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.

Microbiological Methods Used in the Enterics for Global Health Shigella Surveillance Study.

Background: Shigella is a major cause of diarrhea in young children worldwide. Multiple vaccines targeting Shigella are in development, and phase 3 clinical trials are imminent to determine efficacy against shigellosis. Methods: The Enterics for Global Health (EFGH) Shigella surveillance study is designed to determine the incidence of medically attended shigellosis in 6- to 35-month-old children in 7 resource-limited settings. Here, we describe the microbiological methods used to isolate and identify Shigella. We developed a standardized laboratory protocol for isolation and identification of Shigella by culture. This protocol was implemented across all 7 sites, ensuring consistency and comparability of results. Secondary objectives of the study are to determine the antibiotic resistance profiles of Shigella, compare isolation of Shigella from rectal swabs versus whole stool, and compare isolation of Shigella following transport of rectal swabs in Cary-Blair versus a modified buffered glycerol saline transport medium. Conclusions: Data generated from EFGH using culture methods described herein can potentially be used for microbiological endpoints in future phase 3 clinical trials to evaluate vaccines against shigellosis and for other clinical and public health studies focused on these organisms.

Evaluation of Fecal Inflammatory Biomarkers to Identify Bacterial Diarrhea Episodes: Systematic Review and Protocol for the Enterics for Global Health Shigella Surveillance Study.

Background: The measurement of fecal inflammatory biomarkers among individuals presenting to care with diarrhea could improve the identification of bacterial diarrheal episodes that would benefit from antibiotic therapy. We reviewed prior literature in this area and describe our proposed methods to evaluate 4 biomarkers in the Enterics for Global Health (EFGH) Shigella surveillance study. Methods: We systematically reviewed studies since 1970 from PubMed and Embase that assessed the diagnostic characteristics of inflammatory biomarkers to identify bacterial diarrhea episodes. We extracted sensitivity and specificity and summarized the evidence by biomarker and diarrhea etiology. In EFGH, we propose using commercial enzyme-linked immunosorbent assays to test for myeloperoxidase, calprotectin, lipocalin-2, and hemoglobin in stored whole stool samples collected within 24 hours of enrollment from participants in the Bangladesh, Kenya, Malawi, Pakistan, Peru, and The Gambia sites. We will develop clinical prediction scores that incorporate the inflammatory biomarkers and evaluate their ability to identify Shigella and other bacterial etiologies of diarrhea as determined by quantitative polymerase chain reaction (qPCR). Results: Forty-nine studies that assessed fecal leukocytes (n = 39), red blood cells (n = 26), lactoferrin (n = 13), calprotectin (n = 8), and myeloperoxidase (n = 1) were included in the systematic review. Sensitivities were high for identifying Shigella, moderate for identifying any bacteria, and comparable across biomarkers. Specificities varied depending on the outcomes assessed. Prior studies were generally small, identified red and white blood cells by microscopy, and used insensitive gold standard diagnostics, such as conventional bacteriological culture for pathogen detection. Conclusions: Our evaluation of inflammatory biomarkers to distinguish diarrhea etiologies as determined by qPCR will provide an important addition to the prior literature, which was likely biased by the limited sensitivity of the gold standard diagnostics used. We will determine whether point-of-care biomarker tests could be a viable strategy to inform treatment decision making and increase appropriate targeting of antibiotic treatment to bacterial diarrhea episodes.

The Enterics for Global Health (EFGH) Shigella Surveillance Study in Malawi.

Background: Malawi is among 7 countries participating in the Enterics for Global Health (EFGH) Shigella surveillance study, which aims to determine the incidence of medically attended diarrhea attributed to Shigella, a leading bacterial cause of diarrhea in children in low-resource settings. Methods: We describe the EFGH study site in the densely populated informal settlement of Ndirande Township, Blantyre, Malawi. We explore the site's geographical location, demographic characteristics, and the healthcare-seeking behavior of its population, particularly for childhood diarrhea. We also describe the management of childhood diarrhea at the health facility, and the associated challenges to attaining optimum adherence to local and national guidelines at the site. Conclusions: Our overarching aim is to improve global health through understanding and mitigating the impact of diarrhea attributed to Shigella.

Data Management in Multicountry Consortium Studies: The Enterics For Global Health (EFGH) Shigella Surveillance Study Example.

Background: Rigorous data management systems and planning are essential to successful research projects, especially for large, multicountry consortium studies involving partnerships across multiple institutions. Here we describe the development and implementation of data management systems and procedures for the Enterics For Global Health (EFGH) Shigella surveillance study-a 7-country diarrhea surveillance study that will conduct facility-based surveillance concurrent with population-based enumeration and a health care utilization survey to estimate the incidence of Shigella--associated diarrhea in children 6 to 35 months old. Methods: The goals of EFGH data management are to utilize the knowledge and experience of consortium members to collect high-quality data and ensure equity in access and decision-making. During the planning phase before study initiation, a working group of representatives from each EFGH country site, the coordination team, and other partners met regularly to develop the data management systems for the study. Results: This resulted in the Data Management Plan, which included selecting REDCap and SurveyCTO as the primary database systems. Consequently, we laid out procedures for data processing and storage, study monitoring and reporting, data quality control and assurance activities, and data access. The data management system and associated real-time visualizations allow for rapid data cleaning activities and progress monitoring and will enable quicker time to analysis. Conclusions: Experiences from this study will contribute toward enriching the sparse landscape of data management methods publications and serve as a case study for future studies seeking to collect and manage data consistently and rigorously while maintaining equitable access to and control of data.

Diarrhea Case Surveillance in the Enterics for Global Health Shigella Surveillance Study: Epidemiologic Methods.

Background: Shigella is a leading cause of acute watery diarrhea, dysentery, and diarrhea-attributed linear growth faltering, a precursor to stunting and lifelong morbidity. Several promising Shigella vaccines are in development and field efficacy trials will require a consortium of potential vaccine trial sites with up-to-date Shigella diarrhea incidence data. Methods: The Enterics for Global Health (EFGH) Shigella surveillance study will employ facility-based enrollment of diarrhea cases aged 6-35 months with 3 months of follow-up to establish incidence rates and document clinical, anthropometric, and financial consequences of Shigella diarrhea at 7 country sites (Mali, Kenya, The Gambia, Malawi, Bangladesh, Pakistan, and Peru). Over a 24-month period between 2022 and 2024, the EFGH study aims to enroll 9800 children (1400 per country site) between 6 and 35 months of age who present to local health facilities with diarrhea. Shigella species (spp.) will be identified and serotyped from rectal swabs by conventional microbiologic methods and quantitative polymerase chain reaction. Shigella spp. isolates will undergo serotyping and antimicrobial susceptibility testing. Incorporating population and healthcare utilization estimates from contemporaneous household sampling in the catchment areas of enrollment facilities, we will estimate Shigella diarrhea incidence rates. Conclusions: This multicountry surveillance network will provide key incidence data needed to design Shigella vaccine trials and strengthen readiness for potential trial implementation. Data collected in EFGH will inform policy makers about the relative importance of this vaccine-preventable disease, accelerating the time to vaccine availability and uptake among children in high-burden settings.

Exploring Natural Immune Responses to Shigella Exposure Using Multiplex Bead Assays on Dried Blood Spots in High-Burden Countries: Protocol From a Multisite Diarrhea Surveillance Study.

Background: Molecular diagnostics on human fecal samples have identified a larger burden of shigellosis than previously appreciated by culture. Evidence of fold changes in immunoglobulin G (IgG) to conserved and type-specific Shigella antigens could be used to validate the molecular assignment of type-specific Shigella as the etiology of acute diarrhea and support polymerase chain reaction (PCR)-based microbiologic end points for vaccine trials. Methods: We will test dried blood spots collected at enrollment and 4 weeks later using bead-based immunoassays for IgG to invasion plasmid antigen B and type-specific lipopolysaccharide O-antigen for Shigella flexneri 1b, 2a, 3a, and 6 and Shigella sonnei in Shigella-positive cases and age-, site-, and season-matched test-negative controls from all sites in the Enterics for Global Health (EFGH) Shigella surveillance study. Fold antibody responses will be compared between culture-positive, culture-negative but PCR-attributable, and PCR-positive but not attributable cases and test-negative controls. Age- and site-specific seroprevalence distributions will be identified, and the association between baseline antibodies and Shigella attribution will be estimated. Conclusions: The integration of these assays into the EFGH study will help support PCR-based attribution of acute diarrhea to type-specific Shigella, describe the baseline seroprevalence of conserved and type-specific Shigella antibodies, and support correlates of protection for immunity to Shigella diarrhea. These insights can help support the development and evaluation of Shigella vaccine candidates.

Shigella Detection and Molecular Serotyping With a Customized TaqMan Array Card in the Enterics for Global Health (EFGH): Shigella Surveillance Study.

Background: Quantitative polymerase chain reaction (qPCR) targeting ipaH has been proven to be highly efficient in detecting Shigella in clinical samples compared to culture-based methods, which underestimate Shigella burden by 2- to 3-fold. qPCR assays have also been developed for Shigella speciation and serotyping, which is critical for both vaccine development and evaluation. Methods: The Enterics for Global Health (EFGH) Shigella surveillance study will utilize a customized real-time PCR-based TaqMan Array Card (TAC) interrogating 82 targets, for the detection and differentiation of Shigella spp, Shigella sonnei, Shigella flexneri serotypes, other diarrhea-associated enteropathogens, and antimicrobial resistance (AMR) genes. Total nucleic acid will be extracted from rectal swabs or stool samples, and assayed on TAC. Quantitative analysis will be performed to determine the likely attribution of Shigella and other particular etiologies of diarrhea using the quantification cycle cutoffs derived from previous studies. The qPCR results will be compared to conventional culture, serotyping, and phenotypic susceptibility approaches in EFGH. Conclusions: TAC enables simultaneous detection of diarrheal etiologies, the principal pathogen subtypes, and AMR genes. The high sensitivity of the assay enables more accurate estimation of Shigella-attributed disease burden, which is critical to informing policy and in the design of future clinical trials.

Utilizing river and wastewater as a SARS-CoV-2 surveillance tool in settings with limited formal sewage systems.

The COVID-19 pandemic has profoundly impacted health systems globally and robust surveillance has been critical for pandemic control, however not all countries can currently sustain community pathogen surveillance programs. Wastewater surveillance has proven valuable in high-income settings, but less is known about the utility of water surveillance of pathogens in low-income countries. Here we show how wastewater surveillance of SAR-CoV-2 can be used to identify temporal changes and help determine circulating variants quickly. In Malawi, a country with limited community-based COVID-19 testing capacity, we explore the utility of rivers and wastewater for SARS-CoV-2 surveillance. From May 2020-May 2022, we collect water from up to 112 river or defunct wastewater treatment plant sites, detecting SARS-CoV-2 in 8.3% of samples. Peak SARS-CoV-2 detection in water samples predate peaks in clinical cases. Sequencing of water samples identified the Beta, Delta, and Omicron variants, with Delta and Omicron detected well in advance of detection in patients. Our work highlights how wastewater can be used to detect emerging waves, identify variants of concern, and provide an early warning system in settings with no formal sewage systems.

Cross-sectional health centre and community-based evaluation of the impact of pneumococcal and malaria vaccination on antibiotic prescription and usage, febrile illness and antimicrobial resistance in young children in Malawi: the IVAR study protocol.

INTRODUCTION: Vaccination is a potentially critical component of efforts to arrest development and dissemination of antimicrobial resistance (AMR), though little is known about vaccination impact within low-income and middle-income countries. This study will evaluate the impact of vaccination on reducing carriage prevalence of resistant Streptococcus pneumoniae and extended spectrum beta-lactamase-producing Escherichia coli and Klebsiella species. We will leverage two large ongoing cluster-randomised vaccine evaluations in Malawi assessing; first, adding a booster dose to the 13-valent pneumococcal conjugate vaccine (PCV13) schedule, and second, introduction of the RTS,S/AS01 malaria vaccine. METHODS AND ANALYSIS: Six cross-sectional surveys will be implemented within primary healthcare centres (n=3000 users of outpatient facilities per survey) and their local communities (n=700 healthy children per survey): three surveys in Blantyre district (PCV13 component) and three surveys in Mangochi district (RTS,S/AS01 component). We will evaluate antibiotic prescription practices and AMR carriage in children ≤3 years. For the PCV13 component, surveys will be conducted 9, 18 and 33 months following a 3+0 to 2+1 schedule change. For the RTS,S/AS01 component, surveys will be conducted 32, 44 and 56 months post-RTS,S/AS01 introduction. Six health centres in each study component will be randomly selected for study inclusion. Between intervention arms, the primary outcome will be the difference in penicillin non-susceptibility prevalence among S. pneumoniae nasopharyngeal carriage isolates in healthy children. The study is powered to detect an absolute change of 13 percentage points (ie, 35% vs 22% penicillin non-susceptibility). ETHICS AND DISSEMINATION: This study has been approved by the Kamuzu University of Health Sciences (Ref: P01-21-3249), University College London (Ref: 18331/002) and University of Liverpool (Ref: 9908) Research Ethics Committees. Parental/caregiver verbal or written informed consent will be obtained prior to inclusion or recruitment in the health centre-based and community-based activities, respectively. Results will be disseminated via the Malawi Ministry of Health, WHO, peer-reviewed publications and conference presentations.

Utilizing river and wastewater as a SARS-CoV-2 surveillance tool to predict trends and identify variants of concern in settings with limited formal sewage systems.

The COVID-19 pandemic continues to impact health systems globally and robust surveillance is critical for pandemic control, however not all countries can sustain community surveillance programs. Wastewater surveillance has proven valuable in high-income settings, but little is known about how river and informal sewage in low-income countries can be used for environmental surveillance of SARS-CoV-2. In Malawi, a country with limited community-based COVID-19 testing capacity, we explored the utility of rivers and wastewater for SARS-CoV-2 surveillance. From May 2020 - January 2022, we collected water from up to 112 river or informal sewage sites/month, detecting SARS-CoV-2 in 8.3% of samples. Peak SARS-CoV-2 detection in water samples predated peaks in clinical cases. Sequencing of water samples identified the Beta, Delta, and Omicron variants, with Delta and Omicron detected well in advance of detection in patients. Our work highlights wastewater can be used for detecting emerging waves, identifying variants of concern and function as an early warning system in settings with no formal sewage systems.

A comparison of four epidemic waves of COVID-19 in Malawi; an observational cohort study.

BACKGROUND: Compared to the abundance of clinical and genomic information available on patients hospitalised with COVID-19 disease from high-income countries, there is a paucity of data from low-income countries. Our aim was to explore the relationship between viral lineage and patient outcome. METHODS: We enrolled a prospective observational cohort of adult patients hospitalised with PCR-confirmed COVID-19 disease between July 2020 and March 2022 from Blantyre, Malawi, covering four waves of SARS-CoV-2 infections. Clinical and diagnostic data were collected using an adapted ISARIC clinical characterization protocol for COVID-19. SARS-CoV-2 isolates were sequenced using the MinION™ in Blantyre. RESULTS: We enrolled 314 patients, good quality sequencing data was available for 55 patients. The sequencing data showed that 8 of 11 participants recruited in wave one had B.1 infections, 6/6 in wave two had Beta, 25/26 in wave three had Delta and 11/12 in wave four had Omicron. Patients infected during the Delta and Omicron waves reported fewer underlying chronic conditions and a shorter time to presentation. Significantly fewer patients required oxygen (22.7% [17/75] vs. 58.6% [140/239], p < 0.001) and steroids (38.7% [29/75] vs. 70.3% [167/239], p < 0.001) in the Omicron wave compared with the other waves. Multivariable logistic-regression demonstrated a trend toward increased mortality in the Delta wave (OR 4.99 [95% CI 1.0-25.0 p = 0.05) compared to the first wave of infection. CONCLUSIONS: Our data show that each wave of patients hospitalised with SARS-CoV-2 was infected with a distinct viral variant. The clinical data suggests that patients with severe COVID-19 disease were more likely to die during the Delta wave.

Emergence of a multidrug-resistant and virulent Streptococcus pneumoniae lineage mediates serotype replacement after PCV13: an international whole-genome sequencing study.

BACKGROUND: Serotype 24F is one of the emerging pneumococcal serotypes after the introduction of pneumococcal conjugate vaccine (PCV). We aimed to identify lineages driving the increase of serotype 24F in France and place these findings into a global context. METHODS: Whole-genome sequencing was performed on a collection of serotype 24F pneumococci from asymptomatic colonisation (n=229) and invasive disease (n=190) isolates among individuals younger than 18 years in France, from 2003 to 2018. To provide a global context, we included an additional collection of 24F isolates in the Global Pneumococcal Sequencing (GPS) project database for analysis. A Global Pneumococcal Sequence Cluster (GPSC) and a clonal complex (CC) were assigned to each genome. Phylogenetic, evolutionary, and spatiotemporal analysis were conducted using the same 24F collection and supplemented with a global collection of genomes belonging to the lineage of interest from the GPS project database (n=25 590). FINDINGS: Serotype 24F was identified in numerous countries mainly due to the clonal spread of three lineages: GPSC10 (CC230), GPSC16 (CC156), and GPSC206 (CC7701). GPSC10 was the only multidrug-resistant lineage. GPSC10 drove the increase in 24F in France and had high invasive disease potential. The international dataset of GPSC10 (n=888) revealed that this lineage expressed 16 other serotypes, with only six included in 13-valent PCV (PCV13). All serotype 24F isolates were clustered in a single clade within the GPSC10 phylogeny and long-range transmissions were detected from Europe to other continents. Spatiotemporal analysis showed GPSC10-24F took 3-5 years to spread across France and a rapid change of serotype composition from PCV13 serotype 19A to 24F during the introduction of PCV13 was observed in neighbouring country Spain. INTERPRETATION: Our work reveals that GPSC10 alone is a challenge for serotype-based vaccine strategy. More systematic investigation to identify lineages like GPSC10 will better inform and improve next-generation preventive strategies against pneumococcal diseases. FUNDING: Bill & Melinda Gates Foundation, Wellcome Sanger Institute, and the US Centers for Disease Control and Prevention.

A comparison of four epidemic waves of COVID-19 in Malawi; an observational cohort study.

Background: Compared to the abundance of clinical and genomic information available on patients hospitalised with COVID-19 disease from high-income countries, there is a paucity of data from low-income countries. Our aim was to explore the relationship between viral lineage and patient outcome. Methods: We enrolled a prospective observational cohort of adult patients hospitalised with PCR-confirmed COVID-19 disease between July 2020 and March 2022 from Blantyre, Malawi, covering four waves of SARS-CoV-2 infections. Clinical and diagnostic data were collected using an adapted ISARIC clinical characterization protocol for COVID-19. SARS-CoV-2 isolates were sequenced using the MinIONâ"¢ in Blantyre. Results: We enrolled 314 patients, good quality sequencing data was available for 55 patients. The sequencing data showed that 8 of 11 participants recruited in wave one had B.1 infections, 6/6 in wave two had Beta, 25/26 in wave three had Delta and 11/12 in wave four had Omicron. Patients infected during the Delta and Omicron waves reported fewer underlying chronic conditions and a shorter time to presentation. Significantly fewer patients required oxygen (22.7% [17/75] vs. 58.6% [140/239], p<0.001) and steroids (38.7% [29/75] vs. 70.3% [167/239], p<0.001) in the Omicron wave compared with the other waves. Multivariable logistic-regression demonstrated a trend toward increased mortality in the Delta wave (OR 4.99 [95% CI 1.0-25.0 p=0.05) compared to the first wave of infection. Conclusions: Our data show that each wave of patients hospitalised with SARS-CoV-2 was infected with a distinct viral variant. The clinical data suggests that patients with severe COVID-19 disease were more likely to die during the Delta wave. Summary: We used genome sequencing to identify the variants of SARS-CoV-2 causing disease in Malawi, and found that each of the four waves was caused by a distinct variant. Clinical investigation suggested that the Delta wave had the highest mortality.

A Streptococcus pneumoniae lineage usually associated with pneumococcal conjugate vaccine (PCV) serotypes is the most common cause of serotype 35B invasive disease in South Africa, following routine use of PCV.

Pneumococcal serotype 35B is an important non-conjugate vaccine (non-PCV) serotype. Its continued emergence, post-PCV7 in the USA, was associated with expansion of a pre-existing 35B clone (clonal complex [CC] 558) along with post-PCV13 emergence of a non-35B clone previously associated with PCV serotypes (CC156). This study describes lineages circulating among 35B isolates in South Africa before and after PCV introduction. We also compared 35B isolates belonging to a predominant 35B lineage in South Africa (GPSC5), with isolates belonging to the same lineage in other parts of the world. Serotype 35B isolates that caused invasive pneumococcal disease in South Africa in 2005-2014 were characterized by whole-genome sequencing (WGS). Multi-locus sequence types and global pneumococcal sequence clusters (GPSCs) were derived from WGS data of 63 35B isolates obtained in 2005-2014. A total of 262 isolates that belong to GPSC5 (115 isolates from South Africa and 147 from other countries) that were sequenced as part of the global pneumococcal sequencing (GPS) project were included for comparison. Serotype 35B isolates from South Africa were differentiated into seven GPSCs and GPSC5 was most common (49 %, 31/63). While 35B was the most common serotype among GPSC5/CC172 isolates in South Africa during the PCV13 period (66 %, 29/44), 23F was the most common serotype during both the pre-PCV (80 %, 37/46) and PCV7 period (32 %, 8/25). Serotype 35B represented 15 % (40/262) of GPSC5 isolates within the global GPS database and 75 % (31/40) were from South Africa. The predominance of the GPSC5 lineage within non-vaccine serotype 35B, is possibly unique to South Africa and warrants further molecular surveillance of pneumococci.

Comparative Genomics of Disease and Carriage Serotype 1 Pneumococci.

The isolation of Streptococcus pneumoniae serotypes in systemic tissues of patients with invasive disease versus the nasopharynx of healthy individuals with asymptomatic carriage varies widely. Some serotypes are hyper-invasive, particularly serotype 1, but the underlying genetics remain poorly understood due to the rarity of carriage isolates, reducing the power of comparison with invasive isolates. Here, we use a well-controlled genome-wide association study to search for genetic variation associated with invasiveness of serotype 1 pneumococci from a serotype 1 endemic setting in Africa. We found no consensus evidence that certain genomic variation is overrepresented among isolates from patients with invasive disease than asymptomatic carriage. Overall, the genomic variation explained negligible phenotypic variability, suggesting a minimal effect on the disease status. Furthermore, changes in lineage distribution were seen with lineages replacing each other over time, highlighting the importance of continued pathogen surveillance. Our findings suggest that the hyper-invasiveness is an intrinsic property of the serotype 1 strains, not specific for a "disease-associated" subpopulation disproportionately harboring unique genomic variation.

Effect of 3 Days of Oral Azithromycin on Young Children With Acute Diarrhea in Low-Resource Settings: A Randomized Clinical Trial.

Importance: World Health Organization (WHO) guidelines do not recommend routine antibiotic use for children with acute watery diarrhea. However, recent studies suggest that a significant proportion of such episodes have a bacterial cause and are associated with mortality and growth impairment, especially among children at high risk of diarrhea-associated mortality. Expanding antibiotic use among dehydrated or undernourished children may reduce diarrhea-associated mortality and improve growth. Objective: To determine whether the addition of azithromycin to standard case management of acute nonbloody watery diarrhea for children aged 2 to 23 months who are dehydrated or undernourished could reduce mortality and improve linear growth. Design, Setting, and Participants: The Antibiotics for Children with Diarrhea (ABCD) trial was a multicountry, randomized, double-blind, clinical trial among 8266 high-risk children aged 2 to 23 months presenting with acute nonbloody diarrhea. Participants were recruited between July 1, 2017, and July 10, 2019, from 36 outpatient hospital departments or community health centers in a mixture of urban and rural settings in Bangladesh, India, Kenya, Malawi, Mali, Pakistan, and Tanzania. Each participant was followed up for 180 days. Primary analysis included all randomized participants by intention to treat. Interventions: Enrolled children were randomly assigned to receive either oral azithromycin, 10 mg/kg, or placebo once daily for 3 days in addition to standard WHO case management protocols for the management of acute watery diarrhea. Main Outcomes and Measures: Primary outcomes included all-cause mortality up to 180 days after enrollment and linear growth faltering 90 days after enrollment. Results: A total of 8266 children (4463 boys [54.0%]; mean [SD] age, 11.6 [5.3] months) were randomized. A total of 20 of 4133 children in the azithromycin group (0.5%) and 28 of 4135 children in the placebo group (0.7%) died (relative risk, 0.72; 95% CI, 0.40-1.27). The mean (SD) change in length-for-age z scores 90 days after enrollment was -0.16 (0.59) in the azithromycin group and -0.19 (0.60) in the placebo group (risk difference, 0.03; 95% CI, 0.01-0.06). Overall mortality was much lower than anticipated, and the trial was stopped for futility at the prespecified interim analysis. Conclusions and Relevance: The study did not detect a survival benefit for children from the addition of azithromycin to standard WHO case management of acute watery diarrhea in low-resource settings. There was a small reduction in linear growth faltering in the azithromycin group, although the magnitude of this effect was not likely to be clinically significant. In low-resource settings, expansion of antibiotic use is not warranted. Adherence to current WHO case management protocols for watery diarrhea remains appropriate and should be encouraged. Trial Registration: ClinicalTrials.gov Identifier: NCT03130114.

Genomic investigation of a suspected Klebsiella pneumoniae outbreak in a neonatal care unit in sub-Saharan Africa.

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.

Distinct clinical and immunological profiles of patients with evidence of SARS-CoV-2 infection in sub-Saharan Africa.

Although the COVID-19 pandemic has left no country untouched there has been limited research to understand clinical and immunological responses in African populations. Here we characterise patients hospitalised with suspected (PCR-negative/IgG-positive) or confirmed (PCR-positive) COVID-19, and healthy community controls (PCR-negative/IgG-negative). PCR-positive COVID-19 participants were more likely to receive dexamethasone and a beta-lactam antibiotic, and survive to hospital discharge than PCR-negative/IgG-positive and PCR-negative/IgG-negative participants. PCR-negative/IgG-positive participants exhibited a nasal and systemic cytokine signature analogous to PCR-positive COVID-19 participants, predominated by chemokines and neutrophils and distinct from PCR-negative/IgG-negative participants. PCR-negative/IgG-positive participants had increased propensity for Staphylococcus aureus and Streptococcus pneumoniae colonisation. PCR-negative/IgG-positive individuals with high COVID-19 clinical suspicion had inflammatory profiles analogous to PCR-confirmed disease and potentially represent a target population for COVID-19 treatment strategies.