Abstract
Understanding the global burden of enterotoxigenic E. coli (ETEC) and Shigella diarrhea as well as estimating the cost effectiveness of vaccines to control these two significant pathogens have been hindered by the lack of a diagnostic test that is rapid, simple, sensitive, and can be applied to the endemic countries. We previously developed a simple and rapid assay, Rapid Loop mediated isothermal amplification based Diagnostic Test (RLDT) for the detection of ETEC and Shigella spp. (Shigella). In this study, the RLDT assay was evaluated in comparison with quantitative PCR (qPCR), culture and conventional PCR for the detection of ETEC and Shigella. This validation was performed using previously collected stool samples from endemic countries, from the travelers to the endemic countries, as well as samples from a controlled human infection model study of ETEC. The performance of RLDT from dried stool spots was also validated. RLDT resulted in excellent sensitivity and specificity compared to qPCR (99% and 99.2% respectively) ranging from 92.3 to 100% for the individual toxin genes of ETEC and 100% for Shigella. Culture was less sensitive compared to RLDT. No significant differences were noted in the performance of RLDT using samples from various sources or stool samples from moderate to severe diarrhea or asymptomatic infections. RLDT performed equally well in detection of ETEC and Shigella from the dried stool samples on filter papers. This study established that RLDT is sufficiently sensitive and specific to be used as a simple and rapid diagnostic assay to detect ETEC and Shigella in endemic countries to determine disease burden of these pathogens in the national and subnational levels. This information will be important to guide public health and policy makers to prioritize resources for accelerating the development and introduction of effective preventative and/or treatment interventions against these enteric infections.
Highlights
Enterotoxigenic Escherichia coli (ETEC) and Shigella spp. (Shigella) remain among the most common bacterial causes of diarrhea-associated morbidity and mortality in the children living in the low-and middle-income countries (LMIC) [1,2] as well as in the travelers and military personnel from the high-income countries [3].In spite of the high impact of these enteric pathogens on child health [4,5,6] a simple, sensitive and specific diagnostic test for ETEC and Shigella is lacking
We developed a simple and rapid diagnostic assay called the RLDT (Rapid Loop-mediated isothermal amplification based Diagnostic Test) for detection of ETEC and Shigella
Our data showed that the RLDT assay exhibited high sensitivity and specificity for detection of ETEC and Shigella, with its result available within 50 minutes
Summary
Enterotoxigenic Escherichia coli (ETEC) and Shigella spp. (Shigella) remain among the most common bacterial causes of diarrhea-associated morbidity and mortality in the children living in the low-and middle-income countries (LMIC) [1,2] as well as in the travelers and military personnel from the high-income countries [3].In spite of the high impact of these enteric pathogens on child health [4,5,6] a simple, sensitive and specific diagnostic test for ETEC and Shigella is lacking. As promising vaccine candidates for ETEC and Shigella move toward field trials in endemic areas, an improved understanding of the epidemiology of both pathogens and identification of high transmission hotspots of these diseases will be critical to plan Phase III trials to assess the potential benefits of vaccine use [8] In this regard, a sensitive, simple and rapid diagnostic method which can be applied in the resource poor settings, where these diseases are endemic is essential. One may draw an analogy with rotavirus where a simple diagnostic test allowed many countries to assess their disease burden and determine the effectiveness of vaccine introduction making it possible to accelerate introduction of the vaccine in the critically required countries in LMICs [10,11] Such a simple, rapid and as well as sensitive diagnostic assay which can be applied in resource poor endemic settings with limited laboratory infrastructure is currently lacking for ETEC and Shigella. Rapid and as well as sensitive diagnostic assay which can be applied in resource poor endemic settings with limited laboratory infrastructure is currently lacking for ETEC and Shigella. [1,7,12]
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