Phenotypic and molecular characterization of ß-lactamase-producing Klebsiella species among children discharged from hospital in Western Kenya.

BACKGROUND: The emergence and spread of ß-lactamase-producing Klebsiella spp. has been associated with a substantial healthcare burden resulting in therapeutic failures. We sought to describe the proportion of phenotypic resistance to commonly used antibiotics, characterize ß-lactamase genes among isolates with antimicrobial resistance (AMR), and assess the correlates of phenotypic AMR in Klebsiella spp. isolated from stool or rectal swab samples collected from children being discharged from hospital. METHODS: We conducted a cross-sectional study involving 245 children aged 1-59 months who were being discharged from hospitals in western Kenya between June 2016 and November 2019. Whole stool or rectal swab samples were collected and Klebsiella spp. isolated by standard microbiological culture. ß-lactamase genes were detected by PCR whilst phenotypic antimicrobial susceptibility was determined using the disc diffusion technique following standard microbiology protocols. Descriptive analyses were used to characterize phenotypic AMR and carriage of ß-lactamase-producing genes. The modified Poisson regression models were used to assess correlates of phenotypic beta-lactam resistance. RESULTS: The prevalence of ß-lactamase carriage among Klebsiella spp. isolates at hospital discharge was 62.9% (154/245). Antibiotic use during hospitalization (adjusted prevalence ratio [aPR] = 4.51; 95%CI: 1.79-11.4, p < 0.001), longer duration of hospitalization (aPR = 1.42; 95%CI: 1.14-1.77, p < 0.002), and access to treated water (aPR = 1.38; 95%CI: 1.12-1.71, p < 0.003), were significant predictors of phenotypically determined ß-lactamase. All the 154 ß-lactamase-producing Klebsiella spp. isolates had at least one genetic marker of ß-lactam/third-generation cephalosporin resistance. The most prevalent genes were blaCTX-M 142/154 (92.2%,) and blaSHV 142/154 (92.2%,) followed by blaTEM 88/154 (57.1%,) and blaOXA 48/154 (31.2%,) respectively. CONCLUSION: Carriage of ß-lactamase producing Klebsiella spp. in stool is common among children discharged from hospital in western Kenya and is associated with longer duration of hospitalization, antibiotic use, and access to treated water. The findings emphasize the need for continued monitoring of antimicrobial susceptibility patterns to inform the development and implementation of appropriate treatment guidelines. In addition, we recommend measures beyond antimicrobial stewardship and infection control within hospitals, improved sanitation, and access to safe drinking water to mitigate the spread of ß-lactamase-producing Klebsiella pathogens in these and similar settings.

Clinical epidemiology of COVID-19 among hospitalized children in rural western Kenya.

The epidemiology of pediatric COVID-19 in sub-Saharan Africa and the role of fecal-oral transmission in SARS-CoV-2 are poorly understood. Among children and adolescents in Kenya, we identify correlates of COVID-19 infection, document the clinical outcomes of infection, and evaluate the prevalence and viability of SARS-CoV-2 in stool. We recruited a prospective cohort of hospitalized children aged two months to 15 years in western Kenya between March 1 and June 30 2021. Children with SARS-CoV-2 were followed monthly for 180-days after hospital discharge. Bivariable logistic regression analysis was used to identify the clinical and sociodemographics correlates of SARS-CoV-2 infection. We also calculated the prevalence of SARS-CoV-2 detection in stool of confirmed cases. Of 355 systematically tested children, 55 (15.5%) were positive and were included in the cohort. The commonest clinical features among COVID-19 cases were fever (42/55, 76%), cough (19/55, 35%), nausea and vomiting (19/55, 35%), and lethargy (19/55, 35%). There were no statistically significant difference in baseline sociodemographic and clinical characteristics between SARS-CoV-2 positive and negative participants. Among positive participants, 8/55 (14.5%, 95%CI: 5.3%-23.9%) died; seven during the inpatient period. Forty-nine children with COVID-19 had stool samples or rectal swabs available at baseline, 9 (17%) had PCR-positive stool or rectal swabs, but none had SARS-CoV-2 detected by culture. Syndromic identification of COVID-19 is particularly challenging among children as the presenting symptoms and signs mirror other common pediatric diseases. Mortality among children hospitalized with COVID-19 was high in this cohort but was comparable to mortality seen with other common illnesses in this setting. Among this small set of children with COVID-19 we detected SARS-CoV-2 DNA, but were not able to culture viable SARs-CoV-2 virus, in stool. This suggests that fecal transmission may not be a substantial risk in children recently diagnosed and hospitalized with COVID-19 infection.

Plasmid-mediated quinolone resistance genes detected in Ciprofloxacin non-susceptible Escherichia coli and Klebsiella isolated from children under five years at hospital discharge, Kenya.

BACKGROUND: The increasing spread of fluoroquinolone resistant enteric bacteria is a global public health concern. Children recently discharged from the hospital are at high risk of carriage of antimicrobial resistance (AMR) due to frequent exposure to antimicrobials during inpatient stays. This study aimed to determine the prevalence, correlates of ciprofloxacin (CIP) non-susceptibility, and distribution of plasmid-mediated quinolone resistance (PMQR) genes in Escherichia coli (E. coli) and Klebsiella spp isolated from children under five years being discharged from two Kenyan Hospitals. METHODS: E. coli and Klebsiella spp were isolated from fecal samples from children discharged from hospital and subjected to antimicrobial susceptibility testing (AST) by disc diffusion and E-test. CIP non-susceptible isolates were screened for seven PMQR genes using multiplex polymerase chain reaction (PCR). Poisson regression was used to determine the association between the carriage of CIP non-susceptible isolates and patient characteristics. RESULTS: Of the 280 CIP non-susceptible isolates: 188 E. coli and 92 Klebsiella spp isolates identified among 266 discharged children, 195 (68%) were CIP-non-susceptible with minimum inhibitory concentrations (MICs) of ≥ 1 µg/mL. Among these 195 isolates, 130 (67%) had high-level CIP MIC = ≥ 32 µg/mL). Over 80% of the isolates had at least one PMQR gene identified: aac(6')lb-cr (60%), qnrB (24%), oqxAB (22%), qnrS (16%), and qepA (6%), however, qnrA was not identified in any isolates tested. Co-carriage of qnrB with acc(6')-lb-cr was the most predominant accounting for 20% of all the isolates. Ceftriaxone use during hospital admission and the presence of extended spectrum beta-lactamase (ESBL) production were significantly associated with the carriage of CIP non-susceptible E. coli and Klebsiella spp. CONCLUSION: CIP non-susceptibility is common among E. coli and Klebsiella spp isolated from hospital discharged children in Kenya. Carriage and co-carriage of PMQR, including the newly identified qepA gene, were frequently observed. These findings suggest that children leaving the hospital may serve as an important reservoir for transmission of resistant E. coli and Klebsiella spp to the community. Enhanced surveillance for AMR determinants is critical to inform interventions to control antimicrobial-resistant bacteria.

The Childhood Acute Illness and Nutrition (CHAIN) network nested case-cohort study protocol: a multi-omics approach to understanding mortality among children in sub-Saharan Africa and South Asia.

Introduction: Many acutely ill children in low- and middle-income settings have a high risk of mortality both during and after hospitalisation despite guideline-based care. Understanding the biological mechanisms underpinning mortality may suggest optimal pathways to target for interventions to further reduce mortality. The Childhood Acute Illness and Nutrition (CHAIN) Network ( www.chainnnetwork.org) Nested Case-Cohort Study (CNCC) aims to investigate biological mechanisms leading to inpatient and post-discharge mortality through an integrated multi-omic approach. Methods and analysis; The CNCC comprises a subset of participants from the CHAIN cohort (1278/3101 hospitalised participants, including 350 children who died and 658 survivors, and 270/1140 well community children of similar age and household location) from nine sites in six countries across sub-Saharan Africa and South Asia. Systemic proteome, metabolome, lipidome, lipopolysaccharides, haemoglobin variants, toxins, pathogens, intestinal microbiome and biomarkers of enteropathy will be determined. Computational systems biology analysis will include machine learning and multivariate predictive modelling with stacked generalization approaches accounting for the different characteristics of each biological modality. This systems approach is anticipated to yield mechanistic insights, show interactions and behaviours of the components of biological entities, and help develop interventions to reduce mortality among acutely ill children. Ethics and dissemination. The CHAIN Network cohort and CNCC was approved by institutional review boards of all partner sites. Results will be published in open access, peer reviewed scientific journals and presented to academic and policy stakeholders. Data will be made publicly available, including uploading to recognised omics databases. Trial registration NCT03208725.

Antimicrobial resistance including Extended Spectrum Beta Lactamases (ESBL) among E. coli isolated from kenyan children at hospital discharge.

BACKGROUND: Children who have been discharged from hospital in sub-Saharan Africa remain at substantial risk of mortality in the post-discharge period. Antimicrobial resistance (AMR) may be an important factor. We sought to determine the prevalence and risk factors associated with AMR in commensal Escherichia coli (E. coli) from Kenyan children at the time of discharge. METHODOLOGY/PRINCIPLE FINDINGS: Fecal samples were collected from 406 children aged 1-59 months in western Kenya at the time of discharge from hospital and cultured for E. coli. Susceptibility to ampicillin, ceftriaxone, cefotaxime, ceftazidime, cefoxitin, imipenem, ciprofloxacin, gentamicin, combined amoxicillin/clavulanic acid, trimethoprim-sulfamethoxazole, azithromycin, and chloramphenicol was determined by disc diffusion according to guidelines from the Clinical and Laboratory Standards Institute (CLSI). Poisson regression was used to determine associations between participant characteristics and the presence of extended-spectrum beta-lactamases (ESBL) producing E. coli. Non-susceptibility to ampicillin (95%), gentamicin (44%), ceftriaxone (46%), and the presence of ESBL (44%) was high. Receipt of antibiotics during the hospitalization was associated with the presence of ESBL (aPR = 2.23; 95% CI: 1.29-3.83) as was being hospitalized within the prior year (aPR = 1.32 [1.07-1.69]). Open defecation (aPR = 2.02; 95% CI: 1.39-2.94), having a toilet shared with other households (aPR = 1.49; 95% CI: 1.17-1.89), and being female (aPR = 1.42; 95% CI: 1.15-1.76) were associated with carriage of ESBL E. coli. CONCLUSIONS/SIGNIFICANCE: AMR is common among isolates of E. coli from children at hospital discharge in Kenya, including nearly half having detectable ESBL.

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.

Azithromycin for the prevention of rehospitalisation and death among Kenyan children being discharged from hospital: a double-blind, placebo-controlled, randomised controlled trial.

BACKGROUND: Mass drug administration of azithromycin to children in sub-Saharan Africa has been shown to improve survival in high-mortality settings. The period after hospital discharge is a time of elevated risk unaddressed by current interventions and might provide an opportunity for targeting empirical azithromycin administration. We aimed to assess the efficacy of azithromycin administered at hospital discharge on risk of death and rehospitalisation in Kenyan children younger than 5 years. METHODS: In this double-blind, placebo-controlled randomised trial, children were randomly assigned (1:1) to receive a 5-day course of azithromycin (oral suspension 10 mg/kg on day 1, followed by 5mg/kg per day on days 2-5) or identically appearing and tasting placebo at discharge from four hospitals in western Kenya. Children were eligible if they were aged 1-59 months at hospital discharge, weighed at least 2 kg, and had been admitted to hospital for any medical reason other than trauma, poisoning, or congenital anomaly. The primary outcome was death or rehospitalisation in the subsequent 6-month period in a modified intention-to-treat population, compared by randomisation group with Cox proportional hazards regression and Kaplan-Meier. Azithromycin resistance in Escherichia coli isolates from a random subset of children was compared by randomisation group with generalised estimating equations. This trial is registered with ClinicalTrials.gov, NCT02414399. FINDINGS: Between June 28, 2016, and Nov 4, 2019, 1400 children were enrolled in the trial at discharge from hospital, with 703 (50·2%) randomly assigned to azithromycin and 697 (49·8%) to placebo. Among the 1398 children included in the modified intention-to-treat analysis (702 in the azithromycin group and 696 in the placebo group), the incidence of death or rehospitalisation was 20·4 per 100 child-years in the azithromycin group and 22·5 per 100 child-years in the placebo group (adjusted hazard ratio 0·91, 95·5% CI 0·64-1·29, p=0·58). Azithromycin resistance was common in commensal E coli isolates from enrolled children before randomisation (37·7% of 406 isolates) despite only 3·7% of children having received a macrolide antibiotic during the hospitalisation. Azithromycin resistance was slightly higher at 3 months after randomisation in the azithromycin group (26·9%) than in the placebo group (19·1%; adjusted prevalence ratio 1·41, 95% CI 0·95-2·09, p=0·088), with no difference observed at 6 months (1·17, 0·78-1·76, p=0·44). INTERPRETATION: We did not observe a significant benefit of a 5-day course of azithromycin delivered to children younger than 5 years at hospital discharge despite the overall high risk of mortality and rehospitalisation. These findings highlight the need for more research into mechanisms and interventions for prevention of morbidity and mortality in the post-discharge period. FUNDING: Eunice Kennedy Shriver National Institute of Child Health & Human Development.

Prevalence and Correlates of Cryptosporidium Infections in Kenyan Children With Diarrhea and Their Primary Caregivers.

BACKGROUND: Cryptosporidium is a leading cause of diarrhea in Sub-Saharan Africa and is associated with substantial morbidity and mortality in young children. METHODS: We analyzed data from children aged 6-71 months presenting to 2 public hospitals in Western Kenya with acute diarrhea and their primary caregivers, including detection of Cryptosporidium by quantitative polymerase chain reaction (PCR) and immunoassay analysis in stool samples from both children and their caregivers. Associations between potential transmission sources and child/caregiver Cryptosporidium infection were evaluated using prevalence ratios (PRs). Secondary analyses evaluated host and clinical risk factors of child/caregiver Cryptosporidium infection. RESULTS: Among 243 child-caregiver pairs enrolled, 77 children (32%) and 57 caregivers (23%) had Cryptosporidium identified by either immunoassay or PCR. Twenty-six of the 243 child-caregiver pairs (11%) had concordant detection of Cryptosporidium. Cryptosporidium infection in children was associated with detection of Cryptosporidium in caregivers (adjusted PR [aPR], 1.8; 95% CI, 1.2 to 2.6; P = .002) and unprotected water source (aPR, 2.0; 95% CI, 1.3 to 3.2; P = .003). Risk factors for Cryptosporidium detection in caregivers included child Cryptosporidium infection (aPR, 2.0; 95% CI, 1.3 to 3.0; P = .002) as well as cow (aPR, 3.1; 95% CI, 1.4 to 7.0; P = .02) and other livestock ownership (aPR, 2.6; 95% CI, 1.1 to 6.3; P = .03) vs no livestock ownership. Recent diarrhea in caregivers and children was independently associated with child and caregiver Cryptosporidium infections, respectively. CONCLUSIONS: Our results are consistent with the hypothesis that Cryptosporidium transmission can occur directly between child-caregiver dyads as well as through other pathways involving water and livestock. Additional research into caregivers as a source of childhood Cryptosporidium infection is warranted.

Correlates of multi-drug non-susceptibility in enteric bacteria isolated from Kenyan children with acute diarrhea.

BACKGROUND: Reduced antimicrobial susceptibility threatens treatment efficacy in sub-Saharan Africa, where data on the burden and correlates of antibiotic resistance among enteric pathogens are limited. METHODS: Fecal samples from children aged 6 mos-15 yrs presenting with acute diarrhea in western Kenya were cultured for bacterial pathogens. HIV-uninfected children with identified Shigella or Salmonella species or pathogenic Escherichia coli (EPEC, ETEC, EAEC or EIEC) were included in this cross-sectional sub-study. Non-susceptibility to ampicillin, ceftriaxone, ciprofloxacin, cotrimoxazole, and tetracycline was determined using MicroScan Walkaway40 Plus. Multivariable log-binomial regression was used to identify correlates of multi-drug non-susceptibility (MDNS, non-susceptibility to ≥ 3 of these antibiotics). RESULTS: Of 292 included children, median age was 22.5 mos. MDNS was identified in 62.5% of 318 isolates. Non-susceptibility to cotrimoxazole (92.8%), ampicillin (81.3%), and tetracycline (75.0%) was common. Young age (6-24 mos vs. 24-59 mos adjusted prevalence ratio [aPR] = 1.519 [95% confidence interval: 1.19, 1.91]), maternal HIV (aPR = 1.29 [1.01, 1.66]); and acute malnutrition (aPR = 1.28 [1.06, 1.55]) were associated with higher prevalence of MDNS, as were open defecation (aPR = 2.25 [1.13, 4.50]), household crowding (aPR = 1.29 [1.08, 1.53]) and infrequent caregiver hand-washing (aPR = 1.50 [1.15, 1.95]). CONCLUSIONS: Young age, HIV exposure, acute malnutrition and poor sanitation may increase risk of antibiotic non-susceptible enteric pathogen infections among children in Kenya.

Azithromycin to prevent post-discharge morbidity and mortality in Kenyan children: a protocol for a randomised, double-blind, placebo-controlled trial (the Toto Bora trial).

INTRODUCTION: Child mortality due to infectious diseases remains unacceptably high in much of sub-Saharan Africa. Children who are hospitalised represent an accessible population at particularly high risk of death, both during and following hospitalisation. Hospital discharge may be a critical time point at which targeted use of antibiotics could reduce morbidity and mortality in high-risk children. METHODS AND ANALYSIS: In this randomised, double-blind, placebo-controlled trial (Toto Bora Trial), 1400 children aged 1-59 months discharged from hospitals in Western Kenya, in Kisii and Homa Bay, will be randomised to either a 5-day course of azithromycin or placebo to determine whether a short course of azithromycin reduces rates of rehospitalisation and/or death in the subsequent 6-month period. The primary analysis will be modified intention-to-treat and will compare the rates of rehospitalisation or death in children treated with azithromycin or placebo using Cox proportional hazard regression. The trial will also evaluate the effect of a short course of azithromycin on enteric and nasopharyngeal infections and cause-specific morbidities. We will also identify risk factors for postdischarge morbidity and mortality and subpopulations most likely to benefit from postdischarge antibiotic use. Antibiotic resistance in Escherichia coli and Streptococcus pneumoniae among enrolled children and their primary caregivers will also be assessed, and cost-effectiveness analyses will be performed to inform policy decisions. ETHICS AND DISSEMINATION: Study procedures were reviewed and approved by the institutional review boards of the Kenya Medical Research Institute, the University of Washington and the Kenyan Pharmacy and Poisons Board. The study is being externally monitored, and a data safety and monitoring committee has been assembled to monitor patient safety and to evaluate the efficacy of the intervention. The results of this trial will be published in peer-reviewed scientific journals and presented at relevant academic conferences and to key stakeholders. TRIAL REGISTRATION NUMBER: NCT02414399.