Amin Md Ariful, Akhtar Marjahan, Afrad Mokibul Hassan, Rahman Sadia Isfat Ara, Parvin Nasrin, Akter Afroza, Tauheed Imam, Amin Mohammad Ashraful, Ryan Edward T, Khan Ashraful Islam, Chowdhury Fahima, Bhuiyan Taufiqur Rahman, Begum Yasmin Ara, Qadri Firdausi
Infectious Diseases Division, International Centre for Diarrhoeal Disease Research Bangladesh (icddr,b), Dhaka, Bangladesh.
Infectious Diseases Division, International Centre for Diarrhoeal Disease Research Bangladesh (icddr,b), Dhaka, Bangladesh.
J Microbiol Methods. 2025 Aug 6;237:107215. doi: 10.1016/j.mimet.2025.107215.
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.
产肠毒素大肠杆菌（ETEC）具有定植因子（CFs），可在小肠定植。在本研究中，作为孟加拉国常规系统性ETEC监测的一部分，由于用于斑点印迹分析的单克隆抗体供应有限，我们首次使用多重PCR检测法对从住院腹泻患者中分离出的ETEC的CFs进行了评估。在研究期间（2023 - 2024年）共纳入了5914例腹泻患者，通过针对不耐热毒素（LT）和耐热毒素（ST）的PCR检测，发现了531例ETEC感染患者。在我们的研究结果中，40%的ETEC分离株ST呈阳性，其次是38%的LT + ST阳性和22%的LT阳性。在ETEC阳性病例中，使用新建立的多重PCR检测法的总体CF检测率为51%。CFA/I + CS21、CS6 ± CS21、CS5 + CS6以及CS1 + CS3 + CS21是该时期最常见的CFs。为了进行验证，将多重PCR检测法与被视为金标准的传统斑点印迹法进行了比较。两种检测法同时用于评估在一部分ETEC菌株（n = 157）中检测到的CFs频率。我们观察到斑点印迹法和PCR法之间具有显著的一致性。此外，与斑点印迹法相比，PCR检测法在携带CFA/I或CS6的ETEC菌株中多鉴定出了11%的CS21。总体而言，我们的研究结果表明，与斑点印迹分析相比，多重PCR方法在检测ETEC CFs方面既具有优势又节省时间。
