Population Enumeration and Household Utilization Survey Methods in the Enterics for Global Health (EFGH): Shigella Surveillance Study

Background: Accurate estimation of diarrhea incidence from facility-based surveillance requires estimating the population at risk and accounting for case patients who do not seek care. The Enterics for Global Health (EFGH) Shigella surveillance study will characterize population denominators and healthcare-seeking behavior proportions to calculate incidence rates of Shigella diarrhea in children aged 6–35 months across 7 sites in Africa, Asia, and Latin America. Methods: The Enterics for Global Health (EFGH) Shigella surveillance study will use a hybrid surveillance design, supplementing facility-based surveillance with population-based surveys to estimate population size and the proportion of children with diarrhea brought for care at EFGH health facilities. Continuous data collection over a 24 month period captures seasonality and ensures representative sampling of the population at risk during the period of facility-based enrollments. Study catchment areas are broken into randomized clusters, each sized to be feasibly enumerated by individual field teams. Conclusions: The methods presented herein aim to minimize the challenges associated with hybrid surveillance, such as poor parity between survey area coverage and facility coverage, population fluctuations, seasonal variability, and adjustments to care-seeking behavior

"Candidatus Campylobacter infans" detection is not associated with diarrhea in children under the age of 2 in Peru.

A working hypothesis is that less common species of Campylobacter (other than C. jejuni and C. coli) play a role in enteric disease among children in low resource settings and explain the gap between the detection of Campylobacter using culture and culture independent methods. "Candidatus Campylobacter infans" (C. infans), was recently detected in stool samples from children and hypothesized to play a role in Campylobacter epidemiology in low- and middle-income countries (LMIC). This study determined the prevalence of C. infans in symptomatic and asymptomatic stool samples from children living in Iquitos, Peru. Stool samples from 215 children with diarrhea and 50 stool samples from children without diarrhea under the age of two were evaluated using a multiplex qPCR assay to detect Campylobacter spp. (16S rRNA), Campylobacter jejuni / Campylobacter coli (cadF gene), C. infans (lpxA), and Shigella spp. (ipaH). C. infans was detected in 7.9% (17/215) symptomatic samples and 4.0% (2/50) asymptomatic samples. The association between diarrhea and the presence of these targets was evaluated using univariate logistic regressions. C. infans was not associated with diarrhea. Fifty-one percent (75/146) of Campylobacter positive fecal samples were negative for C. jejuni, C. coli, and C. infans via qPCR. Shotgun metagenomics confirmed the presence of C. infans among 13 out of 14 positive C. infans positive stool samples. C infans explained only 20.7% of the diagnostic gap in stools from children with diarrhea and 16.7% of the gap in children without diarrhea. We posit that poor cadF primer performance better explains the observed gap than the prevalence of atypical non-C. jejuni/coli species

Shigella Detection and Molecular Serotyping With a Customized TaqMan Array Card in the Enterics for Global Health (EFGH): Shigella Surveillance Study.

BACKGROUND: Quantitative polymerase chain reaction (qPCR) targeting ipaH has been proven to be highly efficient in detecting Shigella in clinical samples compared to culture-based methods, which underestimate Shigella burden by 2- to 3-fold. qPCR assays have also been developed for Shigella speciation and serotyping, which is critical for both vaccine development and evaluation. METHODS: The Enterics for Global Health (EFGH) Shigella surveillance study will utilize a customized real-time PCR-based TaqMan Array Card (TAC) interrogating 82 targets, for the detection and differentiation of Shigella spp, Shigella sonnei, Shigella flexneri serotypes, other diarrhea-associated enteropathogens, and antimicrobial resistance (AMR) genes. Total nucleic acid will be extracted from rectal swabs or stool samples, and assayed on TAC. Quantitative analysis will be performed to determine the likely attribution of Shigella and other particular etiologies of diarrhea using the quantification cycle cutoffs derived from previous studies. The qPCR results will be compared to conventional culture, serotyping, and phenotypic susceptibility approaches in EFGH. CONCLUSIONS: TAC enables simultaneous detection of diarrheal etiologies, the principal pathogen subtypes, and AMR genes. The high sensitivity of the assay enables more accurate estimation of Shigella-attributed disease burden, which is critical to informing policy and in the design of future clinical trials

Microbiological Methods Used in the Enterics for Global Health <i>Shigella</i> Surveillance Study.

BackgroundShigella is a major cause of diarrhea in young children worldwide. Multiple vaccines targeting Shigella are in development, and phase 3 clinical trials are imminent to determine efficacy against shigellosis.MethodsThe Enterics for Global Health (EFGH) Shigella surveillance study is designed to determine the incidence of medically attended shigellosis in 6- to 35-month-old children in 7 resource-limited settings. Here, we describe the microbiological methods used to isolate and identify Shigella. We developed a standardized laboratory protocol for isolation and identification of Shigella by culture. This protocol was implemented across all 7 sites, ensuring consistency and comparability of results. Secondary objectives of the study are to determine the antibiotic resistance profiles of Shigella, compare isolation of Shigella from rectal swabs versus whole stool, and compare isolation of Shigella following transport of rectal swabs in Cary-Blair versus a modified buffered glycerol saline transport medium.ConclusionsData generated from EFGH using culture methods described herein can potentially be used for microbiological endpoints in future phase 3 clinical trials to evaluate vaccines against shigellosis and for other clinical and public health studies focused on these organisms

<i>Shigella</i> Detection and Molecular Serotyping With a Customized TaqMan Array Card in the Enterics for Global Health (EFGH): <i>Shigella</i> Surveillance Study.

BackgroundQuantitative polymerase chain reaction (qPCR) targeting ipaH has been proven to be highly efficient in detecting Shigella in clinical samples compared to culture-based methods, which underestimate Shigella burden by 2- to 3-fold. qPCR assays have also been developed for Shigella speciation and serotyping, which is critical for both vaccine development and evaluation.MethodsThe Enterics for Global Health (EFGH) Shigella surveillance study will utilize a customized real-time PCR-based TaqMan Array Card (TAC) interrogating 82 targets, for the detection and differentiation of Shigella spp, Shigella sonnei, Shigella flexneri serotypes, other diarrhea-associated enteropathogens, and antimicrobial resistance (AMR) genes. Total nucleic acid will be extracted from rectal swabs or stool samples, and assayed on TAC. Quantitative analysis will be performed to determine the likely attribution of Shigella and other particular etiologies of diarrhea using the quantification cycle cutoffs derived from previous studies. The qPCR results will be compared to conventional culture, serotyping, and phenotypic susceptibility approaches in EFGH.ConclusionsTAC enables simultaneous detection of diarrheal etiologies, the principal pathogen subtypes, and AMR genes. The high sensitivity of the assay enables more accurate estimation of Shigella-attributed disease burden, which is critical to informing policy and in the design of future clinical trials

Microbiological Methods Used in the Enterics for Global Health <i>Shigella</i> Surveillance Study

Funder: UKRI; DOI: https://doi.org/10.13039/100014013Abstract Background Shigella is a major cause of diarrhea in young children worldwide. Multiple vaccines targeting Shigella are in development, and phase 3 clinical trials are imminent to determine efficacy against shigellosis. Methods The Enterics for Global Health (EFGH) Shigella surveillance study is designed to determine the incidence of medically attended shigellosis in 6- to 35-month-old children in 7 resource-limited settings. Here, we describe the microbiological methods used to isolate and identify Shigella. We developed a standardized laboratory protocol for isolation and identification of Shigella by culture. This protocol was implemented across all 7 sites, ensuring consistency and comparability of results. Secondary objectives of the study are to determine the antibiotic resistance profiles of Shigella, compare isolation of Shigella from rectal swabs versus whole stool, and compare isolation of Shigella following transport of rectal swabs in Cary-Blair versus a modified buffered glycerol saline transport medium. Conclusions Data generated from EFGH using culture methods described herein can potentially be used for microbiological endpoints in future phase 3 clinical trials to evaluate vaccines against shigellosis and for other clinical and public health studies focused on these organisms. </jats:sec

Microbiological Methods Used in the Enterics for Global Health Shigella Surveillance Study.

Funder: UKRI; DOI: https://doi.org/10.13039/100014013BACKGROUND: Shigella is a major cause of diarrhea in young children worldwide. Multiple vaccines targeting Shigella are in development, and phase 3 clinical trials are imminent to determine efficacy against shigellosis. METHODS: The Enterics for Global Health (EFGH) Shigella surveillance study is designed to determine the incidence of medically attended shigellosis in 6- to 35-month-old children in 7 resource-limited settings. Here, we describe the microbiological methods used to isolate and identify Shigella. We developed a standardized laboratory protocol for isolation and identification of Shigella by culture. This protocol was implemented across all 7 sites, ensuring consistency and comparability of results. Secondary objectives of the study are to determine the antibiotic resistance profiles of Shigella, compare isolation of Shigella from rectal swabs versus whole stool, and compare isolation of Shigella following transport of rectal swabs in Cary-Blair versus a modified buffered glycerol saline transport medium. CONCLUSIONS: Data generated from EFGH using culture methods described herein can potentially be used for microbiological endpoints in future phase 3 clinical trials to evaluate vaccines against shigellosis and for other clinical and public health studies focused on these organisms

Diarrhea Case Surveillance in the Enterics for Global Health <i>Shigella</i> Surveillance Study: Epidemiologic Methods.

BackgroundShigella is a leading cause of acute watery diarrhea, dysentery, and diarrhea-attributed linear growth faltering, a precursor to stunting and lifelong morbidity. Several promising Shigella vaccines are in development and field efficacy trials will require a consortium of potential vaccine trial sites with up-to-date Shigella diarrhea incidence data.MethodsThe Enterics for Global Health (EFGH) Shigella surveillance study will employ facility-based enrollment of diarrhea cases aged 6-35 months with 3 months of follow-up to establish incidence rates and document clinical, anthropometric, and financial consequences of Shigella diarrhea at 7 country sites (Mali, Kenya, The Gambia, Malawi, Bangladesh, Pakistan, and Peru). Over a 24-month period between 2022 and 2024, the EFGH study aims to enroll 9800 children (1400 per country site) between 6 and 35 months of age who present to local health facilities with diarrhea. Shigella species (spp.) will be identified and serotyped from rectal swabs by conventional microbiologic methods and quantitative polymerase chain reaction. Shigella spp. isolates will undergo serotyping and antimicrobial susceptibility testing. Incorporating population and healthcare utilization estimates from contemporaneous household sampling in the catchment areas of enrollment facilities, we will estimate Shigella diarrhea incidence rates.ConclusionsThis multicountry surveillance network will provide key incidence data needed to design Shigella vaccine trials and strengthen readiness for potential trial implementation. Data collected in EFGH will inform policy makers about the relative importance of this vaccine-preventable disease, accelerating the time to vaccine availability and uptake among children in high-burden settings