Prospective Cohort Study of Cryptosporidium Infection and Shedding in Infants and Their Households.

BACKGROUND: Cryptosporidium spp. are responsible for significant diarrheal morbidity and mortality in under-5 children. There is no vaccine; thus, a focus on prevention is paramount. Prior studies suggest that person-to-person spread may be an important pathway for transmission to young children. Here we describe a longitudinal cohort study of 100 families with infants to determine rates of cryptosporidiosis within households during the coronavirus disease 2019 (COVID-19) pandemic. METHODS: Families living in Mirpur, Bangladesh, with 1 infant aged 6-8 months were enrolled and followed with weekly illness survey and stool testing for Cryptosporidium for 8 months. RESULTS: From December 2020 to August 2021, 100 families were enrolled. Forty-four percent of index children and 35% of siblings had at least 1 Cryptosporidium infection. Shedding of Cryptosporidium occurred for a mean (standard deviation) of 19 (8.3) days in index infants, 16.1 (11.6) days in children 1-5 years, and 16.2 (12.8) days in adults. A longer duration of Cryptosporidium shedding was associated with growth faltering in infants. There was a spike in Cryptosporidium cases in May 2021, which coincided with a spike in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cases in the region. CONCLUSIONS: In this intensive, longitudinal study of Cryptosporidium infection in families we found high rates of cryptosporidiosis in infants and children, and prolonged parasite shedding, especially among malnourished children. These data support that transmission within the household is an important route of exposure for young infants and that treatment of nondiarrheal infection to interrupt person-to-person transmission within the home may be essential for preventing cryptosporidiosis in infants.

Prospective cohort study of Cryptosporidium infection and shedding in infants and their households.

Background: Cryptosporidium spp are responsible for significant diarrheal morbidity and mortality in under-five children. There is no vaccine, thus a focus on prevention is paramount. Prior studies suggest that person-to-person spread may be an important pathway for transmission to young children. Here we describe a longitudinal cohort study of 100 families with infants to determine rates of cryptosporidiosis within households during the COVID-19 pandemic. Methods: Families living in Mirpur, Bangladesh with one infant age 6-8 months were enrolled and followed with weekly illness survey and stool testing for Cryptosporidium for 8 months. Results: From December 2020 to August 2021, 100 families were enrolled. Forty-four percent of index children, and 35% of siblings had at least one Cryptosporidium infection. Shedding of Cryptosporidium occurred for a mean of 19 days (sd 8.3 days) in index infants, 16.1 days (sd 11.6) in children 1-5 years, and 16.2 days (sd 12.8) in adults. A longer duration of Cryptosporidium shedding was associated with growth faltering in infants. There was a spike in Cryptosporidium cases in May 2021, which coincided with a spike in SARS-CoV-2 cases in the region. Conclusion: In this intensive, longitudinal study of Cryptosporidium infection in families we found high rates of cryptosporidiosis in infants and children, and prolonged parasite shedding, especially among malnourished children. These data support that transmission within the household is an important route of exposure for young infants, and that treatment of non-diarrheal infection to interrupt person-to-person transmission within the home may be essential for preventing cryptosporidiosis in infants. summary: Cryptosporidiosis is a leading cause of morbidity and mortality among children. We followed 100 families with infants living in Bangladesh and studied the incidence of Cryptosporidium infection. We found prolonged Cryptosporidium shedding in stool was common among infants and adults.

Effects of routine early treatment with PCSK9 inhibitors in patients undergoing primary percutaneous coronary intervention for ST-segment elevation myocardial infarction: a randomised, double-blind, sham-controlled trial.

BACKGROUND: In patients with ST-segment elevation myocardial infarction (STEMI), early initiation of high-intensity statin therapy, regardless of low-density lipoprotein (LDL) cholesterol levels, is the standard of practice worldwide.  Aims: We sought to determine the effect of a similar early initiation strategy, using a proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor added to the high-intensity statin, on LDL cholesterol in acute STEMI. METHODS: In a randomised, double-blind trial we assigned 68 patients with STEMI undergoing primary percutaneous coronary intervention (PCI) to early treatment with alirocumab 150 mg subcutaneously or to a matching sham control. The first injection was given before primary PCI regardless of the baseline LDL level, then at 2 and 4 weeks. The primary outcome was the percent reduction in direct LDL cholesterol up to 6 weeks, analysed using a linear mixed model.   Results: High-intensity statin use was 97% and 100% in the alirocumab and sham-control groups, respectively. At a median of 45 days, the primary outcome of LDL cholesterol decreased by 72.9% with alirocumab (2.97 mmol/L to 0.75 mmol/L) versus 48.1% with the sham control (2.87 mmol/L to 1.30 mmol/L), for a mean between-group difference of -22.3% (p<0.001). More patients achieved the European Society of Cardiology/European Atherosclerosis Society dyslipidaemia guideline target of LDL ≤1.4 mmol/L in the alirocumab group (92.1% vs 56.7%; p<0.001). Within the first 24 hours, LDL declined slightly more rapidly in the alirocumab group than in the sham-control group (-0.01 mmol/L/hour; p=0.03) with similar between-group mean values.  Conclusions: In this randomised trial of routine early initiation of PCSK9 inhibitors in patients undergoing primary PCI for STEMI, alirocumab reduced LDL cholesterol by 22% compared with sham control on a background of high-intensity statin therapy. A large trial is needed to determine if this simplified approach followed by long-term therapy improves cardiovascular outcomes in patients with acute STEMI. (ClinicalTrials.gov: NCT03718286).

The rise to power of the microbiome: power and sample size calculation for microbiome studies.

A priori power and sample size calculations are crucial to appropriately test null hypotheses and obtain valid conclusions from all clinical studies. Statistical tests to evaluate hypotheses in microbiome studies need to consider intrinsic features of microbiome datasets that do not apply to classic sample size calculation. In this review, we summarize statistical approaches to calculate sample sizes for typical microbiome study scenarios, including those that hypothesize microbiome features to be the outcome, the exposure or the mediator, and provide relevant R scripts to conduct some of these calculations. This review is intended to be a resource to facilitate the conduct of sample size calculations that are based on testable hypotheses across several dimensions of the microbiome. Implementation of these methods will improve the quality of human or animal microbiome studies, enabling reliable conclusions that will generalize beyond the study sample.

Supplementation with a probiotic mixture accelerates gut microbiome maturation and reduces intestinal inflammation in extremely preterm infants.

Probiotics are increasingly administered to premature infants to prevent necrotizing enterocolitis and neonatal sepsis. However, their effects on gut microbiome assembly and immunity are poorly understood. Using a randomized intervention trial in extremely premature infants, we tested the effects of a probiotic product containing four strains of Bifidobacterium species autochthonous to the infant gut and one Lacticaseibacillus strain on the compositional and functional trajectory of microbiome. Daily administration of the mixture accelerated the transition into a mature, term-like microbiome with higher stability and species interconnectivity. Besides infant age, Bifidobacterium strains and stool metabolites were the best predictors of microbiome maturation, and structural equation modeling confirmed probiotics as a major determinant for the trajectory of microbiome assembly. Bifidobacterium-driven microbiome maturation was also linked to an anti-inflammatory intestinal immune milieu. This demonstrates that Bifidobacterium strains are ecosystem engineers that lead to an acceleration of microbiome maturation and immunological consequences in extremely premature infants.

Multi-strain probiotics for extremely preterm infants: a randomized controlled trial.

BACKGROUND: Effects of probiotics on intestinal microbiota and feeding tolerance remain unclear in extremely low-birth-weight (ELBW) infants. METHODS: ELBW infants were randomly assigned to receive probiotics or no intervention. Stool samples were collected prior to, 2 and 4 weeks after initiation, and 2 weeks after probiotics cessation for infants in the probiotics group, and at matched postnatal age time points for infants in the control group. RESULTS: Of the 102 infants assessed for eligibility, sixty-two were included. Infants who received probiotics reached full enteral feeds sooner (Mean difference (MD) -1.8; 95% CI:-3.7 to -0.01 day), had a tendency toward lower incidence of hematochezia before hospital discharge (22.6% vs 3.2%; P = 0.053), and were less likely to require extensively hydrolyzed- or amino acids-based formulas to alleviate signs of cow's milk protein intolerance in the first 6 months of life (19.4% vs 51.6%; P = 0.008). Infants on probiotics were more likely to receive wide-spectrum antibiotics (64.5% vs 32.2%; P = 0.01). Multi-strain probiotics resulted in significant increase in fecal Bifidobacterium (P < 0.001) and Lactobacillus (P = 0.005), and marked reduction in fecal candida abundance (P = 0.04). CONCLUSION: Probiotics sustained intestinal Bifidobacterium and reduced time to achieve full enteral feeds in extremely preterm infants. Probiotics might improve tolerance for cow's milk protein supplements. CLINICAL TRIAL REGISTRATION: This trial has been registered at www. CLINICALTRIALS: gov (identifier NCT03422562). IMPACT: Probiotics may help extremely preterm infants achieve full enteral feeds sooner. Probiotics may improve tolerance for cow's milk protein supplements. Multi-strain probiotics can sustain intestinal Bifidobacterium and Lactobacillus until hospital discharge.

Small Intestine Bacterial Overgrowth in Bangladeshi Infants Is Associated With Growth Stunting in a Longitudinal Cohort.

INTRODUCTION: Small intestine bacterial overgrowth (SIBO) is common in children from low-income countries and has been cross-sectionally associated with growth stunting. We sought to determine whether SIBO was associated with poor growth and neurodevelopmental in a longitudinal analysis. METHODS: We measured SIBO by glucose hydrogen breath test (GHBT) at 18, 52, 78, and 104 weeks of life in a prospective longitudinal birth cohort of Bangladeshi children. Sociodemographic information and measures of enteric inflammation were analyzed as covariates. Diarrheal samples were tested for enteropathogens using polymerase chain reaction. Regression models were created using standardized mean GHBT area under the H2 curve (AUC) to determine associations with linear growth and cognitive, language, and motor scores on the Bayley-III Scales of Infant and Toddler Development at 2 years. We also investigated associations between GHBT AUC and enteropathogen exposure. RESULTS: A 1-ppm increase in standardized mean GHBT AUC was associated with a 0.01-SD decrease in length-for-age Z score (P = 0.03) and a 0.11-point decrease in Bayley language score (P = 0.05) at 2 years of age in adjusted analysis. Enteroaggregative Escherichia coli, Enteropathogenic Escherichia coli, Giardia, and Enterocytozoon bieneusi were associated with increased GHBT AUC, whereas Clostridium difficile, norovirus GI, sapovirus, rotavirus, and Cryptosporidium were associated with decreased GHBT AUC. None were consistent across all 4 time points. DISCUSSION: SIBO in the first 2 years of life is associated with growth stunting and decreased language ability in Bangladeshi infants and may represent a modifiable risk factor in poor growth and neurodevelopment in low-income countries.

Use of Molecular Methods To Detect Shigella and Infer Phenotypic Resistance in a Shigella Treatment Study.

Molecular diagnostic methods improve the detection of Shigella, yet their ability to detect Shigella drug resistance on direct stool specimens is less clear. We tested 673 stool specimens from a Shigella treatment study in Bangladesh, including 154 culture-positive stool specimens and their paired Shigella isolates. We utilized a TaqMan array card that included quantitative PCR (qPCR) assays for 24 enteropathogens and 36 antimicrobial resistance (AMR) genes. Shigella was detected by culture in 23% of stool specimens (154/673), while qPCR detected Shigella at diarrhea-associated quantities in 49% (329/673; P < 0.05). qPCR for AMR genes on the Shigella isolates yielded >94% sensitivity and specificity compared with the phenotypic susceptibility results for azithromycin and ampicillin. The performance for trimethoprim-sulfamethoxazole susceptibility was less robust, and the assessment of ciprofloxacin was limited because most isolates were resistant. The detection of AMR genes in direct stool specimens generally yielded low specificities for predicting the resistance of the paired isolate, whereas the sensitivity and negative predictive values for predicting susceptibility were often higher. For example, the detection of ermB or mphA in stool yielded a specificity of 56% but a sensitivity of 91% and a negative predictive value of 91% versus the paired isolate's azithromycin resistance result. Patients who received azithromycin prior to presentation were universally culture negative (0/112); however, qPCR still detected Shigella at diarrhea-associated quantities in 34/112 (30%). In sum, molecular diagnostics on direct stool specimens greatly increase the diagnostic yield for Shigella, including in the setting of prior antibiotics. The molecular detection of drug resistance genes in direct stool specimens had low specificity for confirming resistance but could potentially "rule out" macrolide resistance.

Plasma VP8∗-Binding Antibodies in Rotavirus Infection and Oral Vaccination in Young Bangladeshi Children.

BACKGROUND: Despite the availability and success of live-attenuated oral vaccines, rotavirus (RV) remains the leading cause of pediatric gastroenteritis worldwide. Next-generation vaccines targeting RV VP8∗ are under evaluation, but the role of VP8∗-specific antibodies in human immunity to RV and their potential as immune correlates of protection remains underexplored. METHODS: We measured plasma RV VP8∗-binding antibodies in 2 cohorts of young children in Dhaka, Bangladesh. Plasma from a cohort study of 137 unvaccinated children aged 6-24 months old hospitalized with acute gastroenteritis was assessed for VP8∗ antibody seropositivity. VP8∗ antibodies were compared with the current standard for RV immunity, total RV-specific IgA (RV-IgA). Additionally, VP8∗ antibody responses were measured as part of an immunogenicity trial of a monovalent, oral, live-attenuated RV vaccine (Rotarix). RESULTS: Fewer children with acute RV gastroenteritis were seropositive for VP8∗-binding IgA or IgG antibodies at hospital admission compared with RV-IgA, suggesting that the absence of VP8∗-binding antibodies more accurately predicts susceptibility to RV gastroenteritis than RV-IgA in unvaccinated children. However, when present, these antibodies appeared insufficient to protect fully from disease and no threshold antibody level for protection was apparent. In vaccinated children, these antibodies were very poorly induced by Rotarix vaccine, suggesting that VP8∗-specific antibodies alone are not necessary for clinical protection following oral vaccination. CONCLUSIONS: This work suggests that VP8∗-binding antibodies may not be sufficient or necessary for protection from RV gastroenteritis following prior RV infection or oral vaccination; the role of VP8∗ antibodies induced by parenteral vaccination with non-replicating vaccines remains to be determined.

Diarrheal Pathogens Associated With Growth and Neurodevelopment.

BACKGROUND: Diarrheal pathogens have been associated with linear growth deficits. The effect of diarrheal pathogens on growth is likely due to inflammation, which also adversely affects neurodevelopment. We hypothesized that diarrheagenic pathogens would be negatively associated with both growth and neurodevelopment. METHODS: We conducted a longitudinal birth cohort study of 250 children with diarrheal surveillance and measured pathogen burden in diarrheal samples using quantitative polymerase chain reaction. Pathogen attributable fraction estimates of diarrhea over the first 2 years of life, corrected for socioeconomic variables, were used to predict both growth and scores on the Bayley-III Scales of Infant and Toddler Development. RESULTS: One hundred eighty children were analyzed for growth and 162 for neurodevelopmental outcomes. Rotavirus, Campylobacter, and Shigella were the leading causes of diarrhea in year 1 while Shigella, Campylobacter, and heat-stable toxin-producing enterotoxigenic Escherichia coli were the leading causes in year 2. Norovirus was the only pathogen associated with length-for-age z score at 24 months and was positively associated (regression coefficient [RC], 0.42 [95% confidence interval {CI}, .04 to .80]). Norovirus (RC, 2.46 [95% CI, .05 to 4.87]) was also positively associated with cognitive scores while sapovirus (RC, -2.64 [95% CI, -4.80 to -.48]) and typical enteropathogenic E. coli (RC, -4.14 [95% CI, -8.02 to -.27]) were inversely associated. No pathogens were associated with language or motor scores. Significant maternal, socioeconomic, and perinatal predictors were identified for both growth and neurodevelopment. CONCLUSIONS: Maternal, prenatal, and socioeconomic factors were common predictors of growth and neurodevelopment. Only a limited number of diarrheal pathogens were associated with these outcomes.

Clinical Outcomes of Drug-resistant Shigellosis Treated With Azithromycin in Bangladesh.

BACKGROUND: Azithromycin is frequently used to treat shigellosis; however, clinical outcomes are uncertain. METHODS: We performed an observational cohort study in Bangladesh of patients with invasive diarrhea treated empirically with azithromycin. Susceptibility testing was performed by broth microdilution and disk diffusion post hoc on all Shigella isolates and clinical response was correlated with in vitro susceptibility. RESULTS: There were 149 Shigella culture-positive patients in the primary analysis. Infection with Shigella with decreased susceptibility to azithromycin was significantly associated with persistence of diarrhea at day 5 (31% vs 12%; relative risk [RR], 2.66; 95% confidence interval [CI], 1.34-5.28), culture positivity at day 5 or 6 (35% vs 5%; RR, 5.26; 95% CI, 1.84-14.85), and a higher rate of overnight hospitalization (58% vs 39%; RR, 1.49; 95% CI, 1.06-2.09). Shigella flexneri was more common than Shigella sonnei (58% vs 36%); however, S. sonnei constituted most of the isolates with decreased susceptibility to azithromycin (67%) and most of the multidrug-resistant strains (54%); thus, poor clinical outcomes were associated with S. sonnei. The current epidemiological cutoff for S. flexneri of ≥16 µg/mL to define decreased susceptibility to azithromycin was clinically predictive of poor outcome. Patients with S. sonnei and a low MIC (4 µg/mL) still had elevated rates of persistent diarrhea and culture positivity. CONCLUSIONS: This study documents worse clinical outcomes for S. flexneri with decreased susceptibility to azithromycin, as well as S. sonnei, and supports the utility of susceptibility testing and clinical breakpoints for azithromycin. S. sonnei is an emerging drug-resistant threat. CLINICAL TRIALS REGISTRATION: NCT03778125.

Rapid assessment of tetanus vaccine-induced immunity in Bangladesh and the Gambia.

We have developed recombinant fragment C based rapid point of care dipstick devices to assess tetanus immunization status using plasma or whole blood. The devices demonstrated specificity of 0.90 and sensitivity of 0.90 (whole blood)/0.94 (plasma) at field sites in Bangladesh and The Gambia when compared to a commercial ELISA with the immune cut-off titer set as ≥0.1IU/mL.