Eric R. Houpt

Introduction: Globally, enteric pathogens have been associated with poor child growth and development. Our aim was to assess for predictors, consequences and potential mechanisms of enteric pathogen burden morbidity among children in rural Tanzania. Methods: We performed a secondary data analysis of 1140 children from the Early-Life Interventions for Childhood Growth and Development in Tanzania (ELICIT) study, assessing stool samples from ages 6, 12 and 18 months using TaqMan Array Cards for enteric pathogens. We examined risk factors for infections and associations of pathogen burden with serum biomarkers of nutrition and inflammation and anthropometry and cognitive development outcomes (using the Malawi Developmental Assessment Tool, MDAT). Results: Over the three time points, children had median (IQR) pathogen counts of 2 (0–7) at 6 months, 3 (0–8) at 12 months and 2 (0–8) at 18 months—for an overall mean of 7.79 total pathogen detections (SD 2.64, range 1–18). Assessed as an outcome, pathogen burden was associated with lower socioeconomic status, higher household occupancy and housing chickens. Assessed as a predictor (and adjusted for sex, socioeconomic status, birth month and age), each additional pathogen was associated with 0.03 lower length-for-age z-scores (p=0.008) and 0.04 lower weight-for-age z-scores (WAZ) (pShigella infections individually. Conclusions: Despite the ubiquity of pathogen carriage, we noted important predictors and outcomes of greater pathogen burden—underscoring ongoing importance of identifying interventions to reduce pathogen exposure among children in developing areas. Trail registration number: NCT03268902 .

Background: Understanding diarrhea etiology is critical for understanding vaccine impact, but causal attribution is difficult. We evaluated the sensitivity of an existing attributable incidence estimator to unadjusted confounding and study design. Methods: We used MAL-ED data to estimate attributable incidence by regressing diarrhea incidence on pathogen quantity and calculating attributable fractions. The partially adjusted model used mixed-effects logistic regression adjusted for co-infection, sex, test batch, and age. We evaluated the magnitude of confounding by prior immunity, antibiotic use, socioeconomic status, and breastfeeding. To understand the impact of post-diarrheal shedding, we compared estimates including and excluding stools collected ±7, 14, or 28 days from an episode. We conducted matched nested case-control and case-crossover analyses and used Poisson regression to calculate risk-based estimates of incidence. Results: 40,406 stools samples from 1715 children were included (6625 diarrheal, 33,781 control). Antibiotic use had the greatest confounding impact, and directionality of bias was dependent on pathogen type. Shedding following diarrheal episodes did not substantially or consistently bias estimates. Nested case-control and case-crossover study designs produced similar attributable incidence estimates, and risk-based estimates were lower than odds-based estimates, ranging from 33% (Shigella) to 49% (tEPEC) episodes per 100 child-years fewer than the partially adjusted model.

Background: The leading causes of fever requiring hospital admission in rural sub-Saharan Africa remain understudied, particularly using modern molecular diagnostics. Methods: We enrolled 258 patients with acute fever (cases) and 48 controls admitted to 1 hospital in Tanzania for 1 year starting in February 2023. Blood was tested by blood culture, polymerase chain reaction (PCR) for 35 pathogens, and serology for 6 pathogens. Results: The leading pathogen detected in cases was measles, with 70 of 258 cases detected by PCR, 74 of 238 by immunoglobulin M serology, and 91 allowing either method. Other pathogens were detected in <5% of cases including, in descending order, Plasmodium, Schistosoma, and Bartonella, among others. Measles was the only pathogen clearly associated with cases versus controls. Of 91 measles cases, only 58 (64%) were clinically suspected and only 56 (62%) met the World Health Organization definition of a clinical measles case, largely because 27 (30%) measles cases did not have recorded rash. Outcomes were poorer for measles without rash versus with rash, with a longer hospital stay (mean, 5.5 vs 3.3 days; P = .010), less full recovery (48% vs 75%; P = .016), and high mortality (7% vs 2%; P = .20). Increased laboratory testing would have detected measles 2 months prior to the first clinical suspicion. Conclusions: Measles was found to be the leading cause of fever during 1 year of acute febrile illness surveillance in rural Tanzania. Measles can present without notable rash in these settings and has poor outcomes. Increasing laboratory test availability and clinician suspicion for measles is a priority.

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of childhood and travelers’ diarrhea. The vaccine candidate ETVAX encompasses several ETEC colonization factors (CFs) with a hybrid LT (heat-labile toxin)/cholera toxin B subunit adjuvanted with a double-mutant LT. Stool samples from a Phase 2b ETVAX trial were tested by a PCR-based customized TaqMan Array Card (TAC), including three ETEC toxin genes (LT and heat-stable toxins, STh and STp) and 18 ETEC CFs. Stool samples were also tested with the molecular platform Amplidiag and culture, followed by GM1-enzyme-linked immunosorbent assay (ELISA) and inhibition GM1-ELISA for LT and ST and dot blot for CFs of ETECs identified among six culture isolates (maximum). Compared with Amplidiag, TAC yielded 89.4% sensitivity (320/358) and 96.4% specificity (405/420) for ETEC detection. The two methods demonstrated a good quantitative correlation (quantification cycle R ² = 0.827, P < 0.05). Compared with culture, TAC and Amplidiag each exhibited 96.8% (184/190) sensitivity and identified an additional of 151 and 174 PCR positives in 588 culture-negative stools, respectively. The concordance of stool TAC versus ELISA of ETEC colonies for LT and STh/STp was 85.5% (165/193). TAC demonstrated 98% sensitivity and 92% specificity versus the dot blot results of 793 colonies for the ETVAX CFs CFA/I, CS3, CS5, and CS6. Overall ETEC was detected by TAC in 335 (43.1%) and by Amplidiag in 358 (46.0%) of specimens compared to 190 (24.4%) by culture. We conclude that molecular diagnostic approaches of TAC or Amplidiag increase the detection of ETEC compared with culture, and TAC can also provide vaccine-subtype ETEC data. CLINICAL TRIALS: This study was registered with ClinicalTrials.gov as NCT03729219 . IMPORTANCE: Enterotoxigenic Escherichia coli (ETEC) is an important cause of childhood and travelers’ diarrhea. Vaccines in development utilize specific toxins and colonization factors (CFs) as antigens. Therefore, clinical microbiologic diagnostic methods are needed to discriminate specific toxins and CFs, both for vaccine trials and to guide epidemiology. In this work, we assessed the diagnostic performance of several methods for ETEC: a PCR-based customized TaqMan Array Card (TAC) and the molecular platform Amplidiag on stool and E. coli culture, followed by GM1-enzyme-linked immunosorbent assay for toxins and dot blot for CFs. Stool samples from a Phase 2b ETEC vaccine trial were used. Overall, ETEC was detected by TAC in 335 (43.1%) and by Amplidiag in 358 samples (46.0%) compared to 190 (24.4%) by culture. TAC additionally provided CF data with 98% sensitivity and 92% specificity. We conclude that the molecular diagnostic approaches of TAC or Amplidiag increase the detection of ETEC compared with culture.

Introduction: Biannual (ie, every 6 months) mass drug administration of azithromycin has reduced childhood mortality in Niger, but its effects on specific respiratory pathogens are not fully elucidated. Methods: Across 2 years of the Macrolides Oraux pour Réduire les Décès avec un Oeil sur la Résistance (MORDOR) study in Niger, we evaluated 1468 nasopharyngeal swabs from children who presented for care with respiratory symptoms. Swabs were tested by quantitative PCR using a customised TaqMan Array Card that included assays for 19 respiratory pathogens. Results: Nasopharyngeal detection of Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae was common in both azithromycin and placebo communities. The prevalence was reduced in children from azithromycin communities for just two pathogens: Bordetella pertussis and H. influenzae type b (Hib). These children had a 49% and 65% reduction in the prevalence of B. pertussis and Hib in nasopharyngeal swabs, respectively, compared with children from the control communities (prevalence ratios 0.51, 95% CI 0.35, 0.75; and 0.35, 95% CI 0.17, 0.71). Conclusions: Biannual administration of azithromycin to communities in Niger was associated with lower prevalence of B. pertussis and Hib compared with placebo. These reductions may explain some of the childhood mortality benefit of azithromycin. Trial registration number: NCT02048007.

Oropouche virus is an arbovirus endemic to the Americas. Periodic outbreaks have occurred since its description in 1955. In late 2023, an outbreak occurred in Peru, centered in and around Iquitos in the Eastern Peruvian Amazon. An existing acute febrile illness (AFI) surveillance program was able to document its emergence and characterize arthralgia and dysuria and the absence of diarrhea as distinctive clinical features of Oropouche virus-associated febrile illness relative to other causes of AFI. Sequencing of isolates from the outbreak demonstrated that strains from this region were distinct from those causing disease in Brazil, despite the large-scale movement of people along the Amazon corridor, but highly similar to strains from Colombia and Ecuador. Our findings suggest that the current outbreak in South America is fundamentally multifocal in origin and not the result of geographic spread from Brazil, which experienced an outbreak between 2022 and 2024.

Background: Small vulnerable newborns (SVNs), including those born preterm, small for gestational age, or with low birth weight, are at higher risk of neonatal mortality and long-term health complications. Early exposure to maternal vaginal microbiota and breastfeeding plays a critical role in the development of the neonatal microbiota and immune system, especially in low-resource settings like Burkina Faso, where neonatal mortality rates remain high. Objectives: The DenBalo study aims to investigate the role of maternal and neonatal factors, such as vaginal and gut microbiota, immune development, and early nutrition, in shaping health outcomes in SVNs and healthy infants. Methods: This prospective cohort observational study will recruit 141 mother-infant pairs (70 SVNs and 71 healthy controls) from four health centers in Bobo-Dioulasso, Burkina Faso. The mother-infant pairs will be followed for six months with anthropometric measurements and biospecimen collections, including blood, breast milk, saliva, stool, vaginal swabs, and placental biopsies. Multi-omics approaches, encompassing metagenomics, metabolomics, proteomics, and immune profiling, will be used to assess vaginal and gut microbiota composition and functionality, immune cell maturation, and cytokine levels at critical developmental stages. Conclusions: This study will generate comprehensive data on how microbiota, metabolomic, and proteomic profiles, along with immune system development, differ between SVNs and healthy infants. These findings will guide targeted interventions to improve neonatal health outcomes and reduce mortality, particularly in vulnerable populations.

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. 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. 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.

Background: Early childhood enteric infection with Shigella/EIEC, enteroaggregative E. coli (EAEC), Campylobacter, and Giardia has been associated with reduced child growth, yet a recent randomized trial of antimicrobial therapy to reduce these infections did not improve growth outcomes. To interrogate this discrepancy, we measured the enteric infections from this study. Methods: We leveraged the Early Life Interventions for Childhood Growth and Development in Tanzania (ELICIT) trial, a randomized double-blind placebo-controlled trial of antimicrobial therapy with azithromycin and nitazoxanide provided quarterly to infants from 6 to 15 months of age. We tested 5,479 stool samples at time points across the study for 34 enteropathogens using quantitative PCR. Results: There was substantial carriage of enteropathogens in stool. Azithromycin administration led to reductions in Campylobacter jejuni/coli, enteroaggregative E. coli, and Shigella/EIEC (absolute risk difference ranged from -0.06 to 0.24) 2 weeks after treatment however there was no effect after 3 months. There was no difference in Giardia after nitazoxanide administration (ARR 0.03 at the 12 month administration). When examining the effect of azithromycin versus placebo on the subset of children infected with specific pathogens at the time of treatment, a small increase in weight-for-age Z score was seen only in those infected with Campylobacter jejuni/coli (0.10 Z score, 95% CI -0.01–0.20; length-for-age Z score 0.07, 95% CI -0.06–0.20). Conclusion: The antimicrobial intervention of quarterly azithromycin plus or minus nitazoxanide led to only transient decreases in enteric infections with Shigella/EIEC, enteroaggregative E. coli (EAEC), Campylobacter, and Giardia. There was a trend towards improved growth in children infected with Campylobacter that received quarterly azithromycin.