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IntroductionSalmonellaTyphi andSalmonellaParatyphi, fecal-oral transmitted bacterium have temporally and geographically heterogeneous pathways of transmission. Previous work in Kathmandu Nepal implicated stone waterspouts as a dominant transmission pathway after 77% of samples tested positive forS. Typhi and 70% forS. Paratyphi. Due to a falling water table, these spouts no longer provide drinking water, but typhoid fever persists, and the question of the disease’s dominant pathway of transmission remains unanswered.MethodsWe used environmental surveillance to detectS. Typhi and Paratyphi DNA from potential sources of transmission. We collected 1L drinking water samples from a population-based random sample of households between February and October 2019 Between November 2019 and July 2021, we collected monthly 50 mL river water samples from 19 sites along the rivers leading through the Kathmandu and Kavre Districts of Nepal. We processed drinking water samples using a single qpcr and processed river water samples using differential centrifugation and qPCR at time 0 and after 16 hours of culture enrichment. A 3-cycle threshold (Ct) decrease ofS. Typhi orS. Paratyphi, pre- and post-enrichment, was used as evidence of growth. We also performed structured observations of human-environment interactions to understand pathways of potential exposure.ResultsAmong 370 drinking water samples,S. Typhi was detected in 7 samples (1.8%) andS. Paratyphi A was detected in 4 (1.0%) samples. Among 381 river water samples,S. Typhi was detected in 171 (45%) andS. Paratyphi A was detected in 152 (42%) samples. Samples located upstream of the Kathmandu city center were positive forS. Typhi 12% of the time while samples from locations in and downstream the city had bacterial DNA detected 58% and 67% of the time respectively. Individuals were observed bathing in the rivers, washing clothes, and washing vegetables for sale in Kathmandu markets.ImplicationsThese results suggest that drinking water was not the dominant pathway of transmission ofS. Typhi andS. Paratyphi A in the Kathmandu Valley in 2019. The high degree of river water contamination and its use for washing vegetables raises the possibility that river systems srepresent an important source of typhoid exposure in Kathmandu.Author SummaryUnderstanding the dominant route of transmission of a pathogen is important for designing and implementing effective control strategies. Salmonella Typhi and Paratyphi which cause typhoid and paratyphoid fever infect an estimated 10 million people and kill more than 100,000 annually. In Kathmandu prior work suggested that stone spouts where people collected drinking water were contaminated and driving transmission of the diseases. However, many of these spouts no longer function, and people are still getting sick. We tested drinking water from households in this area as well as local river water and found that 13 drinking water samples were positive for S.Typhi and 15 were positive for S. Paratyphi and many river samples tested positive for these bacterium. River water samples were not often positive upstream of Kathmandu city center (12% positive for S.Typhi) but were often positive within the city center (58% positive for S.Typhi) and in rural areas up to 10 km downstream of the city (67% positive for S.Typhi). During sample collection, individuals were observed interacting with rivers by walking in them, washing clothes and washing vegetables for sale in markets. This study shows that drinking water may not be primary driver of typhoid transmission in the Kathmandu valley, but that sewage contaminated river water may be a foci of transmission into the wider population.

Environmental surveillance, using detection ofSalmonellaTyphi DNA, has emerged as a potentially useful tool to identify typhoid-endemic settings; however, it is relatively costly and requires molecular diagnostic capacity. We sought to determine whetherS. Typhi bacteriophages are abundant in water sources in a typhoid-endemic setting, using low-cost assays. We collected drinking and surface water samples from urban, peri-urban and rural areas in 4 regions of Nepal. We performed a double agar overlay withS. Typhi to assess the presence of bacteriophages. We isolated and tested phages against multiple strains to assess their host range. We performed whole genome sequencing of isolated phages, and generated phylogenies using conserved genes.S. Typhi-specific bacteriophages were detected in 54.9% (198/361) of river water samples and 6.3% (1/16) drinking water samples from the Kathmandu Valley and Kavrepalanchok. Water samples collected within or downstream of population-dense areas were more likely to be positive (72.6%, 193/266) than those collected upstream from population centers (5.3%, 5/95) (p=0.005). In urban Biratnagar and rural Dolakha, where typhoid incidence is low, only 6.7% (1/15, Biratnagar) and 0% (0/16, Dolakha) samples contained phages. AllS. Typhi phages were unable to infect otherSalmonellaand non-Salmonellastrains, nor a Vi-knockoutS. Typhi strain. Representative strains fromS. Typhi lineages were variably susceptible to the isolated phages. Phylogenetic analysis showed thatS. Typhi phages belonged to two different viral families (AutographiviridaeandSiphoviridae) and clustered in three distinct groups.S. Typhi bacteriophages were highly abundant in surface waters of typhoid-endemic communities but rarely detected in low typhoid burden communities. Bacteriophages recovered were specific forS. Typhi and required Vi polysaccharide for infection. Screening small volumes of water with simple, low-cost plaque assays enables detection ofS. Typhi phages and should be further evaluated as a scalable tool for typhoid environmental surveillance.HighlightsTyphoid phages are detectable in surface water using simple assays, in communities with high typhoid burden.Bacteriophages are highly specific forS. Typhi and required Vi polysaccharide for infection.S. Typhi phages have a broad lytic activity against theS. Typhi strains circulating in Nepal.Phage plaque assay can be used as a low-cost tool to identify communities where typhoid is endemic.The high abundance of phages in river water suggest that this could be an alternative to molecular methods for environmental surveillance for typhoid.

IntroductionScrub typhus is an acute febrile illness caused by the bacteriumOrientia tsutsugamushi. Characterizing the population-level burden of scrub typhus is challenging due to the lack of accessible and accurate diagnostics. In this study, we present a novel approach that utilizes information on antibody dynamics after infection to generate population-level scrub typhus seroincidence estimates from cross-sectional serosurveys.MethodsWe use data from three cohorts of scrub typhus patients enrolled in Chiang Rai, Thailand, and Vellore, India, and representative population data from two serosurveys in and around the Kathmandu valley, Nepal, and Vellore, India. The samples were tested for IgM and IgG responses toOrientia tsutsugamushi-derived recombinant 56-kDa antigen using commercial ELISA kits. We used Bayesian hierarchical models to fit two-phase models to the antibody responses from scrub typhus cases and used the joint distributions of the peak antibody titers and decay rates to estimate population-level incidence rates in the cross-sectional serosurveys. We compared this new method to a traditional cut-off-based approach for estimating seroincidence.ResultsMedian IgG antibodies persisted above OD 1.7 for 22 months, while IgM displayed longer persistence than expected, with 50% of participants having an OD >1 for 5 months. We estimated an overall seroincidence of 18 per 1000 person-years (95% CI: 16-21) in India and 4 per 1000 person-years (95% CI: 3-6) in Nepal. Among 18 to 29-year-olds, the seroincidence was 8 per 1000 person-years (95% CI 4 -16) in India and 9 per 1000 person-years (95% CI: 6-14) in Nepal. In both India and Nepal, seroincidence was higher in urban and periurban settings compared to rural areas. Compared to our method, seroincidence estimates derived from age-dependent IgG-seroprevalence without accounting for antibody decay underestimated the disease burden by 50%. By incorporating antibody dynamics, the approach described here provides more accurate age-specific infection risk estimates, emphasizing the importance of considering both IgG and IgM decay patterns in scrub typhus seroepidemiology.ConclusionThe sero-surveillance approach developed in this study efficiently generates population-level scrub typhus seroincidence estimates from cross-sectional serosurveys. This methodology offers a valuable new tool for informing targeted prevention and control strategies, ultimately contributing to a more effective response to scrub typhus in endemic regions worldwide.

We apply a new serosurveillance tool to estimate typhoidal Salmonella burden from dried blood spots in Juba, South Sudan, finding a seroincidence rate of 35 per 100 person-years and cumulative incidence of 78% over four years.

Background Characterizing healthcare-seeking patterns for acute febrile illness is critical for generating population-based enteric fever incidence estimates from facility-based surveillance data. Methods We used a hybrid model in the Surveillance for Enteric Fever in Asia Project (SEAP) to assess incidence of enteric fever at 6 study hospitals in 3 countries. We recruited individuals presenting to the hospitals and obtained blood cultures to evaluate for enteric fever. For this analysis, we undertook cluster random household surveys in Dhaka, Bangladesh (2 sites); Karachi, Pakistan; Kathmandu, Nepal; and Kavrepalanchok, Nepal between January 2017 and February 2019, to ascertain care-seeking behavior for individuals with 1) fever for ≥3 consecutive days within the past 8 weeks; or 2) fever resulting in hospitalization within the past year. We also collected data about disease severity and household demographics and assets. We used mixed-effect multivariable logistic regression models to identify determinants of healthcare seeking at study hospitals and determinants of culture-confirmed enteric fever. Results We enrolled 31 841 households (53 926 children) in Bangladesh, 25 510 households (84 196 children and adults) in Nepal, and 21 310 households (108 031 children and adults) in Pakistan. Children Conclusions Age, household wealth, and disease severity are important determinants of healthcare seeking for acute febrile illness and enteric fever risk in these communities, and should be incorporated into estimation models for enteric fever incidence.

Though bulk stool remains the gold standard specimen type for enteropathogen diagnosis, rectal swabs may offer comparable sensitivity with greater ease of collection for select pathogens. This study sought to evaluate the validity and reproducibility of rectal swabs as a sample collection method for the molecular diagnosis of Giardia duodenalis. Paired rectal swab and bulk stool samples were collected from 86 children ages 0-4 years living in southwest Niger, with duplicate samples collected among a subset of 50 children. Infection was detected using a previously validated real-time PCR diagnostic targeting the small subunit ribosomal RNA gene. Giardia duodenalis was detected in 65.5% (55/84) of bulk stool samples and 44.0% (37/84) of swab samples. The kappa evaluating test agreement was 0.81 (95% CI: 0.54-1.00) among duplicate stool samples (N = 49) and 0.75 (95% CI: 0.47-1.00) among duplicate rectal swabs (N = 48). Diagnostic sensitivity was 93% (95% CI: 84-98) by bulk stool and 63% (95% CI: 49-75) by rectal swabs. When restricting to the lowest three quartiles of bulk stool quantitation cycle values (an indication of relatively high parasite load), sensitivity by rectal swabs increased to 78.0% (95% CI: 64-89, P < 0.0001). These findings suggest that rectal swabs provide less sensitive and reproducible results than bulk stool for the real-time PCR diagnosis of G. duodenalis. However, their fair sensitivity for higher parasite loads suggests that swabs may be a useful tool for detecting higher burden infections when stool collection is excessively expensive or logistically challenging.

The incidence of enteric fever, an invasive bacterial infection caused by typhoidal Salmonellae (Salmonella enterica serovars Typhi and Paratyphi), is largely unknown in regions without blood culture surveillance. The aim of this study was to evaluate whether new diagnostic serological markers for typhoidal Salmonella can reliably estimate population-level incidence.We collected longitudinal blood samples from patients with blood culture-confirmed enteric fever enrolled from surveillance studies in Bangladesh, Nepal, Pakistan, and Ghana between 2016 and 2021 and conducted cross-sectional serosurveys in the catchment areas of each surveillance site. We used ELISAs to measure quantitative IgA and IgG antibody responses to hemolysin E and S Typhi lipopolysaccharide. We used Bayesian hierarchical models to fit two-phase power-function decay models to the longitudinal antibody responses among enteric fever cases and used the joint distributions of the peak antibody titres and decay rate to estimate population-level incidence rates from cross-sectional serosurveys.The longitudinal antibody kinetics for all antigen-isotypes were similar across countries and did not vary by clinical severity. The seroincidence of typhoidal Salmonella infection among children younger than 5 years ranged between 58·5 per 100 person-years (95% CI 42·1-81·4) in Dhaka, Bangladesh, to 6·6 per 100 person-years (4·3-9·9) in Kavrepalanchok, Nepal, and followed the same rank order as clinical incidence estimates.The approach described here has the potential to expand the geographical scope of typhoidal Salmonella surveillance and generate incidence estimates that are comparable across geographical regions and time.BillMelinda Gates Foundation.For the Nepali, Bengali and Urdu translations of the abstract see Supplementary Materials section.

BackgroundThe incidence of enteric fever, an invasive bacterial infection caused by typhoidal Salmonellae, is largely unknown in regions lacking blood culture surveillance. New serologic markers have proven accurate in diagnosing enteric fever, but whether they could be used to reliably estimate population-level incidence is unknown.MethodsWe collected longitudinal blood samples from blood culture-confirmed enteric fever cases enrolled from surveillance studies in Bangladesh, Nepal, Pakistan, and Ghana and conducted cross-sectional serosurveys in the catchment areas of each surveillance site. We used ELISAs to measure quantitative IgA and IgG antibody responses to Hemolysin E (HlyE) and S. Typhi lipopolysaccharide (LPS). We used Bayesian hierarchical models to fit two-phase power-function decay models to the longitudinal antibody responses among enteric fever cases and used the joint distributions of the peak antibody titers and decay rate to estimate population-level incidence rates from cross-sectional serosurveys.FindingsThe longitudinal antibody kinetics for all antigen-isotypes were similar across countries and did not vary by clinical severity. The seroincidence of typhoidal Salmonella infection among children InterpretationThe approach described here has the potential to expand the geographic scope of typhoidal Salmonella surveillance and generate incidence estimates that are comparable across geographic regions and time.FundingThis work was supported by the Bill and Melinda Gates Foundation (INV-000572).Research in contextEvidence before this studyPrevious studies have identified serologic responses to two antigens (Hemolysin E [HlyE] and Salmonella lipopolysaccharide [LPS]) as promising diagnostic markers of acute typhoidal Salmonella infection. We reviewed the evidence for seroepidemiology tools for enteric fever available as of November 01, 2021, by searching the National Library of Medicine article database and medRxiv for preprint publications, published in English, using the terms “enteric fever”, “typhoid fever”, “Salmonella Typhi”, “Salmonella Paratyphi”, “typhoidal Salmonella”, “Hemolysin E”, “Salmonella lipopolysaccharide”, “seroconversion”, “serosurveillance”, “seroepidemiology”, “seroprevalence” and “seropositivity.” We found no studies using HlyE or LPS as markers to measure the incidence or prevalence of enteric fever in a population. Anti-Vi IgG responses were used as a marker of population seroprevalence in cross-sectional studies conducted in South Africa, Fiji, and Nepal, but were not used to calculate population-based incidence estimates.Added value of this studyWe developed and validated a method to estimate typhoidal Salmonella incidence in cross-sectional population samples using antibody responses measured from dried blood spots. First, using longitudinal dried blood spots collected from over 1400 blood culture-confirmed cases in four countries, we modeled the longitudinal dynamics of antibody responses for up to two years following infection, accounting for heterogeneity in antibody responses and age-dependence. We found that longitudinal antibody responses were highly consistent across four countries on two continents and did not differ by clinical severity. We then used these antibody kinetic parameters to estimate incidence in population-based samples in six communities across the four countries, where concomitant population-based incidence was measured using blood cultures. Seroincidence estimates were much higher than blood-culture-based case estimates across all six sites, suggestive of a high incidence of asymptomatic or unrecognized infections. Still, the rank order of seroincidence and culture-based incidence rates were the same, with the highest rates in Bangladesh and lowest in Ghana.Implications of all the available evidenceMany at-risk low- and middle-income countries lack data on typhoid incidence needed to inform and evaluate vaccine introduction. Even in countries where incidence estimates are available, data are typically geographically and temporally sparse due to the resources necessary to initiate and sustain blood culture surveillance. We found that typhoidal Salmonella infection incidence can be estimated from community-based serosurveys using dried blood spots, representing an efficient and scalable approach for generating the typhoid burden data needed to inform typhoid control programs in resource-constrained settings.

Enteric fever remains a major cause of morbidity in developing countries with poor sanitation conditions that enable fecal contamination of water distribution systems. Historical evidence has shown that contamination of water systems used for household consumption or agriculture are key transmission routes for Salmonella Typhi and Salmonella Paratyphi A. The World Health Organization now recommends that typhoid conjugate vaccines (TCV) be used in settings with high typhoid incidence; consequently, governments face a challenge regarding how to prioritize typhoid against other emerging diseases. A key issue is the lack of typhoid burden data in many low- and middle-income countries where TCV could be deployed. Here we present an argument for utilizing environmental sampling for the surveillance of enteric fever organisms to provide data on community-level typhoid risk. Such an approach could complement traditional blood culture-based surveillance or even replace it in settings where population-based clinical surveillance is not feasible. We review historical studies characterizing the transmission of enteric fever organisms through sewage and water, discuss recent advances in the molecular detection of typhoidal Salmonella in the environment, and outline challenges and knowledge gaps that need to be addressed to establish environmental sampling as a tool for generating actionable data that can inform public health responses to enteric fever.

Precise enteric fever disease burden data are needed to inform prevention and control measures, including the use of newly available typhoid vaccines. We established the Surveillance for Enteric Fever in Asia Project (SEAP) to inform these strategies.From September, 2016, to September, 2019, we conducted prospective clinical surveillance for Salmonella enterica serotype Typhi (S Typhi) and Paratyphi (S Paratyphi) A, B, and C at health facilities in predetermined catchment areas in Dhaka, Bangladesh; Kathmandu and Kavrepalanchok, Nepal; and Karachi, Pakistan. Patients eligible for inclusion were outpatients with 3 or more consecutive days of fever in the last 7 days; inpatients with suspected or confirmed enteric fever; patients with blood culture-confirmed enteric fever from the hospital laboratories not captured by inpatient or outpatient enrolment and cases from the laboratory network; and patients with non-traumatic ileal perforation under surgical care. We used a hybrid surveillance model, pairing facility-based blood culture surveillance with community surveys of health-care use. Blood cultures were performed for enrolled patients. We calculated overall and age-specific typhoid and paratyphoid incidence estimates for each study site. Adjusted estimates accounted for the sensitivity of blood culture, the proportion of eligible individuals who consented and provided blood, the probability of care-seeking at a study facility, and the influence of wealth and education on care-seeking. We additionally calculated incidence of hospitalisation due to typhoid and paratyphoid.A total of 34 747 patients were enrolled across 23 facilitates (six tertiary hospitals, surgical wards of two additional hospitals, and 15 laboratory network sites) during the study period. Of the 34 303 blood cultures performed on enrolled patients, 8705 (26%) were positive for typhoidal Salmonella. Adjusted incidence rates of enteric fever considered patients in the six tertiary hospitals. Adjusted incidence of S Typhi, expressed per 100 000 person-years, was 913 (95% CI 765-1095) in Dhaka. In Nepal, the adjusted typhoid incidence rates were 330 (230-480) in Kathmandu and 268 (202-362) in Kavrepalanchok. In Pakistan, the adjusted incidence rates per hospital site were 176 (144-216) and 103 (85-126). The adjusted incidence rates of paratyphoid (of which all included cases were due to S Paratyphi A) were 128 (107-154) in Bangladesh, 46 (34-62) and 81 (56-118) in the Nepal sites, and 23 (19-29) and 1 (1-1) in the Pakistan sites. Adjusted incidence of hospitalisation was high across sites, and overall, 2804 (32%) of 8705 patients with blood culture-confirmed enteric fever were hospitalised.Across diverse communities in three south Asian countries, adjusted incidence exceeded the threshold for "high burden" of enteric fever (100 per 100 000 person-years). Incidence was highest among children, although age patterns differed across sites. The substantial disease burden identified highlights the need for control measures, including improvements to water and sanitation infrastructure and the implementation of typhoid vaccines.BillMelinda Gates Foundation.

Background Stool consistency is an important diagnostic criterion in both research and clinical medicine and is often used to define diarrheal disease. Methods We examine the pediatric enteric virome across stool consistencies to evaluate differences in richness and community composition using fecal samples collected from children aged 0 to 5 years participating in a clinical trial in the Amhara region of Ethiopia. The consistency of each sample was graded according to the modified Bristol Stool Form Scale for children (mBSFS-C) before a portion of stool was preserved for viral metagenomic analysis. Stool samples were grouped into 29 pools according to stool consistency type. Differential abundance was determined using negative-binomial modeling. Results Of 446 censused children who were eligible to participate, 317 presented for the study visit examination and 269 provided stool samples. The median age of children with stool samples was 36 months. Species richness was highest in watery-consistency stool and decreased as stool consistency became firmer (Spearman’s r = − 0.45, p = 0.013). The greatest differential abundance comparing loose or watery to formed stool was for norovirus GII (7.64, 95% CI 5.8, 9.5) followed by aichivirus A (5.93, 95% CI 4.0, 7.89) and adeno-associated virus 2 (5.81, 95%CI 3.9, 7.7). Conclusions In conclusion, we documented a difference in pediatric enteric viromes according to mBSFS-C stool consistency category, both in species richness and composition. Electronic supplementary material The online version of this article (10.1186/s12879-019-3674-3) contains supplementary material, which is available to authorized users.

Background: Precise enteric fever disease burden data are needed to inform prevention and control measures, including the use of newly available typhoid vaccines. To inform these strategies, the Surveillance for Enteric Fever in Asia Project (SEAP) conducted prospective, facility-based surveillance for Salmonella Typhi and S . Paratyphi A from 2016-2019. Methods: To generate population-based incidence estimates of enteric fever, we conducted prospective clinical surveillance at five hospitals in pre-determined catchment areas in Dhaka, Bangladesh, Kathmandu and Kavrepalanchok, Nepal, and Karachi, Pakistan. We employed a hybrid surveillance model, pairing facility-based blood culture surveillance with community surveys of healthcare utilization. Blood cultures were performed for enrolled patients. We calculated overall and age-specific typhoid and paratyphoid incidence estimates for each study site. Adjusted estimates accounted for the sensitivity of blood culture, the proportion of eligible individuals who consented and provided blood, the probability of care-seeking at a study facility, and the influence of wealth and education on care-seeking. Findings: Of the 33,136 blood cultures performed on enrolled patients, 8,705 (26%) were positive for typhoidal Salmonella . Adjusted incidence of S . Typhi, expressed per 100,000 person-years, was 1,110 (95% CI: 949-1,305) in Dhaka. In Nepal, the adjusted typhoid incidence rates were 330 (95% CI: 232-476) in Kathmandu and 271 (95% CI:205-365) in Kavrepalanchok. In Pakistan, the adjusted incidence rates per hospital site were 195 (95% CI:163-236) and 126 (95% CI:106-151). Although lower than the rates of typhoid, the adjusted incidence rates of paratyphoid were 150 ( 95% CI: 127-177) in Bangladesh, 49 (95% CI: 38-65) and 82 (95% CI: 57-116) in the Nepal sites, and 25 (95% CI: 21-31) and 1 (95% CI: 1-1) in the Pakistan sites. Adjusted incidence of hospitalization was high across sites, and overall 32% of case patients were hospitalized. Interpretation: Across diverse communities in three South Asian countries, adjusted incidence exceeded the threshold for “high burden” of enteric fever (100 per 100,000 person-years). Incidence was highest among children, although age patterns differed across sites. The substantial disease burden identified highlights the need for control measures, including improvements to water and sanitation infrastructure and the implementation of typhoid vaccines. Funding: This work was supported by a grant from the Bill and Melinda Gates Foundation (grant number INV-008335). Declaration of Interest: None to declare. Ethical Approval: We obtained written informed consent from all eligible participants and the parents/guardians of participants aged

BackgroundImplementation of population-based surveys is resource intensive and logistically demanding, especially in areas with rapidly changing demographics and incomplete or no enumeration of the underlying population and their residences. To remove the need for pre-enumeration and to simplify field logistics for the population healthcare utilization survey used for the Surveillance for Enteric Fever in Asia Project in Nepal, we incorporated a geographic information system–based geosurvey and field mapping system into a single-stage cluster sampling approach.MethodsA survey was administered to ascertain healthcare-seeking behavior in individuals with recent suspected enteric fever. Catchment areas were based on residential addresses of enteric fever patients using study facilities; clusters were randomly selected from digitally created grids using available satellite images and all households within clusters were offered enrollment. A tablet-compatible geosurvey and mapping system that allowed for data-syncing and use in areas without cellular data was created using the ArcGIS suite of software.ResultsBetween January 2017 and November 2018, we surveyed 25 521 households in Nepal (16 769 in urban Kathmandu and 8752 in periurban Kavrepalanchok), representing 84 202 individuals. Overall, the survey participation rate was 90.9%, with geographic heterogeneity in participation rates within each catchment area. Areas with higher average household wealth had lower participation rates.ConclusionA geographic information system–based geosurvey and field mapping system allowed creation of a virtual household map at the same time as survey administration, enabling a single-stage cluster sampling method to assess healthcare utilization in Nepal for the Surveillance for Enteric Fever in Asia Project . This system removed the need for pre-enumeration of households in sampling areas, simplified logistics and could be replicated in future community surveys.

Background Typhoid fever is endemic in the urban Kathmandu Valley of Nepal; however, there have been no population-based studies of typhoid outside of this community in the past 3 decades. Whether typhoid immunization should be prioritized in periurban and rural communities has been unclear. Methods We performed population-based surveillance for enteric fever in 1 urban catchment (Kathmandu) and 1 periurban and rural catchment (Kavrepalanchok) as part of the Surveillance for Enteric Fever in Asia Project (SEAP). We recruited individuals presenting to outpatient and emergency departments at 2 study hospitals with suspected enteric fever and performed blood cultures. Additionally, we conducted a household survey in each catchment area to characterize care seeking for febrile illness. We evaluated spatial heterogeneity in febrile illness, care seeking, and enteric fever incidence. Results Between September 2016 and September 2019, we enrolled 5736 participants with suspected enteric fever at 2 study hospitals. Among these, 304 (5.3%) were culture positive for Salmonella Typhi (249 [81.9%]) or Paratyphi A (55 [18.1%]). Adjusted typhoid incidence in Kathmandu was 484 per 100 000 person-years and in Kavrepalanchok was 615 per 100 000 person-years. While all geographic areas for which estimates could be made had incidence >200 per 100 000 person-years, we observed spatial heterogeneity with up to 10-fold variation in incidence between communities. Conclusions In urban, periurban, and rural communities in and around Kathmandu, we measured a high but heterogenous incidence of typhoid. These findings provide some support for the introduction of conjugate vaccines in Nepal, including outside urban areas, alongside other measures to prevent enteric fever.

Background Enteric fever, a bacterial infection caused by Salmonella enterica serotypes Typhi and Paratyphi A, frequently presents as a nonlocalizing febrile illness that is difficult to distinguish from other infectious causes of fever. Blood culture is not widely available in endemic settings and, even when available, results can take up to 5 days. We evaluated the diagnostic performance of clinical features, including both reported symptoms and clinical signs, of enteric fever among patients participating in the Surveillance for Enteric Fever in Asia Project (SEAP), a 3-year surveillance study in Bangladesh, Nepal, and Pakistan. Methods Outpatients presenting with ≥3 consecutive days of reported fever and inpatients with clinically suspected enteric fever from all 6 SEAP study hospitals were eligible to participate. We evaluated the diagnostic performance of select clinical features against blood culture results among outpatients using mixed-effect regression models with a random effect for study site hospital. We also compared the clinical features of S. Typhi to S. Paratyphi A among both outpatients and inpatients. Results We enrolled 20 899 outpatients, of whom 2116 (10.1%) had positive blood cultures for S. Typhi and 297 (1.4%) had positive cultures for S. Paratyphi A. The sensitivity of absence of cough was the highest among all evaluated features, at 65.5% (95% confidence interval [CI], 55.0–74.7), followed by measured fever at presentation at 59.0% (95% CI, 51.6–65.9) and being unable to complete normal activities for 3 or more days at 51.0% (95% CI, 23.8–77.6). A combined case definition of 3 or more consecutive days of reported fever and 1 or more of the following (a) either the absence of cough, (b) fever at presentation, or (c) 3 or more consecutive days of being unable to conduct usual activity--yielded a sensitivity of 94.6% (95% CI, 93.4–95.5) and specificity of 13.6% (95% CI, 9.8–17.5). Conclusions Clinical features do not accurately distinguish blood culture–confirmed enteric fever from other febrile syndromes. Rapid, affordable, and accurate diagnostics are urgently needed, particularly in settings with limited or no blood culture capacity.

BackgroundAntibiotic use prior to seeking care at a hospital may reduce the sensitivity of blood culture for enteric fever, with implications for both clinical care and surveillance. The Surveillance for Enteric Fever in Asia Project (SEAP) is a prospective study of enteric fever incidence in Nepal, Bangladesh, and Pakistan. Nested within SEAP, we evaluated the accuracy of self-reported antibiotic use and investigated the association between antibiotic use and blood culture positivity.MethodsBetween November 2016 and April 2019, we collected urine samples among a subset of SEAP participants to test for antibiotic use prior to the hospital visit using an antibacterial activity assay. All participants were asked about recent antibiotic use and had a blood culture performed. We used mixed-effect logit models to evaluate the effect of antimicrobial use on blood culture positivity, adjusted for markers of disease severity.ResultsWe enrolled 2939 patients with suspected enteric fever. Antibiotics were detected in 39% (1145/2939) of urine samples. The correlation between measured and reported antibiotic use was modest (κ = 0.72). After adjusting for disease severity, patients with antibiotics in their urine were slightly more likely to be blood culture positive for enteric fever; however, the effect was not statistically significant (prevalence ratio, 1.22 [95% confidence interval, .99–1.50]).ConclusionsThe reliability of self-reported prior antibiotic use was modest among individuals presenting with fever to tertiary hospitals. While antibiotics are likely to reduce the sensitivity of blood culture, our findings indicate that there is still considerable value in performing blood culture for individuals reporting antibiotic use.

Building on previous multicountry surveillance studies of typhoid and others salmonelloses such as the Diseases of the Most Impoverished program and the Typhoid Surveillance in Africa Project, several ongoing blood culture surveillance studies are generating important data about incidence, severity, transmission, and clinical features of invasive Salmonella infections in sub-Saharan Africa and South Asia. These studies are also characterizing drug resistance patterns in their respective study sites. Each study answers a different set of research questions and employs slightly different methodologies, and the geographies under surveillance differ in size, population density, physician practices, access to healthcare facilities, and access to microbiologically safe water and improved sanitation. These differences in part reflect the heterogeneity of the epidemiology of invasive salmonellosis globally, and thus enable generation of data that are useful to policymakers in decision-making for the introduction of typhoid conjugate vaccines (TCVs). Moreover, each study is evaluating the large-scale deployment of TCVs, and may ultimately be used to assess post-introduction vaccine impact. The data generated by these studies will also be used to refine global disease burden estimates. It is important to ensure that lessons learned from these studies not only inform vaccination policy, but also are incorporated into sustainable, low-cost, integrated vaccine-preventable disease surveillance systems. ispartof: CLINICAL INFECTIOUS DISEASES vol:71 issue:Suppl 2 pages:S102-S110 ispartof: location:United States status: published

Pit latrines are promoted in resource-limited settings, but unpleasant odours may deter their use. In this study, latrines in rural Ethiopia were randomized to the addition of cooking ash, the addition of boiling water or neither. Study staff ranked odour on a 6-point scale before and approximately 24 h after intervention. Following intervention, odour grades were on average 0.2 points lower (95% confidence interval [CI] 0.7 lower to 0.3 higher) in ash-treated latrines and 0.4 points lower (95% CI 0.9 lower to 0.1 higher) in boiled water–treated latrines, although the difference between the three groups was not statistically significant (p = 0.21). Larger studies might detect a smaller difference.

The transmission of trachoma, caused by repeat infections with Chlamydia trachomatis, and many enteropathogens are linked to water quantity. We hypothesized that children living further from a water source would have higher exposure to C. trachomatis and enteric pathogens as determined by antibody responses. We used a multiplex bead assay to measure IgG antibody responses to C. trachomatis, Giardia intestinalis, Cryptosporidium parvum, Entamoeba histolytica, Salmonella enterica, Campylobacter jejuni, enterotoxigenic Escherichia coli (ETEC) and Vibrio cholerae in eluted dried blood spots collected from 2267 children ages 0–9 years in 40 communities in rural Ethiopia in 2016. Linear distance from the child’s house to the nearest water source was calculated. We derived seroprevalence cutoffs using external negative control populations, if available, or by fitting finite mixture models. We used targeted maximum likelihood estimation to estimate differences in seroprevalence according to distance to the nearest water source. Seroprevalence among 1–9-year-olds was 43% for C. trachomatis, 28% for S. enterica, 70% for E. histolytica, 54% for G. intestinalis, 96% for C. jejuni, 76% for ETEC and 94% for C. parvum. Seroprevalence increased with age for all pathogens. Median distance to the nearest water source was 473 meters (IQR 268, 719). Children living furthest from a water source had a 12% (95% CI: 2.6, 21.6) higher seroprevalence of S. enterica and a 12.7% (95% CI: 2.9, 22.6) higher seroprevalence of G. intestinalis compared to children living nearest. Seroprevalence for C. trachomatis and enteropathogens was high, with marked increases for most enteropathogens in the first two years of life. Children living further from a water source had higher seroprevalence of S. enterica and G. intestinalis indicating that improving access to water in the Ethiopia’s Amhara region may reduce exposure to these enteropathogens in young children., Author summary Trachoma, an infection of the eye caused by the bacteria Chlamydia trachomatis, and many diarrhea-causing infections are associated with access to water for washing hands and faces. Measuring these different pathogens in a population is challenging and rarely are multiple infections measured at the same time. Here, we used an integrated approach to simultaneously measure antibody responses to C. trachomatis, Giardia intestinalis, Cryptosporidium parvum, Entamoeba histolytica, Salmonella enterica, Campylobacter jejuni, enterotoxigenic Escherichia coli (ETEC) and Vibrio cholerae among young children residing in rural Ethiopia. We found that the seroprevalence of all pathogens increased with age and that seropositivity to more than one pathogen was common. Children living further from a water source were more likely to be exposed to S. enterica and G. intestinalis. Integrated sero-surveillance is a promising avenue to explore the complexities of multi-pathogen exposure as well as to investigate associations between water, sanitation, and hygiene related exposures and disease transmission.

IntroductionFacial hygiene promotion and environmental improvements are central components of the global trachoma elimination strategy despite a lack of experimental evidence supporting the effectiveness of water, sanitation and hygiene (WASH) measures for reducing trachoma transmission. The objective of the WUHA (WASH Upgrades for Health in Amhara) trial is to evaluate if a comprehensive water improvement and hygiene education programme reduces the prevalence of ocular chlamydia infection in rural Africa.Methods and analysisForty study clusters, each of which had received at least annual mass azithromycin distributions for the 7 years prior to the start of the study, are randomised in a 1:1 ratio to the WASH intervention arm or a delayed WASH arm. The WASH package includes a community water point, community-based hygiene promotion workers, household wash stations, household WASH education books, household soap distribution and a primary school hygiene curriculum. Educational activities emphasise face-washing and latrine use. Mass antibiotic distributions are not provided during the first 3 years but are provided annually over the final 4 years of the trial. Annual monitoring visits are conducted in each community. The primary outcome is PCR evidence of ocular chlamydia infection among children aged 0–5 years, measured in a separate random sample of children annually over 7 years. A secondary outcome is improvement of the clinical signs of trachoma between the baseline and final study visits as assessed by conjunctival photography. Laboratory workers and photo-graders are masked to treatment allocation.Ethics and disseminationStudy protocols have been approved by human subjects review boards at the University of California, San Francisco, Emory University, the Ethiopian Food and Drug Authority, and the Ethiopian Ministry of Innovation and Technology. A data safety and monitoring committee oversees the trial. Results will be disseminated through peer-reviewed publications and presentations.Trial registration number(http://www.clinicaltrials.gov): NCT02754583; Pre-results.

Eukaryotic parasites are significant contributors to childhood illness in Niger. While helminthiases have received national attention through mass deworming efforts, the epidemiology of intestinal protozoa in Niger remains underexamined. This study employed real-time PCR diagnostics to describe the prevalence of two schistosomes, four soil-transmitted helminths, and one protozoan parasite in Boboye Department, Dosso Region. Prevalence was assessed using bulk stool specimens collected from a population-based sample of 86 children residing in 9 communities. Anthropometric measurements were used to calculate child growth z-scores and stool consistency was graded. Helminths were absent from the study population, with the exception of a single Schistosoma haematobium infection (1/86; 1.2%). Giardia duodenalis was the only protozoa present, detected in 65% (56/86) of children. Prevalence of G. duodenalis peaked in 2-year-olds with 88% (15/17) positivity. The population was generally undernourished, though growth indices did not differ significantly between children with and without G. duodenalis infection.

Diarrhea is a leading cause of death among children aged less than five years globally. Most studies of pediatric diarrhea rely on caregiver-reported stool consistency and frequency to define the disease. Research on the validity of caregiver-reported diarrhea is sparse. We collected stool samples from 2,398 children participating in two clinical trials in the Amhara region of Ethiopia. The consistency of each stool sample was graded by the child's caregiver and two trained laboratory technicians according to an illustrated stool consistency scale. We assessed the reliability of graded stool consistency among the technicians, and then compared the caregiver's grade with the technician's grade. We also tested if the illustrated stool consistency scale could improve the validity of caregiver's report. The weighted kappa measuring the agreement between the two laboratory technicians reached 0.90 after 500 stool samples were graded. The sensitivity of caregiver-reported loose or watery stool was 15.5% (95% confidence interval [CI]: 9.7, 24.2) and the specificity was 98.4% (95% CI 97.1, 99.1). With the illustrated scale, the sensitivity was 68.5% (95% CI: 58.5, 77.1) and the specificity was 86.1% (95% CI: 79.3, 90.9). The results indicate that caregiver-reported stool consistency using the terms "loose or watery" does not accurately describe stool consistency as graded by trained laboratory technicians. Given the predominance of using caregiver-reported stool consistency to define diarrheal disease, the low sensitivity identified in this study suggests that the burden of diarrheal disease may be underestimated and intervention effects could be biased. The illustrated scale is a potential low-lost tool to improve the validity of caregiver-reported stool consistency.

Intestinal parasites are important contributors to global morbidity and mortality and are the second most common cause of outpatient morbidity in Ethiopia. This cross-sectional survey describes the prevalence of soil-transmitted helminths and intestinal protozoa in preschool children 0-5 years of age in seven communities in the Amhara region of Ethiopia, and investigates associations between infection, household water and sanitation characteristics, and child growth. Stool samples were collected from children 0-5 years of age, 1 g of sample was preserved in sodium acetate-acetic acid-formalin, and examined for intestinal helminth eggs and protozoa cysts ether-concentration method. A total of 212 samples were collected from 255 randomly selected children. The prevalence of Ascaris lumbricoides, Trichuris trichiura, and hookworm were 10.8% (95% confidence interval [CI] 6.6-15.1), 1.4% (95% CI = 0-3.0), and 0% (95% CI = 0-1.7), respectively. The prevalence of the pathogenic intestinal protozoa Giardia lamblia and Entamoeba histolytica/dispar were 10.4% (95% CI = 6.2-14.6) and 3.3% (95% CI = 0.09-5.7), respectively. Children with A. lumbricoides infections had lower height-for-age z-scores compared with those without, but were not more likely to have stunting. Compared with those without G. lamblia, children with G. lamblia infections had lower weight-for-age and weight-for-height z-scores and were more than five times as likely to meet the z-score definition for wasting (prevalence ratio = 5.42, 95% CI = 2.97-9.89). This article adds to a growing body of research on child growth and intestinal parasitic infections and has implications for their treatment and prevention in preschool-aged children.

Latrines are the most basic form of improved sanitation and are a common public health intervention. Understanding motivations for building and using latrines can help develop effective, sustainable latrine promotion programs. We conducted a mixed-methods study of latrine use in the Amhara region of Ethiopia. We held 15 focus group discussions and surveyed 278 households in five communities. We used the Integrated Behavioral Model for Water, Sanitation, and Hygiene interventions to guide our qualitative analysis. Seventy-one percent of households had a latrine, but coverage varied greatly across communities. Higher household income was not associated with latrine use (odds ratio [OR] = 1.9; 95% confidence interval [CI] = 0.5, 7.7); similarly, cost and availability of materials were not discussed as barriers to latrine use in the focus groups. Male-headed households were more likely to use latrines than households with female heads (OR = 3.5; 95% CI = 1.6, 7.7), and households with children in school were more likely to use latrines than households without children in school (OR = 2.3; 95% CI = 1.6, 3.3). These quantitative findings were confirmed in focus groups, where participants discussed how children relay health messages from school. Participants discussed how women prefer not to use latrines, often finding them strange or even scary. These findings are useful for public health implementation; they imply that community-level drivers are important predictors of household latrine use and that cost is not a significant barrier. These findings confirm that school-aged children may be effective conduits of health messages and suggest that latrines can be better marketed and designed for women.

Background Mass azithromycin distributions have been shown to reduce mortality among pre-school children in sub-Saharan Africa. It is unclear what mediates this mortality reduction, but one possibility is that antibiotics function as growth promoters for young children. Methods and findings 24 rural Ethiopian communities that had received biannual mass azithromycin distributions over the previous four years were enrolled in a parallel-group, cluster-randomized trial. Communities were randomized in a 1:1 ratio to either continuation of biannual oral azithromycin (20mg/kg for children, 1 g for adults) or to no programmatic antibiotics over the 36 months of the study period. All community members 6 months and older were eligible for the intervention. The primary outcome was ocular chlamydia; height and weight were measured as secondary outcomes on children less than 60 months of age at months 12 and 36. Study participants were not masked; anthropometrists were not informed of the treatment allocation. Anthropometric measurements were collected for 282 children aged 0–36 months at the month 12 assessment and 455 children aged 0–59 months at the month 36 assessment, including 207 children who had measurements at both time points. After adjusting for age and sex, children were slightly but not significantly taller in the biannually treated communities (84.0 cm, 95%CI 83.2–84.8, in the azithromycin-treated communities vs. 83.7 cm, 95%CI 82.9–84.5, in the untreated communities; mean difference 0.31 cm, 95%CI -0.85 to 1.47, P = 0.60). No adverse events were reported. Conclusions Periodic mass azithromycin distributions for trachoma did not demonstrate a strong impact on childhood growth. Trial registration The TANA II trial was registered on clinicaltrials.gov #NCT01202331., Author summary Mass distribution of a single dose of the broad-spectrum antibiotic azithromycin twice per year to pre-school children in Sub-Saharan Africa has been shown to reduce childhood mortality. The mechanism by which azithromycin reduces mortality is currently not clear, especially since the antibiotic is not targeted to sick children but rather given to all children in the community whether or not they have an infectious disease. In this study, we report the height and weight of children enrolled in a trial in Ethiopia in which communities were randomized either to twice annual mass azithromycin distributions for blinding trachoma or to no treatments. After accounting for age and sex, children from azithromycin-treated communities were on average slightly taller at the 12- and 36-month study visits than those from untreated communities, but the difference was not statistically significant.

Background Face cleanliness is a core component of the SAFE (Surgery, Antibiotics, Facial cleanliness, and Environmental improvements) strategy for trachoma control. Understanding knowledge, attitudes, and behaviors related to face washing may be helpful for designing effective interventions for improving facial cleanliness. Methods In April 2014, a mixed methods study including focus groups and a quantitative cross-sectional study was conducted in the East Gojjam zone of the Amhara region of Ethiopia. Participants were asked about face washing practices, motivations for face washing, use of soap (which may reduce bacterial load), and fly control strategies. Results Overall, both knowledge and reported practice of face washing was high. Participants reported they knew that washing their own face and their children’s faces daily was important for hygiene and infection control. Although participants reported high knowledge of the importance of soap for face washing, quantitative data revealed strong variations by community in the use of soap for face washing, ranging from 4.4% to 82.2% of households reporting using soap for face washing. Cost and forgetfulness were cited as barriers to the use of soap for face washing. Keeping flies from landing on children was a commonly cited motivator for regular face washing, as was trachoma prevention. Conclusions Interventions aiming to improve facial cleanliness for trachoma prevention should focus on habit formation (to address forgetfulness) and address barriers to the use of soap, such as reducing cost. Interventions that focus solely on improving knowledge may not be effective for changing face-washing behaviors., Author Summary Facial cleanliness is a core component of the SAFE (Surgery, Antibiotics, Facial cleanliness, and Environmental improvements) strategy for trachoma control. We conducted a mixed methods study in a trachoma hyperendemic region of rural Ethiopia to better understand knowledge, attitudes, and behaviors related to face washing. Overall, knowledge of the benefits of face washing was high, and participants reported regularly engaging in face washing practices. However, the use of soap for face washing varied more between communities. Participants cited cost and forgetting to use soap as the primary barriers to using soap for face washing. Trachoma prevention, including keeping flies from landing on children’s faces, was a commonly-cited motivator for face washing discussed in focus groups. Given the near-universal knowledge of the benefits of face washing, interventions focused on changing face washing behavior for trachoma control should focus on habit formation and removal of barriers to the use of soap rather than simply educational interventions.

Background Stool consistency is an important diagnostic criterion in both research and clinical medicine and is often used to define diarrheal disease. Methods We examine the pediatric enteric virome across stool consistency to evaluate differences in richness and community composition using fecal samples collected from children participating in a clinical trial in the Amhara region of Ethiopia. The consistency of each sample was graded according to the modified Bristol Stool Form Scale for children (mBSFS-C) before a portion of stool was preserved for viral metagenomic analysis. Stool samples were grouped into 29 pools according to stool consistency type. Differential abundance was determined using negative-binomial modeling. Results Of 446 censused children who were eligible to participate, 317 presented for the study visit examination and 269 provided stool samples. The mean age of children with stool samples was 2.7 years old. Species richness was highest in watery-consistency stool and decreased as stool consistency became firmer (Spearman’s r = −0.45, P = 0.013).The greatest differential abundance comparing loose or watery to formed stool was for norovirus GII (7.64, 95% CI 5.8, 9.5) followed by aichivirus A (5.93, 95% CI 4.0, 7.89) and adeno-associated virus 2 (5.81, 95% CI 3.9, 7.7). Conclusion We documented a difference in pediatric enteric viromes according to mBSFS-C stool consistency category, both in species richness and composition. Our results suggest that loose or watery stool, as measured by the mBSFS-C, may signal enteric viral infection in young children. Additional studies are warranted to confirm these findings. Disclosures All authors: No reported disclosures.

Background The enteric viruses shed by different populations can be influenced by multiple factors including access to clean drinking water. We describe here the eukaryotic viral genomes in the feces of Ethiopian children participating in a clean water intervention trial. Methodology/principal findings Fecal samples from 269 children with a mean age of 2.7 years were collected from 14 villages in the Amhara region of Ethiopia, half of which received a new hand-dug water well. Feces from these villages were then analyzed in 29 sample pools using viral metagenomics. A total of 127 different viruses belonging to 3 RNA and 3 DNA viral families were detected. Picornaviridae family sequence reads were the most commonly found, originating from 14 enterovirus and 6 parechovirus genotypes plus multiple members of four other picornavirus genera (cosaviruses, saliviruses, kobuviruses, and hepatoviruses). Picornaviruses with nearly identical capsid VP1 were detected in different pools reflecting recent spread of these viral strains. Next in read frequencies and positive pools were sequences from the Caliciviridae family including noroviruses GI and GII and sapoviruses. DNA viruses from multiple genera of the Parvoviridae family were detected (bocaviruses 1-4, bufavirus 3, and dependoparvoviruses), together with four species of adenoviruses and common anelloviruses shedding. RNA in the order Picornavirales and CRESS-DNA viral genomes, possibly originating from intestinal parasites or dietary sources, were also characterized. No significant difference was observed between the number of mammalian viruses shed from children from villages with and without a new water well. Conclusions We describe an approach to estimate the efficacy of potentially virus transmission-reducing interventions and the first complete (DNA and RNA viruses) description of the enteric viromes of East African children. A wide diversity of human enteric viruses was found in both intervention and control groups. Mammalian enteric virome diversity was not reduced in children from villages with a new water well. This population-based sampling also provides a baseline of the enteric viruses present in Northern Ethiopia against which to compare future viromes.

The disposal of human excreta in latrines is an important step in reducing the transmission of diarrhoeal diseases. However, in latrines, flies can access the latrine contents and serve as a mechanical transmitter of diarrhoeal pathogens. Furthermore, the latrine contents can be used as a breeding site for flies, which may further contribute to disease transmission. Latrines do not all produce flies, and there are some which produce only a few, while others can produce thousands. In order to understand the role of the latrine in determining this productivity, a pilot study was conducted, in which fifty latrines were observed in and around Ifakara, Tanzania. The characteristics of the latrine superstructure, use of the latrine, and chemical characteristics of pit latrine contents were compared to the numbers of flies collected in an exit trap placed over the drop hole in the latrine. Absence of a roof was found to have a significant positive association (t=3.17, p=0.003) with the total number of flies collected, and temporary superstructures, particularly as opposed to brick superstructures (z=4.26, p