Evaluation of colonization factors of ETEC isolated from diarrheal patients in Bangladesh: A comparative approach using multiplex PCR and dot blot immunoassay.

Enterotoxigenic Escherichia coli (ETEC) harbors colonization factors (CFs) to colonize the small intestine. In this study, as part of routine systematic ETEC surveillance in Bangladesh, we evaluated CFs from ETEC isolated from hospitalized diarrheal patients using a multiplex PCR assay for the first time, as the availability of the monoclonal antibodies for dot blot assay became limited. A total of 5914 diarrheal patients were enrolled during the study period (2023-2024), and a PCR assay, targeting heat-labile toxin (LT) and heat-stable toxin (ST), found 531 ETEC-infected patients. In our findings, 40 % of the ETEC isolates were positive for ST, followed by 38 % for LT + ST and 22 % for LT. The overall CF detection rate by using newly established multiplex PCR was 51 % among the ETEC-positive cases. CFA/I + CS21, CS6 ± CS21, CS5 + CS6, and CS1 + CS3 + CS21 were the most prevalent CFs in that period. For validation purposes, the multiplex PCR assay was compared to the conventional dot blot method, considered the gold standard. Both assays were used in parallel to assess the frequency of CFs detected in a subset of ETEC strains (n = 157). We observed a significant consistency between the dot blot and the PCR method. Additionally, compared to the dot blot method, the PCR assay identified CS21 in 11 % more ETEC strains harboring CFA/I or CS6. Overall, our findings suggest the multiplex PCR method is advantageous as well as time-efficient for detection of ETEC CFs compared to the dot blot assay.