare seeking for individuals with fever. Significance If environmental detection of Salmonella Typhi correlates with the incidence of typhoid fever estimated through clinical surveillance, typhoid ES may be a powerful and low-cost tool to estimate the local burden of typhoid fever and support the introduction of typhoid conjugate vaccines. Typhoid ES could also allow the impact of vaccination to be assessed and rapidly identify circulation of drug resistant strains. Author Summary Typhoid fever is a bacterial enteric infection that remains a significant health burden to the 6.4 billion people living in low- and middle-income countries. The WHO recently prequalified highly immunogenic typhoid conjugate vaccines (TCVs) and recommended that the introduction of public vaccination programmes should be based on an understanding of the local epidemiology of infection. However, representative data on the community level describing incidence of microbiologically confirmed typhoid cases are limited, reflecting the costs and challenges of implementing blood culture for Salmonella Typhi (S. Typhi). Therefore, cost-effective alternative means of community surveillance are required to assess overall burden of disease. We developed a protocol for typhoid environmental surveillance, including methods for the identification of suitable sampling sites, sample collection and laboratory processing. The detection of S. Typhi in wastewater and sewage samples will be examined against the outcomes of hospital-based surveillance for typhoid fever with healthcare utilisation surveys to evaluate and validate the use of ES as an effective means of assessing community level incidence of typhoid. The findings of this study will inform potential wider implementation of ES for typhoid to support the introduction of TCVs and monitor their impact.

We report the complete genome sequencing and annotation of four Salmonella enterica serovar Enteritidis isolates, two that are representative of the Central/Eastern African clade (CP255 and D7795) and two from the Global Epidemic clade (A1636 and P125109)., We report the complete genome sequencing and annotation of four Salmonella enterica serovar Enteritidis isolates, two that are representative of the Central/Eastern African clade (CP255 and D7795) and two of the Global Epidemic clade (A1636 and P125109).