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

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.

A longitudinal study of the association between domestic contact with livestock and contamination of household point-of-use stored drinking water in rural Siaya County (Kenya).

BACKGROUND: Emerging evidence suggests close domestic proximity of livestock and humans may lead to microbiological contamination of hands, objects, food and water supplies within domestic environments, adversely impacting public health. However, evidence quantifying the relationship between livestock, domestic animals, humans and microbiological contamination of household stored water remains limited. AIM: This longitudinal study aimed to examine the relationship between domestic contact with livestock and domestic animals on microbiological contamination of household Point-of-Use (POU) stored drinking water in rural Kenya and assess the influence of choice of faecal indicator on such associations. METHODOLOGY: A survey was performed in 234 households in Siaya county, Kenya, to observe presence of livestock (cattle, goats, poultry) and domestic animals (cats, dogs) in household compounds, alongside other risk factors for contamination of POU stored drinking water such as sanitation, storage conditions and hygiene practices. Samples from water sources (e.g. piped, spring/wells, boreholes, surface and rainwater) and from POU storage containers were tested for E. coli and intestinal enterococci. Livestock-related risk factors for water contamination were examined through multinomial regression, controlling for confounders. RESULTS: Rainwater was the main POU water source and was found to be highly susceptible to contamination. Multivariate analysis showed greater risk of gross (>100 CFU/100 mL) water contamination (with E. coli) for households where goats were observed, and/or where poultry roosted in proximity to stored household water (relative risk RR = 2.71; p = 0.001 and RR = 2.02; p = 0.012 respectively). Presence of a poultry coop was also associated with elevated intestinal enterococci densities (RR = 4.46; p = 0.001). Associations between contamination and livestock risk factors were thus similar for both bacteria groups, but E. coli counts declined more rapidly following collection from surface waters than enterococci counts (p = 0.024). CONCLUSION: The presence of livestock (particularly goats) and poultry within household compounds increases POU water contamination risk, suggesting the need for improved interventions to address cross-contamination within rural domestic settings. Within Siaya county, more effective community education is needed to raise awareness of POU water quality protection, particularly of rainwater.

Portable Rabies Virus Sequencing in Canine Rabies Endemic Countries Using the Oxford Nanopore MinION.

As countries with endemic canine rabies progress towards elimination by 2030, it will become necessary to employ techniques to help plan, monitor, and confirm canine rabies elimination. Sequencing can provide critical information to inform control and vaccination strategies by identifying genetically distinct virus variants that may have different host reservoir species or geographic distributions. However, many rabies testing laboratories lack the resources or expertise for sequencing, especially in remote or rural areas where human rabies deaths are highest. We developed a low-cost, high throughput rabies virus sequencing method using the Oxford Nanopore MinION portable sequencer. A total of 259 sequences were generated from diverse rabies virus isolates in public health laboratories lacking rabies virus sequencing capacity in Guatemala, India, Kenya, and Vietnam. Phylogenetic analysis provided valuable insight into rabies virus diversity and distribution in these countries and identified a new rabies virus lineage in Kenya, the first published canine rabies virus sequence from Guatemala, evidence of rabies spread across an international border in Vietnam, and importation of a rabid dog into a state working to become rabies-free in India. Taken together, our evaluation highlights the MinION's potential for low-cost, high volume sequencing of pathogens in locations with limited resources.