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1. Multiplex real time PCR panels to identify fourteen colonization factors of enterotoxigenic Escherichia coli (ETEC).

Author

Jie Liu, Sasikorn Silapong, Pimmada Jeanwattanalert, Paphavee Lertsehtakarn, Ladaporn Bodhidatta, Brett Swierczewski, Carl Mason, Annette L McVeigh, Stephen J Savarino, Rosemary Nshama, Esto Mduma, Athanasia Maro, Jixian Zhang, Jean Gratz, and Eric R Houpt

Subjects
Medicine, Science
Abstract

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of childhood diarrhea in low income countries and in travelers to those areas. Inactivated enterotoxins and colonization factors (CFs) are leading vaccine candidates, therefore it is important to determine the prevailing CF types in different geographic locations and populations. Here we developed real time PCR (qPCR) assays for 14 colonization factors, including the common vaccine targets. These assays, along with three enterotoxin targets (STh, STp, and LT) were formulated into three 5-plex qPCR panels, and validated on 120 ETEC isolates and 74 E. coli colony pools. The overall sensitivity and specificity was 99% (199/202) and 99% (2497/2514), respectively, compared to the CF results obtained with conventional PCR. Amplicon sequencing of discrepant samples revealed that the qPCR was 100% accurate. qPCR panels were also performed on nucleic acid extracted from stool and compared to the results of the ETEC isolates or E. coli colony pools cultured from them. 95% (105/110) of the CF detections in the cultures were confirmed in the stool. Additionally, direct testing of stool yielded 30 more CF detections. Among 74 randomly selected E. coli colony pools with paired stool, at least one CF was detected in 63% (32/51) of the colony pools while at least one CF was detected in 78% (47/60) of the stool samples (P = NS). We conclude that these ETEC CF assays can be used on both cultures and stool samples to facilitate better understanding of CF distribution for ETEC epidemiology and vaccine development.

Published
2017
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2. Multiplex real time PCR panels to identify fourteen colonization factors of enterotoxigenic Escherichia coli (ETEC)

Author

Brett E. Swierczewski, Sasikorn Silapong, Pimmada Jeanwattanalert, Stephen J. Savarino, Annette L. McVeigh, Rosemary Nshama, Paphavee Lertsehtakarn, Athanasia Maro, Carl J. Mason, Jixian Zhang, Eric R. Houpt, Ladaporn Bodhidatta, Esto Mduma, Jie Liu, and Jean Gratz

Subjects
0301 basic medicine, Cystic Fibrosis, Pulmonology, lcsh:Medicine, Artificial Gene Amplification and Extension, Enterotoxin, medicine.disease_cause, Toxicology, Pathology and Laboratory Medicine, Polymerase Chain Reaction, Enterotoxins, Feces, 0302 clinical medicine, Limit of Detection, Enterotoxigenic Escherichia coli, Medicine and Health Sciences, Toxins, Multiplex, Colonization, Public and Occupational Health, 030212 general & internal medicine, lcsh:Science, Vaccines, Multidisciplinary, Gene Pool, Vaccination and Immunization, Real-time polymerase chain reaction, Infectious Diseases, Genetic Diseases, Amplicon sequencing, Research Article, Diarrhea, Validation study, Infectious Disease Control, 030106 microbiology, Toxic Agents, Bacterial Toxins, Immunology, Gastroenterology and Hepatology, Biology, Research and Analysis Methods, Microbiology, 03 medical and health sciences, Signs and Symptoms, Autosomal Recessive Diseases, Diagnostic Medicine, Vaccine Development, medicine, Genetics, Humans, Molecular Biology Techniques, Molecular Biology, Clinical Genetics, Evolutionary Biology, Childhood diarrhea, Population Biology, lcsh:R, Biology and Life Sciences, Fibrosis, lcsh:Q, Preventive Medicine, Multiplex Polymerase Chain Reaction, Population Genetics, Developmental Biology
Abstract

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of childhood diarrhea in low income countries and in travelers to those areas. Inactivated enterotoxins and colonization factors (CFs) are leading vaccine candidates, therefore it is important to determine the prevailing CF types in different geographic locations and populations. Here we developed real time PCR (qPCR) assays for 14 colonization factors, including the common vaccine targets. These assays, along with three enterotoxin targets (STh, STp, and LT) were formulated into three 5-plex qPCR panels, and validated on 120 ETEC isolates and 74 E. coli colony pools. The overall sensitivity and specificity was 99% (199/202) and 99% (2497/2514), respectively, compared to the CF results obtained with conventional PCR. Amplicon sequencing of discrepant samples revealed that the qPCR was 100% accurate. qPCR panels were also performed on nucleic acid extracted from stool and compared to the results of the ETEC isolates or E. coli colony pools cultured from them. 95% (105/110) of the CF detections in the cultures were confirmed in the stool. Additionally, direct testing of stool yielded 30 more CF detections. Among 74 randomly selected E. coli colony pools with paired stool, at least one CF was detected in 63% (32/51) of the colony pools while at least one CF was detected in 78% (47/60) of the stool samples (P = NS). We conclude that these ETEC CF assays can be used on both cultures and stool samples to facilitate better understanding of CF distribution for ETEC epidemiology and vaccine development.

Published
2017

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