Impact of Rotavirus Vaccine Introduction and Postintroduction Etiology of Diarrhea Requiring Hospital Admission in Haydom, Tanzania, a Rural African Setting.

Background: No data are available on the etiology of diarrhea requiring hospitalization after rotavirus vaccine introduction in Africa. The monovalent rotavirus vaccine was introduced in Tanzania on 1 January 2013. We performed a vaccine impact and effectiveness study as well as a quantitative polymerase chain reaction (qPCR)-based etiology study at a rural Tanzanian hospital. Methods: We obtained data on admissions among children <5 years to Haydom Lutheran Hospital between 1 January 2010 and 31 December 2015 and estimated the impact of vaccine introduction on all-cause diarrhea admissions. We then performed a vaccine effectiveness study using the test-negative design. Finally, we tested diarrheal specimens during 2015 by qPCR for a broad range of enteropathogens and calculated pathogen-specific attributable fractions (AFs). Results: Vaccine introduction was associated with a 44.9% (95% confidence interval [CI], 17.6%-97.4%) reduction in diarrhea admissions in 2015, as well as delay of the rotavirus season. The effectiveness of 2 doses of vaccine was 74.8% (95% CI, -8.2% to 94.1%) using an enzyme immunoassay-based case definition and 85.1% (95% CI, 26.5%-97.0%) using a qPCR-based case definition. Among 146 children enrolled in 2015, rotavirus remained the leading etiology of diarrhea requiring hospitalization (AF, 25.8% [95% CI, 24.4%-26.7%]), followed by heat-stable enterotoxin-producing Escherichia coli (AF, 18.4% [95% CI, 12.9%-21.9%]), Shigella/enteroinvasive E. coli (AF, 14.5% [95% CI, 10.2%-22.8%]), and Cryptosporidium (AF, 7.9% [95% CI, 6.2%-9.3%]). Conclusions: Despite the clear impact of vaccine introduction in this setting, rotavirus remained the leading etiology of diarrhea requiring hospitalization. Further efforts to maximize vaccine coverage and improve vaccine performance in these settings are warranted.

Giardia hinders growth by disrupting nutrient metabolism independent of inflammatory enteropathy.

Giardia lamblia (Giardia) is among the most common intestinal pathogens in children in low- and middle-income countries (LMICs). Although Giardia associates with early-life linear growth restriction, mechanistic explanations for Giardia-associated growth impairments remain elusive. Unlike other intestinal pathogens associated with constrained linear growth that cause intestinal or systemic inflammation or both, Giardia seldom associates with chronic inflammation in these children. Here we leverage the MAL-ED longitudinal birth cohort and a model of Giardia mono-association in gnotobiotic and immunodeficient mice to propose an alternative pathogenesis of this parasite. In children, Giardia results in linear growth deficits and gut permeability that are dose-dependent and independent of intestinal markers of inflammation. The estimates of these findings vary between children in different MAL-ED sites. In a representative site, where Giardia associates with growth restriction, infected children demonstrate broad amino acid deficiencies, and overproduction of specific phenolic acids, byproducts of intestinal bacterial amino acid metabolism. Gnotobiotic mice require specific nutritional and environmental conditions to recapitulate these findings, and immunodeficient mice confirm a pathway independent of chronic T/B cell inflammation. Taken together, we propose a new paradigm that Giardia-mediated growth faltering is contingent upon a convergence of this intestinal protozoa with nutritional and intestinal bacterial factors.

Etiology of severe invasive infections in young infants in rural settings in sub-Saharan Africa.

BACKGROUND: Serious invasive infections in newborns are a major cause of death. Lack of data on etiological causes hampers progress towards reduction of mortality. This study aimed to identify pathogens responsible for such infections in young infants in sub-Saharan Africa and to describe their antibiotics resistance profile. METHODS: Between September 2016 and April 2018 we implemented an observational study in two rural sites in Burkina Faso and Tanzania enrolling young infants aged 0-59 days old with serious invasive infection. Blood samples underwent blood culture and molecular biology. RESULTS: In total 634 infants with clinical diagnosis of serious invasive infection were enrolled and 4.2% of the infants had a positive blood culture. The most frequent pathogens identified by blood culture were Klebsiella pneumonia and Staphylococcus aureus, followed by Escherichia coli. Gram-negative isolates were only partially susceptible to first line WHO recommended treatment for neonatal sepsis at community level. A total of 18.6% of the infants were PCR positive for at least one pathogen and Escherichia coli and Staphylococcus aureus were the most common bacteria detected. Among infants enrolled, 60/634 (9.5%) died. Positive blood culture but not positive PCR was associated with risk of death. For most deaths, no pathogen was identified either by blood culture or molecular testing, and hence a causal agent remained unclear. Mortality was associated with low body temperature, tachycardia, respiratory symptoms, convulsions, history of difficult feeding, movement only when stimulated or reduced level of consciousness, diarrhea and/or vomiting. CONCLUSION: While Klebsiella pneumonia and Staphylococcus aureus, as well as Escherichia coli were pathogens most frequently identified in infants with clinical suspicion of serious invasive infections, most cases remain without definite diagnosis, making more accurate diagnostic tools urgently needed. Antibiotics resistance to first line antibiotics is an increasing challenge even in rural Africa.

Pharmacogenetic testing for NAT2 genotypes in a Tanzanian population across the lifespan to guide future personalized isoniazid dosing.

Despite updated recommendations for weight-based isoniazid dosing in children with drug-susceptible tuberculosis (TB) and higher dose isoniazid in regimens for adults with drug-resistant TB, individual pharmacokinetic variability can lead to sub-target isoniazid exposure. Host pharmacogenetics and isoniazid exposure remain understudied, especially in the East African population. We therefore employed a real-time polymerase chain reaction (qPCR) assay system to test genomic DNA extracted from saliva samples targeting the NAT2 gene responsible for isoniazid metabolism to describe the frequency of human single nucleotide polymorphisms in NAT2 within populations of children and adults in Tanzania, ascribe those polymorphisms to acetylator phenotype, and correlate to serum isoniazid exposures. In adults treated with higher dose isoniazid, genotypes with a predicted allelic phenotype of slow or intermediate acetylation were able to achieve a 0.41 µg/mL higher Cmax (p = 0.018) and a 2.9h*µg/mL higher AUC0-12 (p = 0.003) per mg/kg increase in isoniazid dosage versus adults with rapid acetylation phenotype. A similar relationship was not found in the younger age population as predicted by timing of NAT2 maturation. This saliva based qPCR assay was fieldable to guide personalized isoniazid dosing in adults but not young children that may not have full NAT2 maturation and activity.

Longitudinal Assessment of Antibiotic Resistance in Fecal Escherichia coli in Tanzanian Children.

Antibiotic-resistant bacterial infections are a major public health problem, and children in low-resource settings represent a particularly high-risk group. Few data are available on the dynamics of and risk factors for gastrointestinal carriage of antibiotic-resistant bacteria in these vulnerable populations. In this study, we described the antibiotic susceptibility profiles of Escherichia coli isolated from stool specimens collected from children aged 6 to 60 months enrolled in a birth cohort study in Haydom, Tanzania. We estimated the association between sociodemographic risk factors, child illnesses, and antibiotic exposure and E. coli drug resistance. Carriage of antibiotic-resistant E. coli was common starting early in life and did not clearly increase with age. The majority of isolates were resistant to ampicillin (749/837; 89.5%), cefazolin (742/837; 88.6%), and cotrimoxazole (721/837; 86.1%). Resistance to amoxicillin/clavulanate (361/836; 43.2%), ampicillin/sulbactam (178/819; 21.7%), nalidixic acid (131/831; 15.8%), and azithromycin (115/837; 13.7%) was also seen. Only 1.8% (15/837) of the pooled E. coli isolates met the criteria for extended-spectrum beta-lactamase production. High antibiotic use (0.26 additional resistant antibiotic classes; 95% CI: 0.05, 0.47) and high income (0.28 additional resistant antibiotic classes; 95% CI: 0.06, 0.50) were associated with the carriage of antibiotic-resistant E. coli, whereas hospital birth, crowding in the home, improved drinking water and sanitation, and common childhood illnesses were not. In this setting, the carriage of antibiotic-resistant E. coli was common. Other than recent antibiotic exposure and high income, individual risk factors for the acquisition and carriage of resistance could not be identified, suggesting that population-level interventions are needed.

Genotypic antimicrobial resistance assays for use on E. coli isolates and stool specimens.

Antimicrobial resistance (AMR) is an emerging public health problem and methods for surveillance are needed. We designed 85 sequence-specific PCR reactions to detect 79 genes or mutations associated with resistance across 10 major antimicrobial classes, with a focus on E. coli. The 85 qPCR assays demonstrated >99.9% concordance with sequencing. We evaluated the correlation between genotypic resistance markers and phenotypic susceptibility results on 239 E. coli isolates. Both sensitivity and specificity exceeded 90% for ampicillin, ceftriaxone, cefepime, imipenem, ciprofloxacin, azithromycin, gentamicin, amikacin, trimethoprim/sulfamethoxazole, tetracycline, and chloramphenicol phenotypic susceptibility results. We then evaluated the assays on direct stool specimens and observed a sensitivity of 97% ± 5 but, as expected, a lower specificity of 75% ± 31 versus the genotype of the E. coli cultured from stool. Finally, the assays were incorporated into a convenient TaqMan Array Card (TAC) format. These assays may be useful for tracking AMR in E. coli isolates or directly in stool for targeted testing of the fecal antibiotic resistome.

Optimization of Quantitative PCR Methods for Enteropathogen Detection.

Detection and quantification of enteropathogens in stool specimens is useful for diagnosing the cause of diarrhea but is technically challenging. Here we evaluate several important determinants of quantification: specimen collection, nucleic acid extraction, and extraction and amplification efficiency. First, we evaluate the molecular detection and quantification of pathogens in rectal swabs versus stool, using paired flocked rectal swabs and whole stool collected from 129 children hospitalized with diarrhea in Tanzania. Swabs generally yielded a higher quantification cycle (Cq) (average 29.7, standard deviation 3.5 vs. 25.3 ± 2.9 from stool, P<0.001) but were still able to detect 80% of pathogens with a Cq < 30 in stool. Second, a simplified total nucleic acid (TNA) extraction procedure was compared to separate DNA and RNA extractions and showed 92% (318/344) sensitivity and 98% (951/968) specificity, with no difference in Cq value for the positive results (ΔCq(DNA+RNA-TNA) = -0.01 ± 1.17, P = 0.972, N = 318). Third, we devised a quantification scheme that adjusts pathogen quantity to the specimen's extraction and amplification efficiency, and show that this better estimates the quantity of spiked specimens than the raw target Cq. In sum, these methods for enteropathogen quantification, stool sample collection, and nucleic acid extraction will be useful for laboratories studying enteric disease.