The epidemiology of fecal carriage of nontyphoidal Salmonella among healthy children and adults in three sites in Kenya.

BACKGROUND: Despite the importance of non-Typhoidal Salmonella (NTS) disease in Africa, epidemiologic data on carriage and transmission are few. These data are important to understand the transmission of NTS in Africa and to design control strategies. METHOD: To estimate the prevalence of stool carriage of NTS in Kenya, we conducted a cross-sectional study in Kilifi, Nairobi, and Siaya, sites with a low, moderate and high incidence of invasive NTS disease, respectively. At each site, we randomly selected 100 participants in each age-group of 0-11 months, 12-59 months, 5-14 years, 15-54 years and ≥55 years. We collected stool, venous blood (for hemoglobin and malaria rapid tests), anthropometric measurements, and administered a questionnaire on Water Access Sanitation and Hygiene (WASH) practices. Stool samples were cultured on selective agar for Salmonella; suspect isolates underwent serotyping and antimicrobial susceptibility testing. RESULT: Overall, 53 (3.5%) isolates of NTS were cultured from 1497 samples. Age-adjusted prevalence was 13.1% (95%CI 8.8-17.4) in Kilifi, 0.4% (95%CI 0-1.3) in Nairobi, and 0.9% (95%CI 0-2.0) in Siaya. Prevalence was highest among those aged 15-54 years (6.2%). Of 53 isolates; 5 were S. Enteritidis, 1 was S. Typhimurium. No S. Typhi was isolated. None of the risk factors were associated with carriage of NTS. All isolates were susceptible to all antibiotics tested, including ampicillin, chloramphenicol, ciprofloxacin and co-trimoxazole. CONCLUSION: Prevalence of fecal carriage was high in Kilifi, an area of low incidence of invasive NTS disease and was low in areas of higher incidence in Nairobi and Siaya. The age-prevalence, risk factors, geographical and serotype distribution of NTS in carriage differs from invasive disease.

BIRC6 modifies risk of invasive bacterial infection in Kenyan children.

Invasive bacterial disease is a major cause of morbidity and mortality in African children. Despite being caused by diverse pathogens, children with sepsis are clinically indistinguishable from one another. In spite of this, most genetic susceptibility loci for invasive infection that have been discovered to date are pathogen specific and are not therefore suggestive of a shared genetic architecture of bacterial sepsis. Here, we utilise probabilistic diagnostic models to identify children with a high probability of invasive bacterial disease among critically unwell Kenyan children with Plasmodium falciparum parasitaemia. We construct a joint dataset including 1445 bacteraemia cases and 1143 severe malaria cases, and population controls, among critically unwell Kenyan children that have previously been genotyped for human genetic variation. Using these data, we perform a cross-trait genome-wide association study of invasive bacterial infection, weighting cases according to their probability of bacterial disease. In doing so, we identify and validate a novel risk locus for invasive infection secondary to multiple bacterial pathogens, that has no apparent effect on malaria risk. The locus identified modifies splicing of BIRC6 in stimulated monocytes, implicating regulation of apoptosis and autophagy in the pathogenesis of sepsis in Kenyan children.

Bacterial infections are a major cause of severe illness and death in African children. Understanding which children are at risk of life-threatening infection and why, is key to designing new tools to help protect them. Some risk is likely inherited, but scientists do not know which genes are responsible. Genome-wide association studies (GWAS) may be one way to identify bacterial infection risk genes. GWAS look for genetic differences associated with a particular disease. But previous GWAS studies have failed to find genes linked with bacterial infections in African children because they were too small. Malaria is another frequent cause of life-threatening illness in African children. It can be hard for clinicians to determine if a child's illness is caused by malaria, a bacterial infection, or both. Many children in Africa have malaria parasites in their blood, but they do not always cause disease. Most children with suspected severe malaria are treated with antibiotics in case of bacterial infection. Clinicians may then conduct further testing to determine the illness's actual cause. Scientists may be able to use this data on children with suspected malaria to study bacterial infections. Gilchrist et al. show that children with an unusual alteration in the BIRC6 gene are at increased risk of bacterial infections. In the experiments, Gilchrist et al. used computer modeling to identify a subset of children with likely bacterial infections among 2,200 children admitted to a hospital in Kenya with a high fever and malaria parasites. By combining information on this subset of children with data on children with confirmed bacterial infections and healthy children, Gilchrist created a sample of 5,400 children for a GWAS. The analyses found that children with a variation in the BIRC6 gene on chromosome 2 had a higher risk of bacterial infections. This genetic change is linked with the production of a modified form of BIRC6 in infection-fighting immune cells called monocytes. More studies will help scientists understand how this change might contribute to severe bacterial infections. Learning more may help scientists develop new treatment strategies and identify children most at risk.

Incidence of chikungunya virus infections among Kenyan children with neurological disease, 2014-2018: A cohort study.

BACKGROUND: Neurological complications due to chikungunya virus (CHIKV) infection have been described in different parts of the world, with children being disproportionately affected. However, the burden of CHIKV-associated neurological disease in Africa is currently unknown and given the lack of diagnostic facilities in routine care it is possible that CHIKV is an unrecognized etiology among children with encephalitis or other neurological illness. METHODS AND FINDINGS: We estimated the incidence of CHIKV infection among children hospitalized with neurological disease in Kilifi County, coastal Kenya. We used reverse transcriptase polymerase chain reaction (RT-PCR) to systematically test for CHIKV in cerebrospinal fluid (CSF) samples from children aged <16 years hospitalized with symptoms of neurological disease at Kilifi County Hospital between January 2014 and December 2018. Clinical records were linked to the Kilifi Health and Demographic Surveillance System and population incidence rates of CHIKV infection estimated. There were 18,341 pediatric admissions for any reason during the 5-year study period, of which 4,332 (24%) had CSF collected. The most common clinical reasons for CSF collection were impaired consciousness, seizures, and coma (47%, 22%, and 21% of all collections, respectively). After acute investigations done for immediate clinical care, CSF samples were available for 3,980 admissions, of which 367 (9.2%) were CHIKV RT-PCR positive. Case fatality among CHIKV-positive children was 1.4% (95% CI 0.4, 3.2). The annual incidence of CHIKV-associated neurological disease varied between 13 to 58 episodes per 100,000 person-years among all children <16 years old. Among children aged <5 years, the incidence of CHIKV-associated neurological disease was 77 per 100,000 person-years, compared with 20 per 100,000 for cerebral malaria and 7 per 100,000 for bacterial meningitis during the study period. Because of incomplete case ascertainment due to children not presenting to hospital, or not having CSF collected, these are likely minimum estimates. Study limitations include reliance on hospital-based surveillance and limited CSF sampling in children in coma or other contraindications to lumbar puncture, both of which lead to under-ascertainment of incidence and of case fatality. CONCLUSIONS: In this study, we observed that CHIKV infections are relatively more common than cerebral malaria and bacterial meningitis among children hospitalized with neurological disease in coastal Kenya. Given the wide distribution of CHIKV mosquito vectors, studies to determine the geographic extent of CHIKV-associated neurological disease in Africa are essential.

The CINAMR (Clinical Information Network-Antimicrobial Resistance) Project: A pilot microbial surveillance using hospitals linked to regional laboratories in Kenya: Study Protocol.

Background: Antimicrobial resistance (AMR) is a global threat and is thought to be acute in low-and middle-income country (LMIC) settings, including in Kenya, but there is limited unbiased surveillance that can provide reliable estimates of its burden. Current efforts to build capacity for microbiology testing in Kenya are unlikely to result in systematic routine microbiological testing in the near term. Therefore, there is little prospect for microbiological support to inform clinical diagnoses nor for indicating the burden of AMR and for guiding empirical choice of antibiotics. Objective: We aim to build on an existing collaboration, the Clinical Information Network (CIN), to pilot microbiological surveillance using a 'hub-and-spoke' model where selected hospitals are linked to high quality microbiology research laboratories. Methods: Children admitted to paediatric wards of 12 participating hospitals will have a sample taken for blood culture at admission before antibiotics are started. Indication for blood culture will be a clinician's prescription of antibiotics. Samples will then be transported daily to the research laboratories for culture and antibiotic susceptibility testing and results relayed back to clinicians for patient management. The surveillance will take place for 6 months in each hospital. Separately, we shall conduct semi-structured interviews with frontline health workers to explore the feasibility and utility of this approach. We will also seek to understand how the availability of microbiology results might inform antibiotic stewardship, and as an interim step to the development of better national or regional laboratories linked to routine surveillance. Conclusions: If feasible, this approach is less costly and periodic 'hub-and-spoke' surveillance can be used to track AMR trends and to broadly guide empirical antibiotic guidance meaning it is likely to be more sustainable than establishing functional microbiological facilities in each hospital in a LMIC setting.

Hepcidin regulation in Kenyan children with severe malaria and non-typhoidal Salmonella bacteremia.

Malaria and invasive non-typhoidal Salmonella (NTS) are life-threatening infections that often co-exist in African children. The iron-regulatory hormone hepcidin is highly upregulated during malaria and controls the availability of iron, a critical nutrient for bacterial growth. We investigated the relationship between Plasmodium falciparum malaria and NTS bacteremia in all pediatric admissions aged <5 years between August 1998 and October 2019 (n=75,034). We then assayed hepcidin and measures of iron status in five groups: (1) children with concomitant severe malarial anemia (SMA) and NTS (SMA+NTS, n=16); and in matched children with (2) SMA (n=33); (3) NTS (n=33); (4) cerebral malaria (CM, n=34); and (5) community-based children. SMA and severe anemia without malaria were associated with a 2-fold or more increased risk of NTS bacteremia, while other malaria phenotypes were not associated with increased NTS risk. Children with SMA had lower hepcidin/ferritin ratios (0.10; interquartile range [IQR]: 0.03-0.19) than those with CM (0.24; IQR: 0.14-0.69; P=0.006) or asymptomatic malaria (0.19; IQR: 0.09-0.46; P=0.01) indicating suppressed hepcidin levels. Children with SMA+NTS had lower hepcidin levels (9.3 ng/mL; IQR: 4.7-49.8) and hepcidin/ferritin ratios (0.03; IQR: 0.01-0.22) than those with NTS alone (105.8 ng/mL; IQR: 17.3-233.3; P=0.02 and 0.31; IQR: 0.06-0.66; P=0.007, respectively). Since hepcidin degrades ferroportin on the Salmonella-containing vacuole, we hypothesize that reduced hepcidin in children with SMA might contribute to NTS growth by modulating iron availability for bacterial growth. Further studies are needed to understand how the hepcidin-ferroportin axis might mediate susceptibility to NTS in severely anemic children.

The Etiology of Pneumonia in HIV-uninfected Children in Kilifi, Kenya: Findings From the Pneumonia Etiology Research for Child Health (PERCH) Study.

BACKGROUND: In the 1980s, Streptococcus pneumoniae and Haemophilus influenzae were identified as the principal causes of severe pneumonia in children. We investigated the etiology of severe childhood pneumonia in Kenya after introduction of conjugate vaccines against H. influenzae type b, in 2001, and S. pneumoniae, in 2011. METHODS: We conducted a case-control study between August 2011 and November 2013 among residents of the Kilifi Health and Demographic Surveillance System 28 days to 59 months of age. Cases were hospitalized at Kilifi County Hospital with severe or very severe pneumonia according to the 2005 World Health Organization definition. Controls were randomly selected from the community and frequency matched to cases on age and season. We tested nasal and oropharyngeal samples, sputum, pleural fluid, and blood specimens and used the Pneumonia Etiology Research for Child Health Integrated Analysis, combining latent class analysis and Bayesian methods, to attribute etiology. RESULTS: We enrolled 630 and 863 HIV-uninfected cases and controls, respectively. Among the cases, 282 (44%) had abnormal chest radiographs (CXR positive), 33 (5%) died in hospital, and 177 (28%) had diagnoses other than pneumonia at discharge. Among CXR-positive pneumonia cases, viruses and bacteria accounted for 77% (95% CrI: 67%-85%) and 16% (95% CrI: 10%-26%) of pneumonia attribution, respectively. Respiratory syncytial virus, S. pneumoniae and H. influenza, accounted for 37% (95% CrI: 31%-44%), 5% (95% CrI: 3%-9%), and 6% (95% CrI: 2%-11%), respectively. CONCLUSIONS: Respiratory syncytial virus was the main cause of CXR-positive pneumonia. The small contribution of H. influenzae type b and pneumococcus to pneumonia may reflect the impact of vaccine introductions in this population.

Clinical features of bacterial meningitis among hospitalised children in Kenya.

BACKGROUND: Diagnosing bacterial meningitis is essential to optimise the type and duration of antimicrobial therapy to limit mortality and sequelae. In sub-Saharan Africa, many public hospitals lack laboratory capacity, relying on clinical features to empirically treat or not treat meningitis. We investigated whether clinical features of bacterial meningitis identified prior to the introduction of conjugate vaccines still discriminate meningitis in children aged ≥60 days. METHODS: We conducted a retrospective cohort study to validate seven clinical features identified in 2002 (KCH-2002): bulging fontanel, neck stiffness, cyanosis, seizures outside the febrile convulsion age range, focal seizures, impaired consciousness, or fever without malaria parasitaemia and Integrated Management of Childhood Illness (IMCI) signs: neck stiffness, lethargy, impaired consciousness or seizures, and assessed at admission in discriminating bacterial meningitis after the introduction of conjugate vaccines. Children aged ≥60 days hospitalised between 2012 and 2016 at Kilifi County Hospital were included in this analysis. Meningitis was defined as positive cerebrospinal fluid (CSF) culture, organism observed on CSF microscopy, positive CSF antigen test, leukocytes ≥50/µL, or CSF to blood glucose ratio <0.1. RESULTS: Among 12,837 admissions, 98 (0.8%) had meningitis. The presence of KCH-2002 signs had a sensitivity of 86% (95% CI 77-92) and specificity of 38% (95% CI 37-38). Exclusion of 'fever without malaria parasitaemia' reduced sensitivity to 58% (95% CI 48-68) and increased specificity to 80% (95% CI 79-80). IMCI signs had a sensitivity of 80% (95% CI 70-87) and specificity of 62% (95% CI 61-63). CONCLUSIONS: A lower prevalence of bacterial meningitis and less typical signs than in 2002 meant the lower performance of KCH-2002 signs. Clinicians and policymakers should be aware of the number of lumbar punctures (LPs) or empirical treatments needed for each case of meningitis. Establishing basic capacity for CSF analysis is essential to exclude bacterial meningitis in children with potential signs.

Clinical features to distinguish meningitis among young infants at a rural Kenyan hospital.

BACKGROUND: Detection of meningitis is essential to optimise the duration and choice of antimicrobial agents to limit mortality and sequelae. In low and middle-income countries most health facilities lack laboratory capacity and rely on clinical features to empirically treat meningitis. OBJECTIVE: We conducted a diagnostic validation study to investigate the performance of clinical features (fever, convulsions, irritability, bulging fontanel and temperature ≥39°C) and WHO-recommended signs (drowsiness, lethargy, unconsciousness, convulsions, bulging fontanel, irritability or a high-pitched cry) in discriminating meningitis in young infants. DESIGN: Retrospective cohort study. SETTING: Kilifi County Hospital. PATIENTS: Infants aged <60 days hospitalised between 2012 and 2016. MAIN OUTCOME MEASURE: Definite meningitis defined as positive cerebrospinal fluid (CSF) culture, microscopy or antigen test, or leucocytes ≥0.05 x 10∧9/L. RESULTS: Of 4809 infants aged <60 days included, 81 (1.7%) had definite meningitis. WHO-recommended signs had sensitivity of 58% (95% CI 47% to 69%) and specificity of 57% (95% CI 56% to 59%) for definite meningitis. Addition of history of fever improved sensitivity to 89% (95% CI 80% to 95%) but reduced specificity to 26% (95% CI 25% to 27%). Presence of ≥1 of 5 previously identified signs had sensitivity of 79% (95% CI 69% to 87%) and specificity of 51% (95% CI 50% to 53%). CONCLUSIONS: Despite a lower prevalence of definite meningitis, the performance of previously identified signs at admission in predicting meningitis was unchanged. Presence of history of fever improves the sensitivity of WHO-recommended signs but loses specificity. Careful evaluation, repeated assessment and capacity for lumbar puncture and CSF microscopy to exclude meningitis in most young infants with potential signs are essential to management in this age group.

Risk of pneumococcal bacteremia in Kenyan children with glucose-6-phosphate dehydrogenase deficiency.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzyme deficiency state in humans. The clinical phenotype is variable and includes asymptomatic individuals, episodic hemolysis induced by oxidative stress, and chronic hemolysis. G6PD deficiency is common in malaria-endemic regions, an observation hypothesized to be due to balancing selection at the G6PD locus driven by malaria. G6PD deficiency increases risk of severe malarial anemia, a key determinant of invasive bacterial disease in malaria-endemic settings. The pneumococcus is a leading cause of invasive bacterial infection and death in African children. The effect of G6PD deficiency on risk of pneumococcal disease is undefined. We hypothesized that G6PD deficiency increases pneumococcal disease risk and that this effect is dependent upon malaria. METHODS: We performed a genetic case-control study of pneumococcal bacteremia in Kenyan children stratified across a period of falling malaria transmission between 1998 and 2010. RESULTS: Four hundred twenty-nine Kenyan children with pneumococcal bacteremia and 2677 control children were included in the study. Among control children, G6PD deficiency, secondary to the rs1050828 G>A mutation, was common, with 11.2% (n = 301 of 2677) being hemi- or homozygotes and 33.3% (n = 442 of 1329) of girls being heterozygotes. We found that G6PD deficiency increased the risk of pneumococcal bacteremia, but only during a period of high malaria transmission (P = 0.014; OR 2.33, 95% CI 1.19-4.57). We estimate that the population attributable fraction of G6PD deficiency on risk of pneumococcal bacteremia in areas under high malaria transmission is 0.129. CONCLUSIONS: Our data demonstrate that G6PD deficiency increases risk of pneumococcal bacteremia in a manner dependent on malaria. At the population level, the impact of G6PD deficiency on invasive pneumococcal disease risk in malaria-endemic regions is substantial. Our study highlights the infection-associated morbidity and mortality conferred by G6PD deficiency in malaria-endemic settings and adds to our understanding of the potential indirect health benefits of improved malaria control.

Pooled testing conserves SARS-CoV-2 laboratory resources and improves test turn-around time: experience on the Kenyan Coast.

Background. International recommendations for the control of the coronavirus disease 2019 (COVID-19) pandemic emphasize the central role of laboratory testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent, at scale. The availability of testing reagents, laboratory equipment and qualified staff are important bottlenecks to achieving this. Elsewhere, pooled testing (i.e. combining multiple samples in the same reaction) has been suggested to increase testing capacities in the pandemic period. Methods. We discuss our experience with SARS-CoV-2 pooled testing using real-time reverse transcription polymerase chain reaction (RT-PCR) on the Kenyan Coast. Results. In mid-May, 2020, our RT-PCR testing capacity for SARS-CoV-2 was improved by ~100% as a result of adoption of a six-sample pooled testing strategy. This was accompanied with a concomitant saving of ~50% of SARS-CoV-2 laboratory test kits at both the RNA extraction and RT-PCR stages. However, pooled testing came with a slight decline of test sensitivity. The RT-PCR cycle threshold value (ΔCt) was ~1.59 higher for samples tested in pools compared to samples tested singly. Conclusions. Pooled testing is a useful strategy to increase SARS-CoV-2 laboratory testing capacity especially in low-income settings.

An optimization of four SARS-CoV-2 qRT-PCR assays in a Kenyan laboratory to support the national COVID-19 rapid response teams.

Background: The global COVID-19 outbreak relies on a quantitative real-time polymerase chain reaction (qRT-PCR) for the detection of severe acute respiratory syndrome coronavirus (SARS-CoV-2), to facilitate the roll-out of patient care and infection control measures. There are several qRT-PCR assays with little evidence on their comparability. We report alterations to the developers' recommendations to sustain the testing capability in our setting, where the supply of testing reagents is limited. Methods: Standards generated from a serially-diluted positive control and previously identified positive/negative samples were used to determine the optimal volumes of the qRT-PCR reagents and to evaluate the validity and performance of four assays: Charité Berlin and European Virus Archive - GLOBAL (EVAg) primer-probe sets, and DAAN and Beijing Genomics Institute (BGI) premixed commercial kits. A multiplex and singleplex RT-PCR kit was used with the two primer-probe sets and the recommended assay volumes of the two premixed kits were altered. Results: In comparison to the multiplex RT-PCR kit, the singleplex RT-PCR kit combined with the primer-probe sets yielded consistent cycle threshold (Ct) values across the different titrations tested. The DAAN premixed kit produced comparable Ct values across the titrations, while the BGI kit showed incomparable Ct values and inconsistent results between batches using the manufacturer's recommended volumes. Conclusion: We achieved a 2.5-fold and 4-fold increase in the number of tests/kit for the premixed kits and the primer-probe sets, respectively. The primer-probe set assays were reliable and consistent, and we preferred a combination of an EVAg and a Berlin target. Any inconclusive result was repeated by different individuals following the same protocol. DAAN was a consistent and reliable assay even at lower concentrations from the stated recommendations. BGI in contrast, required dilution to improve its performance and was hence an assay that was used in combination with EVAg or Berlin targets.

Population-Based Estimate of Melioidosis, Kenya.

Melioidosis is thought to be endemic, although underdiagnosed, in Africa. We identified 5 autochthonous cases of Burkholderia pseudomallei infection in a case series in Kenya. Incidence of B. pseudomallei bacteremia in Kenya's Kilifi County is low, at 1.5 cases per million person-years, but this result might be an underestimate.

Carriage and Acquisition of Extended-spectrum ß-Lactamase-producing Enterobacterales Among Neonates Admitted to Hospital in Kilifi, Kenya.

BACKGROUND: Infections caused by extended-spectrum ß-lactamase-producing Enterobacterales (ESBL-E) among hospitalized neonates in sub-Saharan Africa pose significant clinical challenges. Data on prevalence and acquisition of ESBL-E carriage among hospitalized neonates in the region are few, and risk factors for transmission are not clearly defined. METHODS: In a cohort study of consecutive neonatal admissions to Kilifi County Hospital from July 2013 through August 2014, we estimated ESBL-E carriage prevalence on admission using rectal swab cultures and identified risk factors using logistic regression. Using twice-weekly follow-up swabs, we estimated the incidence and identified risk factors for ESBL-E acquisition in hospital using Poisson regression. RESULTS: The prevalence of ESBL-E carriage at admission was 10% (59/569). Cesarean delivery, older neonatal age, and smaller household size were significant risk factors. Of the 510 infants admitted without ESBL-E carriage, 238 (55%) acquired carriage during their hospital stay. The incidence of acquisition was 21.4% (95% confidence interval, 19.0%-24.0%) per day. The rate was positively associated with the number of known neonatal ESBL-E carriers and with the total number of neonates on the same ward. CONCLUSIONS: Carriage of ESBL-E was common among neonates on admission, and in-hospital acquisition was rapid. The dissemination and selection of ESBL-E appears to be driven by hospital exposures, operative delivery, and neonatal ward patient density. Further attention to infection control, patient crowding, and carriage surveillance is warranted.

Invasive Cronobacter species infection in infants and children admitted to a rural Kenyan hospital with a high prevalence of malnutrition.

For children with acute malnutrition, ready-to-use therapeutic foods (RUTF) are lifesaving treatments. In 2012, detailed testing detected Enterobacteriaceae including Cronobacter species at low levels in RUTF from all UNICEF-approved producers. Cronobacter in milk feeds has previously been associated with severe neonatal infections. Thus, given the susceptibility of severely malnourished children to invasive bacterial infections, concerns arose about the potential for Cronobacter infections from RUTF. This led to widespread production and supply problems in emergency feeding programmes. The KEMRI/Wellcome Trust Research Programme has conducted systematic surveillance for invasive bacterial infections among children admitted to Kilifi County Hospital, Kenya since 1998. 65,426 paediatric blood and cerebrospinal fluid cultures from 52,733 admissions resulted in 3953 with growth of a pathogenic organism. From the 60 Enterobacter and Cronobacter isolates, possible Cronobacter species were initially selected from their original API-20E biochemical profile, which was repeated and then confirmed using ID-32E. Only two isolates were consistent with Cronobacter species, neither case had received RUTF. Serious infection due to Cronobacter species does not have a significant burden in this population. This has important implications for the continued supply, manufacture and monitoring of emergency feeds for malnourished children.

Risk of nontyphoidal Salmonella bacteraemia in African children is modified by STAT4.

Nontyphoidal Salmonella (NTS) is a major cause of bacteraemia in Africa. The disease typically affects HIV-infected individuals and young children, causing substantial morbidity and mortality. Here we present a genome-wide association study (180 cases, 2677 controls) and replication analysis of NTS bacteraemia in Kenyan and Malawian children. We identify a locus in STAT4, rs13390936, associated with NTS bacteraemia. rs13390936 is a context-specific expression quantitative trait locus for STAT4 RNA expression, and individuals carrying the NTS-risk genotype demonstrate decreased interferon-γ (IFNγ) production in stimulated natural killer cells, and decreased circulating IFNγ concentrations during acute NTS bacteraemia. The NTS-risk allele at rs13390936 is associated with protection against a range of autoimmune diseases. These data implicate interleukin-12-dependent IFNγ-mediated immunity as a determinant of invasive NTS disease in African children, and highlight the shared genetic architecture of infectious and autoimmune disease.

Molecular epidemiology of Klebsiella pneumoniae invasive infections over a decade at Kilifi County Hospital in Kenya.

Multidrug resistant (MDR) Klebsiella pneumoniae is a common cause of nosocomial infections worldwide. Recent years have seen an explosion of resistance to extended-spectrum ß-lactamases (ESBLs) and emergence of carbapenem resistance. Here, we examine 198 invasive K. pneumoniae isolates collected from over a decade in Kilifi County Hospital (KCH) in Kenya. We observe a significant increase in MDR K. pneumoniae isolates, particularly to third generation cephalosporins conferred by ESBLs. Using whole-genome sequences, we describe the population structure and the distribution of antimicrobial resistance genes within it. More than half of the isolates examined in this study were ESBL-positive, encoding CTX-M-15, SHV-2, SHV-12 and SHV-27, and 79% were MDR conferring resistance to at least three antimicrobial classes. Although no isolates in our dataset were found to be resistant to carbapenems we did find a plasmid with the genetic architecture of a known New Delhi metallo-ß-lactamase-1 (NDM)-carrying plasmid in 25 isolates. In the absence of carbapenem use in KCH and because of the instability of the NDM-1 gene in the plasmid, the NDM-1 gene has been lost in these isolates. Our data suggests that isolates that encode NDM-1 could be present in the population; should carbapenems be introduced as treatment in public hospitals in Kenya, resistance is likely to ensue rapidly.

Standardization of Laboratory Methods for the PERCH Study.

The Pneumonia Etiology Research for Child Health study was conducted across 7 diverse research sites and relied on standardized clinical and laboratory methods for the accurate and meaningful interpretation of pneumonia etiology data. Blood, respiratory specimens, and urine were collected from children aged 1-59 months hospitalized with severe or very severe pneumonia and community controls of the same age without severe pneumonia and were tested with an extensive array of laboratory diagnostic tests. A standardized testing algorithm and standard operating procedures were applied across all study sites. Site laboratories received uniform training, equipment, and reagents for core testing methods. Standardization was further assured by routine teleconferences, in-person meetings, site monitoring visits, and internal and external quality assurance testing. Targeted confirmatory testing and testing by specialized assays were done at a central reference laboratory.

The Diagnostic Utility of Induced Sputum Microscopy and Culture in Childhood Pneumonia.

BACKGROUND.: Sputum microscopy and culture are commonly used for diagnosing the cause of pneumonia in adults but are rarely performed in children due to difficulties in obtaining specimens. Induced sputum is occasionally used to investigate lower respiratory infections in children but has not been widely used in pneumonia etiology studies. METHODS.: We evaluated the diagnostic utility of induced sputum microscopy and culture in patients enrolled in the Pneumonia Etiology Research for Child Health (PERCH) study, a large study of community-acquired pneumonia in children aged 1-59 months. Comparisons were made between induced sputum samples from hospitalized children with radiographically confirmed pneumonia and children categorized as nonpneumonia (due to the absence of prespecified clinical and laboratory signs and absence of infiltrate on chest radiograph). RESULTS.: One induced sputum sample was available for analysis from 3772 (89.1%) of 4232 suspected pneumonia cases enrolled in PERCH. Of these, sputum from 2608 (69.1%) met the quality criterion of <10 squamous epithelial cells per low-power field, and 1162 (44.6%) had radiographic pneumonia. Induced sputum microscopy and culture results were not associated with radiographic pneumonia, regardless of prior antibiotic use, stratification by specific bacteria, or interpretative criteria used. CONCLUSIONS.: The findings of this study do not support the culture of induced sputum specimens as a diagnostic tool for pneumonia in young children as part of routine clinical practice.

Human genetic and metabolite variation reveals that methylthioadenosine is a prognostic biomarker and an inflammatory regulator in sepsis.

Sepsis is a deleterious inflammatory response to infection with high mortality. Reliable sepsis biomarkers could improve diagnosis, prognosis, and treatment. Integration of human genetics, patient metabolite and cytokine measurements, and testing in a mouse model demonstrate that the methionine salvage pathway is a regulator of sepsis that can accurately predict prognosis in patients. Pathway-based genome-wide association analysis of nontyphoidal Salmonella bacteremia showed a strong enrichment for single-nucleotide polymorphisms near the components of the methionine salvage pathway. Measurement of the pathway's substrate, methylthioadenosine (MTA), in two cohorts of sepsis patients demonstrated increased plasma MTA in nonsurvivors. Plasma MTA was correlated with levels of inflammatory cytokines, indicating that elevated MTA marks a subset of patients with excessive inflammation. A machine-learning model combining MTA and other variables yielded approximately 80% accuracy (area under the curve) in predicting death. Furthermore, mice infected with Salmonella had prolonged survival when MTA was administered before infection, suggesting that manipulating MTA levels could regulate the severity of the inflammatory response. Our results demonstrate how combining genetic data, biomolecule measurements, and animal models can shape our understanding of disease and lead to new biomarkers for patient stratification and potential therapeutic targeting.