Amanda K. DEBES1*, Etienne GUENOU2, Jerome ATEUDJIEU2,3, Paul SCALZO1, Allison SHAFFER1, Malathi RAM1, Jie LIU4, David A SACK1.
1John Hopkins Bloomberg School of Public Health, 615 N. Wolfe Street, E2132, Baltimore, Maryland 21205, United States of America ; 2Meilleur Accès aux Soins de Santé (M.A. SANTE), Yaoundé, Cameroon; Clinical Research Unit, Division of Health Operations Research ; 3Ministère de la Santé, N°8, Rue 3038 quartier du Lac (Yaoundé III), Cameroon ; 4University of Virginia, Charlottesville, Virginia 22908, United States of America
CaHReF 2018, Yaoundé Congres hall, 08 – 11 January 2019 , AUU0120
Background: Diarrhea is one of the leading causes of death in children under 5; in 2012 diarrheal diseases claimed more than 1600 lives under age 5 per day and accounted for 9% of all under 5 deaths, accounting for nearly 600,000 deaths every year (UNICEF). Recent studies, including The Interactions of Malnutrition & Enteric Infections: Consequences for Child Health and Development (MAL-ED) and the Global Enteric Multi-center Study (GEMS) have been conducted to improved diarrheal disease surveillance, among other objectives. GEMS identified four pathogens which caused the majority of moderate-to-severe (MSD) diarrhea in children: Rotavirus, Cryptosporidium, Shigella and Enterotoxigenic E. coli (ETEC) (Kotloff 2013). Molecular methods have been developed for the rapid detection of enteric pathogens, however, the costs associated with establishing molecular capacities, cold chain storage and transport and shipment of specimens in developing countries limit applicability.
Since 2013, we have been conducting hospital-based cholera surveillance in Cameroon where we successfully demonstrated the use of simplified laboratory and surveillance methods to rapidly detect cholera (Debes 2016). We also found extremely high numbers of MSD that was not cholera in origin. This sub-study was nested into a diarrheal surveillance study in Cameroon from 2015-2017, which was not included in the recent multi-center studies. Any child that presented to our surveillance sites reporting >3 loose stools in the previous 24-hours with sudden onset was eligible for enrollment in our nested sub-study.
Hypotheses and Specific Aims: Our hypothesis was that the use of dried filter paper specimen preservation will provide a low-cost, simplified and timely methodology to identify diarrheal diseases causing significant morbidity among children in resource constrained countries. The specific aims were: 1) to evaluate/adapt a simplified specimen preservation methodology to provide a low cost tool to improve detection of diarrheal pathogens of increasing importance in vulnerable populations; 2) To apply this new tool in endemic settings to determine the prevalence of these pathogens among pediatric diarrheal patients presenting to treatment centers; 3) To use quantitative real time PCR to determine the quantity of the bacterial DNA present and how it correlates to clinical severity of illness.
Design: This was an exploratory study for characterization of diarrheal diseases in children presenting with MSD. We defined this as having 3 or more loose stools within 24hours with the presence of dehydration (determined based on established WHO criteria) and/or blood. We recruited at least 62 children in each age group (0-2, 3-5, 5-17) and preserved stool specimens on filter paper. Epidemiological information pertinent to severity of disease was collected at enrollment. We validated the use of filter paper for the preservation of fecal specimens to enable detection of multiple enteric pathogens using spiked stool. We then extracted the stool from patient specimens using the newly evaluated methods. We tested the DNA using currently published as well as recently designed molecular assays for the detection of broad panel of enteric pathogens, particularly those of heightened importance highlighted in recent research, such as Shigella and ETEC. We evaluated current molecular platforms (e.g. Taqman Array Cards) for use with our crude filter paper extracts. We compared the detection of bacterial pathogen to severity of diarrhea, age and sex, among other potential variables of interest.
Potential Impact: The ability to use dried filter paper fecal samples will allow the identification and quantification of the enteric pathogens from children living in resource poor countries. This study will facilitate increased ability to conduct enteric surveillance studies and an overall understanding of disease burden, which is needed to guide vaccine development and intervention guidelines. In addition, this work may yield a more practical and fieldable approach to diarrheal disease diagnosis. Additionally, this methodology eliminates the need for costly storage and maintenance of freezer stocks, while addressing any concerns regarding the potential for wild-type polio in frozen stool specimens.