Longitudinal analysis within one hospital in sub-Saharan Africa over 20 years reveals repeated replacements of dominant clones of Klebsiella pneumoniae and stresses the importance to include temporal patterns for vaccine design considerations.

BACKGROUND: Infections caused by multidrug-resistant gram-negative bacteria present a severe threat to global public health. The WHO defines drug-resistant Klebsiella pneumoniae as a priority pathogen for which alternative treatments are needed given the limited treatment options and the rapid acquisition of novel resistance mechanisms by this species. Longitudinal descriptions of genomic epidemiology of Klebsiella pneumoniae can inform management strategies but data from sub-Saharan Africa are lacking. METHODS: We present a longitudinal analysis of all invasive K. pneumoniae isolates from a single hospital in Blantyre, Malawi, southern Africa, from 1998 to 2020, combining clinical data with genome sequence analysis of the isolates. RESULTS: We show that after a dramatic increase in the number of infections from 2016 K. pneumoniae becomes hyperendemic, driven by an increase in neonatal infections. Genomic data show repeated waves of clonal expansion of different, often ward-restricted, lineages, suggestive of hospital-associated transmission. We describe temporal trends in resistance and surface antigens, of relevance for vaccine development. CONCLUSIONS: Our data highlight a clear need for new interventions to prevent rather than treat K. pneumoniae infections in our setting. Whilst one option may be a vaccine, the majority of cases could be avoided by an increased focus on and investment in infection prevention and control measures, which would reduce all healthcare-associated infections and not just one.

Genomics for antimicrobial resistance surveillance to support infection prevention and control in health-care facilities.

Integration of genomic technologies into routine antimicrobial resistance (AMR) surveillance in health-care facilities has the potential to generate rapid, actionable information for patient management and inform infection prevention and control measures in near real time. However, substantial challenges limit the implementation of genomics for AMR surveillance in clinical settings. Through a workshop series and online consultation, international experts from across the AMR and pathogen genomics fields convened to review the evidence base underpinning the use of genomics for AMR surveillance in a range of settings. Here, we summarise the identified challenges and potential benefits of genomic AMR surveillance in health-care settings, and outline the recommendations of the working group to realise this potential. These recommendations include the definition of viable and cost-effective use cases for genomic AMR surveillance, strengthening training competencies (particularly in bioinformatics), and building capacity at local, national, and regional levels using hub and spoke models.

Genomics for public health and international surveillance of antimicrobial resistance.

Historically, epidemiological investigation and surveillance for bacterial antimicrobial resistance (AMR) has relied on low-resolution isolate-based phenotypic analyses undertaken at local and national reference laboratories. Genomic sequencing has the potential to provide a far more high-resolution picture of AMR evolution and transmission, and is already beginning to revolutionise how public health surveillance networks monitor and tackle bacterial AMR. However, the routine integration of genomics in surveillance pipelines still has considerable barriers to overcome. In 2022, a workshop series and online consultation brought together international experts in AMR and pathogen genomics to assess the status of genomic applications for AMR surveillance in a range of settings. Here we focus on discussions around the use of genomics for public health and international AMR surveillance, noting the potential advantages of, and barriers to, implementation, and proposing recommendations from the working group to help to drive the adoption of genomics in public health AMR surveillance. These recommendations include the need to build capacity for genome sequencing and analysis, harmonising and standardising surveillance systems, developing equitable data sharing and governance frameworks, and strengthening interactions and relationships among stakeholders at multiple levels.

Innovations in genomic antimicrobial resistance surveillance.

Whole-genome sequencing of antimicrobial-resistant pathogens is increasingly being used for antimicrobial resistance (AMR) surveillance, particularly in high-income countries. Innovations in genome sequencing and analysis technologies promise to revolutionise AMR surveillance and epidemiology; however, routine adoption of these technologies is challenging, particularly in low-income and middle-income countries. As part of a wider series of workshops and online consultations, a group of experts in AMR pathogen genomics and computational tool development conducted a situational analysis, identifying the following under-used innovations in genomic AMR surveillance: clinical metagenomics, environmental metagenomics, gene or plasmid tracking, and machine learning. The group recommended developing cost-effective use cases for each approach and mapping data outputs to clinical outcomes of interest to justify additional investment in capacity, training, and staff required to implement these technologies. Harmonisation and standardisation of methods, and the creation of equitable data sharing and governance frameworks, will facilitate successful implementation of these innovations.

Evidence review and recommendations for the implementation of genomics for antimicrobial resistance surveillance: reports from an international expert group.

Nearly a century after the beginning of the antibiotic era, which has been associated with unparalleled improvements in human health and reductions in mortality associated with infection, the dwindling pipeline for new antibiotic classes coupled with the inevitable spread of antimicrobial resistance (AMR) poses a major global challenge. Historically, surveillance of bacteria with AMR typically relied on phenotypic analysis of isolates taken from infected individuals, which provides only a low-resolution view of the epidemiology behind an individual infection or wider outbreak. Recent years have seen increasing adoption of powerful new genomic technologies with the potential to revolutionise AMR surveillance by providing a high-resolution picture of the AMR profile of the bacteria causing infections and providing real-time actionable information for treating and preventing infection. However, many barriers remain to be overcome before genomic technologies can be adopted as a standard part of routine AMR surveillance around the world. Accordingly, the Surveillance and Epidemiology of Drug-resistant Infections Consortium convened an expert working group to assess the benefits and challenges of using genomics for AMR surveillance. In this Series, we detail these discussions and provide recommendations from the working group that can help to realise the massive potential benefits for genomics in surveillance of AMR.

Exploiting genomics for antimicrobial resistance surveillance at One Health interfaces.

The intersection of human, animal, and ecosystem health at One Health interfaces is recognised as being of key importance in the evolution and spread of antimicrobial resistance (AMR) and represents an important, and yet rarely realised opportunity to undertake vital AMR surveillance. A working group of international experts in pathogen genomics, AMR, and One Health convened to take part in a workshop series and online consultation focused on the opportunities and challenges facing genomic AMR surveillance in a range of settings. Here we outline the working group's discussion of the potential utility, advantages of, and barriers to, the implementation of genomic AMR surveillance at One Health interfaces and propose a series of recommendations for addressing these challenges. Embedding AMR surveillance at One Health interfaces will require the development of clear beneficial use cases, especially in low-income and middle-income countries. Evidence of directionality, risks to human and animal health, and potential trade implications were also identified by the working group as key issues. Addressing these challenges will be vital to enable genomic surveillance technology to reach its full potential for assessing the risk of transmission of AMR between the environment, animals, and humans at One Health interfaces.

Global diversity and antimicrobial resistance of typhoid fever pathogens: Insights from a meta-analysis of 13,000 Salmonella Typhi genomes.

Background: The Global Typhoid Genomics Consortium was established to bring together the typhoid research community to aggregate and analyse Salmonella enterica serovar Typhi (Typhi) genomic data to inform public health action. This analysis, which marks 22 years since the publication of the first Typhi genome, represents the largest Typhi genome sequence collection to date (n=13,000). Methods: This is a meta-analysis of global genotype and antimicrobial resistance (AMR) determinants extracted from previously sequenced genome data and analysed using consistent methods implemented in open analysis platforms GenoTyphi and Pathogenwatch. Results: Compared with previous global snapshots, the data highlight that genotype 4.3.1 (H58) has not spread beyond Asia and Eastern/Southern Africa; in other regions, distinct genotypes dominate and have independently evolved AMR. Data gaps remain in many parts of the world, and we show the potential of travel-associated sequences to provide informal 'sentinel' surveillance for such locations. The data indicate that ciprofloxacin non-susceptibility (>1 resistance determinant) is widespread across geographies and genotypes, with high-level ciprofloxacin resistance (≥3 determinants) reaching 20% prevalence in South Asia. Extensively drug-resistant (XDR) typhoid has become dominant in Pakistan (70% in 2020) but has not yet become established elsewhere. Ceftriaxone resistance has emerged in eight non-XDR genotypes, including a ciprofloxacin-resistant lineage (4.3.1.2.1) in India. Azithromycin resistance mutations were detected at low prevalence in South Asia, including in two common ciprofloxacin-resistant genotypes. Conclusions: The consortium's aim is to encourage continued data sharing and collaboration to monitor the emergence and global spread of AMR Typhi, and to inform decision-making around the introduction of typhoid conjugate vaccines (TCVs) and other prevention and control strategies. Funding: No specific funding was awarded for this meta-analysis. Coordinators were supported by fellowships from the European Union (ZAD received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 845681), the Wellcome Trust (SB, Wellcome Trust Senior Fellowship), and the National Health and Medical Research Council (DJI is supported by an NHMRC Investigator Grant [GNT1195210]).

Salmonella Typhi (Typhi) is a type of bacteria that causes typhoid fever. More than 110,000 people die from this disease each year, predominantly in areas of sub-Saharan Africa and South Asia with limited access to safe water and sanitation. Clinicians use antibiotics to treat typhoid fever, but scientists worry that the spread of antimicrobial-resistant Typhi could render the drugs ineffective, leading to increased typhoid fever mortality. The World Health Organization has prequalified two vaccines that are highly effective in preventing typhoid fever and may also help limit the emergence and spread of resistant Typhi. In low resource settings, public health officials must make difficult trade-off decisions about which new vaccines to introduce into already crowded immunization schedules. Understanding the local burden of antimicrobial-resistant Typhi and how it is spreading could help inform their actions. The Global Typhoid Genomics Consortium analyzed 13,000 Typhi genomes from 110 countries to provide a global overview of genetic diversity and antimicrobial-resistant patterns. The analysis showed great genetic diversity of the different strains between countries and regions. For example, the H58 Typhi variant, which is often drug-resistant, has spread rapidly through Asia and Eastern and Southern Africa, but is less common in other regions. However, distinct strains of other drug-resistant Typhi have emerged in other parts of the world. Resistance to the antibiotic ciprofloxacin was widespread and accounted for over 85% of cases in South Africa. Around 70% of Typhi from Pakistan were extensively drug-resistant in 2020, but these hard-to-treat variants have not yet become established elsewhere. Variants that are resistant to both ciprofloxacin and ceftriaxone have been identified, and azithromycin resistance has also appeared in several different variants across South Asia. The Consortium's analyses provide valuable insights into the global distribution and transmission patterns of drug-resistant Typhi. Limited genetic data were available fromseveral regions, but data from travel-associated cases helped fill some regional gaps. These findings may help serve as a starting point for collective sharing and analyses of genetic data to inform local public health action. Funders need to provide ongoing supportto help fill global surveillance data gaps.

Individual and population level costs and health-related quality of life outcomes of third-generation cephalosporin resistant bloodstream infection in Blantyre, Malawi.

Data which accurately enumerate the economic costs of antimicrobial resistance (AMR) in low- and middle- income countries are essential. This study aimed to quantify the impact of third-generation cephalosporin resistant (3GC-R) bloodstream infection (BSI) on economic and health related quality of life outcomes for adult patients in Blantyre, Malawi. Participants were recruited from a prospective, longitudinal cohort study of hospitalised patients with bloodstream infection caused by Enterobacterales at Queen Elizabeth Central Hospital (QECH). Primary costing studies were used to estimate the direct medical costs associated with the inpatient stay. Recruited participants were asked about direct non-medical and indirect costs associated with their admission and their health-related quality of life was measured using the EuroQol EQ-5D questionnaire. Multiple imputation was undertaken to account for missing data. Costs were adjusted to 2019 US Dollars. Cost and microbiology surveillance data from QECH, Blantyre was used to model the annual cost of, and quality-adjusted life years lost to, 3GC-R and 3GC-Susceptible BSI from 1998 to 2030 in Malawi. The mean health provider cost per participant with 3GC-R BSI was US$110.27 (95%CR; 22.60-197.95), higher than for those with 3GC-S infection. Patients with resistant BSI incurred an additional indirect cost of US$155.48 (95%CR; -67.80, 378.78) and an additional direct non-medical cost of US$20.98 (95%CR; -36.47, 78.42). Health related quality of life outcomes were poor for all participants, but participants with resistant infections had an EQ-5D utility score that was 0.167 (95% CR: -0.035, 0.300) lower than those with sensitive infections. Population level burden estimates suggest that in 2016, 3GC-R accounted for 84% of annual societal costs from admission with bloodstream infection and 82% of QALYs lost. 3GC-R bloodstream infection was associated with higher health provider and patient level costs than 3GC-S infection, as well as poorer HRQoL outcomes. We demonstrate a substantial current and future economic burden to society as a result of 3GC-R E. coli and Klebsiella spp. BSI, data urgently needed by policy makers to provide impetus for implementing strategies to reduce AMR.

Investigating One Health risks for human colonisation with extended spectrum ß-lactamase-producing Escherichia coli and Klebsiella pneumoniae in Malawian households: a longitudinal cohort study.

BACKGROUND: Low-income countries have high morbidity and mortality from drug-resistant infections, especially from enteric bacteria such as Escherichia coli. In these settings, sanitation infrastructure is of variable and often inadequate quality, creating risks of extended-spectrum ß-lactamase (ESBL)-producing Enterobacterales transmission. We aimed to describe the prevalence, distribution, and risks of ESBL-producing Enterobacterales colonisation in sub-Saharan Africa using a One Health approach. METHODS: Between April 29, 2019, and Dec 3, 2020, we recruited 300 households in Malawi for this longitudinal cohort study: 100 each in urban, peri-urban, and rural settings. All households underwent a baseline visit and 195 were selected for longitudinal follow-up, comprising up to three additional visits over a 6 month period. Data on human health, antibiotic usage, health-seeking behaviours, structural and behavioural environmental health practices, and animal husbandry were captured alongside human, animal, and environmental samples. Microbiological processing determined the presence of ESBL-producing E coli and Klebsiella pneumoniae, and hierarchical logistic regression was performed to evaluate the risks of human ESBL-producing Enterobacterales colonisation. FINDINGS: A paucity of environmental health infrastructure and materials for safe sanitation was identified across all sites. A total of 11 975 samples were cultured, and ESBL-producing Enterobacterales were isolated from 1190 (41·8%) of 2845 samples of human stool, 290 (29·8%) of 973 samples of animal stool, 339 (66·2%) of 512 samples of river water, and 138 (46·0%) of 300 samples of drain water. Multivariable models illustrated that human ESBL-producing E coli colonisation was associated with the wet season (adjusted odds ratio 1·66, 95% credible interval 1·38-2·00), living in urban areas (2·01, 1·26-3·24), advanced age (1·14, 1·05-1·25), and living in households where animals were observed interacting with food (1·62, 1·17-2·28) or kept inside (1·58, 1·00-2·43). Human ESBL-producing K pneumoniae colonisation was associated with the wet season (2·12, 1·63-2·76). INTERPRETATION: There are extremely high levels of ESBL-producing Enterobacterales colonisation in humans and animals and extensive contamination of the wider environment in southern Malawi. Urbanisation and seasonality are key risks for ESBL-producing Enterobacterales colonisation, probably reflecting environmental drivers. Without adequate efforts to improve environmental health, ESBL-producing Enterobacterales transmission is likely to persist in this setting. FUNDING: Medical Research Council, National Institute for Health and Care Research, and Wellcome Trust. TRANSLATION: For the Chichewa translation of the abstract see Supplementary Materials section.

Clinical predictors of bacteraemia in neonates with suspected early-onset sepsis in Malawi: a prospective cohort study.

OBJECTIVES: We 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. DESIGN: Prospective observational study (May 2018-June 2019). SETTING: Neonatal ward in Queen Elizabeth Central Hospital, the largest government hospital in Malawi. PATIENTS: All neonates with suspected EOS in whom a blood culture was obtained. RESULTS: Out 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 were Staphylococcus aureus, Klebsiella pneumoniae, Enterobacter cloacae, Escherichia coli and Acinetobacter 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 <32 weeks was associated with pathogen-positive blood cultures (<28 weeks: adjusted OR (AOR) 2.72, 95% CI 1.04 to 7.13; 28-32 weeks: AOR 2.26, 95% CI 1.21 to 4.21; p=0.005). Mortality was 17.6% (202/1149) and associated with low birth weight (<1000 g: AOR 47.57, 95% CI 12.59 to 179.81; 1000-1500 g: AOR 11.31, 95% CI 6.97 to 18.36; 1500-2500 g: AOR 2.20, 95% CI 1.42 to 3.39; p<0.001), low Apgar scores at 5 min (0-3: AOR 18.60, 95% CI 8.81 to 39.27; 4-6: AOR 4.41, 95% CI 2.81 to 6.93; p<0.001), positive maternal venereal disease research laboratory status (AOR 2.53, 95% CI 1.25 to 5.12; p=0.001) and congenital anomalies (AOR 7.37, 95% CI 3.61 to 15.05; p<0.001). Prolonged rupture of membranes was inversely associated with mortality (AOR 0.43, 95% CI 0.19 to 0.98; p 0.007). CONCLUSION: In 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.

Case-control investigation of invasive Salmonella disease in Malawi reveals no evidence of environmental or animal transmission of invasive strains, and supports human to human transmission.

BACKGROUND: Invasive Salmonella infections 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, sampling Salmonella isolates from index cases, healthy people, animals, and the household environment. METHODOLOGY: Sixty index cases of human invasive Salmonella infection were recruited (March 2015-Oct 2016). Twenty-eight invasive Non-Typhoidal Salmonella (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. FINDINGS: 1203 samples from 120 households, yielded 43 non-Typhoidal Salmonella (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). Two S. 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. CONCLUSIONS: The 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 of Salmonella in this setting. Healthy typhoid (S. Typhi) carrier state was not detected. The lack of S. Typhi isolates from the household environment suggests that further methodological development is needed to culture S. Typhi from the environment.

Multiplex PCR Assay for Clade Typing of Salmonella enterica Serovar Enteritidis.

Salmonella enterica serovar Enteritidis is one of the most commonly reported serovars of nontyphoidal Salmonella causing human disease and is responsible for both gastroenteritis and invasive nontyphoidal Salmonella (iNTS) disease worldwide. Whole-genome sequence (WGS) comparison of Salmonella Enteritidis isolates from across the world has identified three distinct clades, 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 are 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. The assay described here is limited to previously confirmed S. Enteritidis isolates. IMPORTANCE Challenges in the diagnosis and treatment of invasive Salmonella Enteritidis bloodstream infections in sub-Saharan Africa are responsible for a case fatality rate of approximately 15%. It is important to identify distinct clades of S. Enteritidis in diagnostic laboratories in the African setting to determine the different health outcomes associated with particular outbreaks. Here, we describe the development of a high-quality molecular classification assay for clade typing of S. Enteritidis that is ideal for use in public health laboratories in resource-limited settings.

Effect of resistance to third-generation cephalosporins on morbidity and mortality from bloodstream infections in Blantyre, Malawi: a prospective cohort study.

BACKGROUND: 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. METHODS: 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. FINDINGS: 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. INTERPRETATION: 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. FUNDING: Wellcome Trust and Wellcome Asia and Africa Programme.

Colonization dynamics of extended-spectrum beta-lactamase-producing Enterobacterales in the gut of Malawian adults.

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.

Rainfall anomalies and typhoid fever in Blantyre, Malawi.

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.

Genomic and antigenic diversity of colonizing Klebsiella pneumoniae isolates mirrors that of invasive isolates in Blantyre, Malawi.

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.

A Longitudinal, Observational Study of Etiology and Long-Term Outcomes of Sepsis in Malawi Revealing the Key Role of Disseminated Tuberculosis.

BACKGROUND: Sepsis protocols in sub-Saharan Africa are typically extrapolated from high-income settings, yet sepsis in sub-Saharan Africa is likely caused by distinct pathogens and may require novel treatment strategies. Data to guide such strategies are lacking. We aimed to define causes and modifiable factors associated with sepsis outcomes in Blantyre, Malawi, in order to inform the design of treatment strategies tailored to sub-Saharan Africa. METHODS: We recruited 225 adults who met a sepsis case definition defined by fever and organ dysfunction in an observational cohort study at a single tertiary center. Etiology was defined using culture, antigen detection, serology, and polymerase chain reaction. The effect of treatment on 28-day outcomes was assessed using Bayesian logistic regression. RESULTS: There were 143 of 213 (67%) participants living with human immunodeficiency virus (HIV). We identified a diagnosis in 145 of 225 (64%) participants, most commonly tuberculosis (TB; 34%) followed by invasive bacterial infections (17%), arboviral infections (13%), and malaria (9%). TB was associated with HIV infection, whereas malaria and arboviruses with the absence of HIV infection. Antituberculous chemotherapy was associated with survival (adjusted odds ratio for 28-day death, 0.17; 95% credible interval, 0.05-0.49 for receipt of antituberculous therapy). Of those with confirmed etiology, 83% received the broad-spectrum antibacterial ceftriaxone, but it would be expected to be active in only 24%. CONCLUSIONS: Sepsis in Blantyre, Malawi, is caused by a range of pathogens; the majority are not susceptible to the broad-spectrum antibacterials that most patients receive. HIV status is a key determinant of etiology. Novel antimicrobial strategies for sepsis tailored to sub-Saharan Africa, including consideration of empiric antituberculous therapy in individuals living with HIV, should be developed and trialed.

Spatial and Genomic Data to Characterize Endemic Typhoid Transmission.

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 <15 to >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 <192 m apart. 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.

Incidence of invasive non-typhoidal Salmonella in Blantyre, Malawi between January 2011-December 2019.

Background: The Malawi-Liverpool Wellcome Trust Clinical Research Programme (MLW) has undertaken sentinel surveillance of bloodstream infection and meningitis at Queen Elizabeth Central Hospital (QECH), Blantyre, Malawi for 20 years. Previously, three epidemics of Salmonella bloodstream infection have been identified. Here we provide updated surveillance data on invasive non-typhoidal Salmonella disease from 2011 - 2019. Methods: Surveillance data describing trends in invasive non-typhoidal Salmonella disease and associated antimicrobial susceptibility profiles are presented for the period January 2011 - December 2019. Results: Between January 2011-December 2019, 128,588 blood cultures and 40,769 cerebrospinal fluid cultures were processed at MLW. Overall, 1.00% of these were positive for S. Typhimurium, 0.10% for S. Enteritidis, and 0.05% positive for other Salmonella species. Estimated minimum incidence of invasive non-typhoidal Salmonella (iNTS) disease decreased from 21/100,000 per year in 2011 to 7/100,000 per year in 2019. Over this period, 26 confirmed cases of Salmonella meningitis were recorded (88.5% S. Typhimurium). Between 2011-2019 there was a substantial decrease in proportion of S. Typhimurium (78.5% to 27.7%) and S. Enteritidis (31.8% in 2011 to 0%) that were multidrug-resistant. Resistance to fluoroquinolones and third-generation generation cephalosporins (3GC) remained uncommon, however 3GC increased amongst Salmonella spp. and S. Typhimurium in the latter part of the period. Conclusions: The total number of iNTS bloodstream infections decreased between 2011-2019. Although the number multidrug resistance (MDR) S. Typhimurium and S. Enteritidis isolates has fallen, the number of MDR isolates of other Salmonella spp. has increased, including 3GC isolates.