Diagnosing diarrheal disease is difficult in part due to challenges in obtaining and transporting a bulk stool specimen, particularly in resource-limited settings. We compared the performance of flocked rectal swabs to that of traditional bulk stool samples for enteric pathogen detection using the BioFire™ FilmArray Gastrointestinal panel in children admitted to 4 hospitals in Botswana with community onset severe gastroenteritis. Of the 117-matched flocked rectal swab/stool pairs, we found no significant difference in pathogen detection rates between the flocked rectal swab samples and traditional bulk stool sampling methods for any bacterial (168 versus 167, respectively), viral (94 versus 92, respectively), or protozoan (18 versus 18, respectively) targets. The combination of flocked rectal swab samples with FilmArray testing allows for the rapid diagnosis of infectious gastroenteritis, facilitating a test and treat approach for infections that are life-threatening in many resource-limited settings. Culture recovery rates for bacterial pathogens utilizing this approach need to be assessed.

<p><strong>INTRODUCTION: </strong>The study aim was to determine if rapid enteric diagnostics followed by the provision of targeted antibiotic therapy ('test-and-treat') and/or&nbsp;<em>Lactobacillus reuteri</em> DSM 17938 would improve outcomes in children hospitalised in Botswana with acute gastroenteritis.</p>

<p><strong>METHODS: </strong>This was a multicentre, randomised, factorial, controlled, trial. Children aged 2-60 months admitted for acute non-bloody diarrhoea to four hospitals in southern Botswana were eligible. Participants were assigned to treatment groups by web-based block randomisation. Test-and-treat results were not blinded, but participants and research staff were blinded to <em>L. reuteri</em>/placebo assignment; this was dosed as 1×10⁸ cfu/mL by mouth daily and continued for 60 days. The primary outcome was 60-day age-standardised height (HAZ) adjusted for baseline HAZ. All analyses were by intention to treat. The trial was registered at Clinicaltrials.gov.</p>

<p><strong>RESULTS: </strong>Recruitment began on 12 June 2016 and continued until 24 October 2018. There were 66 participants randomised to the test-and-treat plus <em>L. reuteri&nbsp;</em>group, 68 randomised to the test-and-treat plus placebo group, 69 to the standard care plus <em>L. reuteri&nbsp;</em>group and 69 to the standard care plus placebo group. There was no demonstrable impact of the test-and-treat intervention (mean increase of 0.01 SD, 95% CI -0.14 to 0.16 SD) or the&nbsp;<em>L. reuteri</em> intervention (mean decrease of 0.07 SD, 95% CI -0.22 to 0.08 SD) on adjusted HAZ at 60 days.</p>

<p><strong>CONCLUSIONS: </strong>In children hospitalised for acute gastroenteritis in Botswana, neither a test-and-treat algorithm targeting enteropathogens, nor a 60-day course of <em>L. reuteri&nbsp;</em>DSM 17938, were found to markedly impact linear growth or other important outcomes. We cannot exclude the possibility that test-and-treat will improve the care of children with significant enteropathogens (such as <em>Shigella</em>) in their stool.</p>

<p><strong>TRIAL REGISTRATION NUMBER: </strong>NCT02803827.</p>

<p><strong>INTRODUCTION: </strong>Infections due to extended spectrum beta-lactamase producing organisms (ESBL) have emerged as the leading cause of sepsis among hospitalized neonates in Botswana and much of sub-Saharan Africa and south Asia. Yet, ESBL reservoirs and transmission dynamics within the neonatal intensive care unit (NICU) environment are not well-understood. This study aimed to assess the efficiency and feasibility of a chromogenic-culture-media-based environmental sampling approach to characterize the ESBL bioburden within a NICU.</p>

<p><strong>METHODS: </strong>A series of four point-prevalence surveys were conducted at a 36-bed NICU at a public tertiary referral hospital in Botswana from January-June 2021. Samples were collected on 4 occasions under semi-sterile technique using 1) flocked swabs &amp; templates (flat surfaces); 2) sterile syringe &amp; tubing (water aspiration); and 3) structured swabbing techniques (hands &amp; equipment). Swabs were transported in physiological saline-containing tubes, vortexed, and 10 µL was inoculated onto chromogenic-agar that was selective and differential for ESBL (CHROMagar™ ESBL, Paris, France), and streaking plates to isolate individual colonies. Bacterial colonies were quantified and phenotypically characterized using biochemical identification tests.</p>

<p><strong>RESULTS: </strong>In total, 567 samples were collected, 248 (44%) of which grew ESBL. Dense and consistent ESBL contamination was detected in and around sinks and certain high-touch surfaces, while transient contamination was demonstrated on medical equipment, caregivers/healthcare worker hands, insects, and feeding stations (including formula powder). Results were available within 24-72&nbsp;h of collection. To collect, plate, and analyse 50 samples, we estimated a total expenditure of $269.40 USD for materials and 13.5 cumulative work hours among all personnel.</p>

<p><strong>CONCLUSIONS: </strong>Using basic environmental sampling and laboratory techniques aided by chromogenic culture media, we identified ESBL reservoirs (sinks) and plausible transmission vehicles (medical equipment, infant formula, hands of caregivers/healthcare workers, &amp; insects) in this NICU environment. This strategy was a simple and cost-efficient method to assess ESBL bioburden and may be feasible for use in other settings to support ongoing infection control assessments and outbreak investigations.</p>

<p><strong>BACKGROUND: </strong>There are scant data on the prevalence and clinical course of pertussis disease among infants with pneumonia in low- and middle-income countries. While pertussis vaccination coverage is high (≥90%) among infants in Botswana, human immunodeficiency virus (HIV) infection affects nearly one-third of pregnancies. We aimed to evaluate the prevalence and clinical course of pertussis disease in a cohort of HIV-unexposed uninfected (HUU), HIV-exposed uninfected (HEU), and HIV-infected infants with pneumonia in Botswana.</p>

<p><strong>METHODS: </strong>We recruited children 1-23 months of age with clinical pneumonia at a tertiary care hospital in Gaborone, Botswana between April 2012 and June 2016. We obtained nasopharyngeal swab specimens at enrollment and tested these samples using a previously validated in-house real-time PCR assay that detects a unique sequence of the porin gene of Bordetella pertussis.</p>

<p><strong>RESULTS: </strong>B. pertussis was identified in 1/248 (0.4%) HUU, 3/110 (2.7%) HEU, and 0/33 (0.0%) HIV-infected children. All pertussis-associated pneumonia cases occurred in infants 1-5 months of age (prevalence, 1.0% [1/103] in HUU and 4.8% [3/62] in HEU infants). No HEU infants with pertussis-associated pneumonia were taking cotrimoxazole prophylaxis at the time of hospital presentation. One HUU infant with pertussis-associated pneumonia required intensive care unit admission for mechanical ventilation, but there were no deaths.</p>

<p><strong>CONCLUSIONS: </strong>The prevalence of pertussis was low among infants and young children with pneumonia in Botswana. Although vaccination against pertussis in pregnancy is designed to prevent classical pertussis disease, reduction of pertussis-associated pneumonia might be an important additional benefit.</p>

<p><strong>BACKGROUND: </strong>Nasopharyngeal colonization precedes infections caused by Streptococcus pneumoniae. A more detailed understanding of interactions between S. pneumoniae and the nasopharyngeal microbiota of children could inform strategies to prevent pneumococcal infections.</p>

<p><strong>METHODS: </strong>We collected nasopharyngeal swabs from children 1 to 23 months of age in Botswana between August 2012 and June 2016. We tested samples for S. pneumoniae and common respiratory viruses using PCR. We sequenced the V3 region of the bacterial 16S ribosomal RNA gene and used random forest models to identify clinical variables and bacterial genera that were associated with pneumococcal colonization.</p>

<p><strong>RESULTS: </strong>Mean age of the 170 children included in this study was 8.3 months. Ninety-six (56%) children were colonized with S. pneumoniae. Pneumococcal colonization was associated with older age (P=0.0001), a lack of electricity in the home (P=0.02), and household use of wood as a cooking fuel (P=0.002). Upper respiratory symptoms were more frequent in children with S. pneumoniae colonization (60% vs. 32%; P=0.001). Adjusting for age, nasopharyngeal microbiota composition differed in colonized and noncolonized children (P=0.001). S. pneumoniae colonization was associated with a higher relative abundance of Moraxella (P=0.001) and lower relative abundances of Corynebacterium (P=0.001) and Staphylococcus (P=0.03). A decision tree model containing the relative abundances of bacterial genera had 81% sensitivity and 85% specificity for the determination of S. pneumoniae colonization status.</p>

<p><strong>CONCLUSIONS: </strong>S. pneumoniae colonization is associated with characteristic alterations of the nasopharyngeal microbiota of children. Prospective studies should determine if nasopharyngeal microbial composition alters the risk of pneumococcal colonization and thus could be modified as a novel pneumonia prevention strategy.</p>

<p><strong>BACKGROUND: </strong>Diarrheal disease is a leading cause of death for young children. Most pediatric gastroenteritis is caused by viral pathogens; consequently, current recommendations advocate against routine antibacterial therapy if children present without bloody stools.</p>

<p><strong>METHODS: </strong>In this prospective cohort study, we enrolled children with severe acute gastroenteritis admitted to hospital in Botswana. Details of presenting history, physical examination, and course in the hospital were recorded. Stool samples were characterized using a 15 pathogen polymerase chain reaction assay.</p>

<p><strong>RESULTS: </strong>There were 671 participants with a median age of 8.3 months; 77 (11%) had severe acute malnutrition. Only 74 children had bloody stools, of whom 48 (65%) had a detectable bacterial pathogen, compared to 195 of 592 (33%) of those without. There were 26 deaths (3.9%). Covariates associated with death in multivariable logistic regression were the detection of any of Campylobacter/Shigella/enterotoxigenic Escherichia coli (odds ratio [OR] 2.57, 95% confidence interval [CI] 1.07-6.17), severe acute malnutrition (OR 4.34, 95% CI 1.79-10.5), and antibiotic therapy (OR 8.82, 95% CI 2.03-38.2). There was no significant association between bloody stools and death, and the effect of Campylobacter/Shigella/enterotoxigenic E. coli infection on death was not modified by the presence of bloody stools.</p>

<p><strong>CONCLUSIONS: </strong>Detection of bacterial enteropathogens is associated with increased mortality in children in sub-Saharan Africa. Unfortunately, most children with these infections do not have bloody stools, and bloody dysentery was not found to be associated with worse outcomes. Clinical trials evaluating outcomes associated with more aggressive diagnostic strategies in children presenting with severe acute gastroenteritis in sub-Saharan Africa should be undertaken.</p>

<p><strong>BACKGROUND: </strong>Studies have demonstrated reduced rotavirus vaccine effectiveness (VE) in resource-limited settings. Enteropathogen co-infections in rotavirus cases have been hypothesized to contribute to the lower vaccine effectiveness in such settings. We sought to determine if co-infections affect rotavirus VE in Botswana.</p>

<p><strong>METHODS: </strong>Between June 2013 and April 2015, children &lt;60 months old, presenting with severe gastroenteritis at four hospitals as part of a national rotavirus surveillance were enrolled. Rotavirus EIA positive samples were tested with an in-house real-time polymerase chain reaction (PCR) panel that detected nine pathogens and a commercial 15 multiplex PCR gastrointestinal pathogen panel (GPP). Co-infection was defined as detection of rotavirus plus one of the five pathogens with the highest attributable fractions for diarrhea. Vaccine status was compared between rotavirus case patients and non-rotavirus "test-negative" controls. Vaccine effectiveness was also calculated restricting cases to those with rotavirus as the only pathogen detected.</p>

<p><strong>RESULTS: </strong>242 children tested rotavirus EIA positive and 368 children were negative. Of the 182 rotavirus EIA-positive samples tested with the GPP assay, co-infections were detected in60 (33%). The overall adjusted 2-dose VE was 59% (95% CI 27-77) in the rotavirus co-infection group and 51% (95% CI -14-79) in the rotavirus mono-infection subgroup. Using in-house multiplex PCR panel, out of 213 rotavirus EIA positive subjects, co-infections were detected in 98 samples (46%). The overall adjusted VE for two doses was 48% (95% CI -2-74) and 62% (95% CI 25-80) in rotavirus mono-infection subgroup.</p>

<p><strong>CONCLUSIONS: </strong>We could not find evidence of an effect of enteric co-infections on the effectiveness of rotavirus vaccine.</p>

<p><strong>INTRODUCTION: </strong>Diarrhoeal disease is the second-leading cause of death in young children. Current guidelines recommend treating children with acute non-bloody diarrhea with oral rehydration solutions and zinc, but not antimicrobials. However, in many resource-limited settings, infections with treatable enteric bacterial and protozoan pathogens are common. Probiotics have shown promise as an adjunct treatment for diarrhoea but have not been studied in sub-Saharan Africa.</p>

<p><strong>METHODS: </strong>We conducted a pilot, factorial, randomized, placebo-controlled trial of children aged 2-60 months hospitalized in Botswana for acute non-bloody diarrhoea. A rapid test-and-treat intervention, consisting of multiplex PCR testing of rectal swabs taken at enrolment, accompanied by targeted antimicrobial therapy if treatable pathogens were detected, was compared to the reference standard of no stool testing. Additionally, Lactobacillus reuteri DSM 17938 x 60 days was compared to placebo treatment. The main objective of this pilot study was to assess feasibility. The primary clinical outcome was the increase in age-standardized height (HAZ) at 60 days adjusted for baseline HAZ.</p>

<p><strong>RESULTS: </strong>Seventy-six patients were enrolled over a seven-month study period. We judged that the recruitment rate, lab processing times, communication protocols, provision of specific antimicrobials, and follow-up rates were acceptable. Compared to the reference arm (no stool testing and placebo treatment), the combination of the rapid test-and-treat strategy plus L. reuteri DSM 17938 was associated with an increase of 0.61 HAZ (95% CI 0.09-1.13) and 93% lower odds of recurrent diarrhoea (OR 0.07, 95%CI 0.01-0.61) at 60 days.</p>

<p><strong>DISCUSSION: </strong>We demonstrated that it was feasible to evaluate the study interventions in Botswana. Despite the small sample size, we observed a statistically significant increase in HAZ at 60 days and significantly lower odds of recurrent diarrhoea in children receiving both rapid test-and-treat and L. reuteri. There is sufficient evidence to warrant proceeding with a larger follow-up trial in a similar setting.</p>

<p><strong>BACKGROUND: </strong>Nearly half of child pneumonia deaths occur in sub-Saharan Africa. Microbial communities in the nasopharynx are a reservoir for pneumonia pathogens and remain poorly described in African children.</p>

<p><strong>METHODS: </strong>Nasopharyngeal swabs were collected from children with pneumonia (N=204), children with upper respiratory infection symptoms (N=55), and healthy children (N=60) in Botswana between April 2012 and April 2014. We sequenced the V3 region of the bacterial 16S ribosomal RNA gene and used partitioning around medoids to cluster samples into microbiota biotypes. We then used multivariable logistic regression to examine whether microbiota biotypes were associated with pneumonia and upper respiratory infection symptoms.</p>

<p><strong>RESULTS: </strong>Mean ages of children with pneumonia, children with upper respiratory infection symptoms, and healthy children were 8.2, 11.4, and 8.0 months, respectively. Clustering of nasopharyngeal microbiota identified five distinct biotypes: Corynebacterium/Dolosigranulum-dominant (23%), Haemophilus-dominant (11%), Moraxella-dominant (24%), Staphylococcus-dominant (13%), and Streptococcus-dominant (28%). The Haemophilus-dominant [odds ratio (OR): 13.55, 95% confidence interval (CI): 2.10-87.26], the Staphylococcus-dominant (OR: 8.27, 95% CI: 2.13-32.14), and the Streptococcus-dominant (OR: 39.97, 95% CI: 6.63-241.00) biotypes were associated with pneumonia. The Moraxella-dominant (OR: 3.71, 95% CI: 1.09-12.64) and Streptococcus-dominant (OR: 12.26, 95% CI: 1.81-83.06) biotypes were associated with upper respiratory infection symptoms. In children with pneumonia, HIV infection was associated with a lower relative abundance of Dolosigranulum (P=0.03).</p>

<p><strong>CONCLUSIONS: </strong>Pneumonia and upper respiratory infection symptoms are associated with distinct nasopharyngeal microbiota biotypes in African children. A lower abundance of the commensal genus Dolosigranulum may contribute to the higher pneumonia risk of HIV-infected children.</p>

<p><strong>BACKGROUND: </strong>The highest incidence of childhood acute lower respiratory tract infection (ALRI) is in low- and middle-income countries. Few studies examined whether detection of respiratory viruses predicts ALRI outcomes in these settings.</p>

<p><strong>METHODS: </strong>We conducted prospective cohort and case-control studies of children 1-23 months of age in Botswana. Cases met clinical criteria for pneumonia and were recruited within six hours of presentation to a referral hospital. Controls were children without pneumonia matched to cases by primary care clinic and date of enrollment. Nasopharyngeal specimens were tested for respiratory viruses using polymerase chain reaction. We compared detection rates of specific viruses in matched case-control pairs. We examined the effect of respiratory syncytial virus (RSV) and other respiratory viruses on pneumonia outcomes.</p>

<p><strong>RESULTS: </strong>Between April 2012 and August 2014, we enrolled 310 cases, of which 133 had matched controls. Median ages of cases and controls were 6.1 and 6.4 months, respectively. One or more viruses were detected from 75% of cases and 34% of controls. RSV and human metapneumovirus were more frequent among cases than controls, but only enterovirus/rhinovirus was detected from asymptomatic controls. Compared with non-RSV viruses, RSV was associated with an increased risk of treatment failure at 48 hours [risk ratio (RR): 1.85; 95% confidence interval (CI): 1.20, 2.84], more days of respiratory support [mean difference (MD): 1.26 days; 95% CI: 0.30, 2.22 days], and longer duration of hospitalization [MD: 1.35 days; 95% CI: 0.20, 2.50 days], but lower in-hospital mortality [RR: 0.09; 95% CI: 0.01, 0.80] in children with pneumonia.</p>

<p><strong>CONCLUSIONS: </strong>Respiratory viruses were detected from most children hospitalized with ALRI in Botswana, but only RSV and human metapneumovirus were more frequent than among children without ALRI. Detection of RSV from children with ALRI predicted a protracted illness course but lower mortality compared with non-RSV viruses.</p>