Epidemiology of enterotoxigenic Escherichia coli and impact on the growth of children in the first two years of life in Lima, Peru.

Background: Enterotoxigenic E. coli (ETEC) is a leading cause of diarrheal morbidity and mortality in children, although the data on disease burden, epidemiology, and impact on health at the community level are limited. Methods: In a longitudinal birth cohort study of 345 children followed until 24 months of age in Lima, Peru, we measured ETEC burden in diarrheal and non-diarrheal samples using quantitative PCR (LT, STh, and STp toxin genes), studied epidemiology and measured anthropometry in children. Results: About 70% of children suffered from one or more ETEC diarrhea episodes. Overall, the ETEC incidence rate (IR) was 73 per 100 child-years. ETEC infections began early after birth causing 10% (8.9-11.1) ETEC-attributable diarrheal burden at the population level (PAF) in neonates and most of the infections (58%) were attributed to ST-ETEC [PAF 7.9% (1.9-13.5)] and LT + ST-ETEC (29%) of which all the episodes were associated with diarrhea. ETEC infections increased with age, peaking at 17% PAF (4.6-27.7%; p = 0.026) at 21 to 24 months. ST-ETEC was the most prevalent type (IR 32.1) with frequent serial infections in a child. The common colonization factors in ETEC diarrhea cases were CFA/I, CS12, CS21, CS3, and CS6, while in asymptomatic ETEC cases were CS12, CS6 and CS21. Only few (5.7%) children had repeated infections with the same combination of ETEC toxin(s) and CFs, suggested genotype-specific immunity from each infection. For an average ETEC diarrhea episode of 5 days, reductions of 0.060 weight-for-length z-score (0.007 to 0.114; p = 0.027) and 0.061 weight-for-age z-score (0.015 to 0.108; p = 0.009) were noted in the following 30 days. Conclusion: This study showed that ETEC is a significant pathogen in Peruvian children who experience serial infections with multiple age-specific pathotypes, resulting in transitory growth impairment.

Development of a simple, rapid, and sensitive molecular diagnostic assay for cholera.

Cholera continues to inflict high rates of morbidity and mortality. Prompt identification of cholera cases facilitates rapid outbreak responses in the short term while providing reliable surveillance data to guide long-term policies and interventions. Microbiological stool culture, the current recognized gold standard for diagnosing cholera, has significant limitations. Rapid diagnostic tests (RDTs) represent promising alternatives for diagnosing cholera in areas with limited laboratory infrastructure. However, studies conducted with the current cholera RDTs demonstrated wide variations in sensitivity and specificity. To address this gap in the diagnosis of cholera, we developed a simple, rapid, and sensitive diagnostic assay, "Rapid LAMP based Diagnostic Test (RLDT)." With a novel, simple sample preparation method directly from the fecal samples along with lyophilized reaction strips and using established Loop-mediated Isothermal Amplification (LAMP) platform, cholera toxin gene (ctxA) and O1 (O1rfb) gene could be detected in less than an hour. Cholera RLDT assay is cold chain and electricity-free. To avoid any end-user bias, a battery-operated, handheld reader was used to read the RLDT results. The performance specifications of the cholera RLDT assay, including analytical sensitivity and specificity, were evaluated using direct fecal samples, dried fecal samples on filter paper, and environmental water samples spiked with cholera strain. The limit of detection (LOD) was ~104 CFU/gm of stool for both ctxA and O1 genes, corresponding to about 1 CFU of Vibrio cholerae per reaction within 40 minutes. The LOD was 10 bacteria per ml of environmental water when tested with RLDT directly, without enrichment. Being simple, RLDT has the potential to be applied in resource-poor endemic settings for rapid, sensitive, and reliable diagnosis of cholera.

Evaluation of a simple, rapid and field-adapted diagnostic assay for enterotoxigenic E. coli and Shigella.

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.

Development of a simple, rapid, and sensitive diagnostic assay for enterotoxigenic E. coli and Shigella spp applicable to endemic countries.

Enterotoxigenic E. coli (ETEC) and Shigella spp (Shigella) are complex pathogens. The diagnostic assays currently used to detect these pathogens are elaborate or complicated, which make them difficult to apply in resource poor settings where these diseases are endemic. The culture methods used to detect Shigella are not sensitive, and the methods used to detect ETEC are only available in a few research labs. To address this gap, we developed a rapid and simple diagnostic assay-"Rapid LAMP based Diagnostic Test (RLDT)." The six minutes sample preparation method directly from the fecal samples with lyophilized reaction strips and using established Loop-mediated Isothermal Amplification (LAMP) platform, ETEC [heat labile toxin (LT) and heat stable toxins (STh, and STp) genes] and Shigella (ipaH gene) detection was made simple, rapid (<50 minutes), and inexpensive. This assay is cold chain and electricity free. Moreover, RLDT requires minimal equipment. To avoid any end user's bias, a battery-operated, handheld reader was used to read the RLDT results. The results can be read as positive/negative or as real time amplification depending on the end user's need. The performance specifications of the RLDT assay, including analytical sensitivity and specificity, were evaluated using fecal samples spiked with ETEC and Shigella strains. The limit of detection was ~105 CFU/gm of stool for LT, STh, and STp and ~104 CFU/gm of stool for the ipaH gene, which corresponds to about 23 CFU and 1 CFU respectively for ETEC and Shigella per 25uL reaction within 40 minutes. The RLDT assay from stool collection to result is simple, and rapid and at the same time sufficiently sensitive. RLDT has the potential to be applied in resource poor endemic settings for the rapid diagnosis of ETEC and Shigella.