Rapid Diagnosis and Characterization of Diarrheagenic Escherichia coli In Egyptian Children Using Multiplex PCR

Escherichia coli (E.coli) is the most important etiologic agent of childhood diarrhea and represents a major public health problem in developing countries. Aim of this work was to investigate the role of diarrheagenic E.coli in Egyptian children below 5 years age using multiplex PCR and to evaluate multiplex PCR in rapid diagnosis of enteric infections caused by diarrheagenic E.coli strains. Rectal swabs were taken from 83 children under 5 years age with diarrhea and 33 age-matched controls. All E.coli isolates were O serotyped using E.coli O polyvalent and monovalent antisera and subjected to multiplex PCR assay with specific primers, eae primer of eaeA (gene of intimin of EHEC and EPEC), primer bfpA of bfpA (structural gene for the bundle-forming pilus of EPEC), primers VT1 and VT2 of vt1 and vt2 genes ( genes of shiga toxins 1 and 2 of EHEC respectively), primer LT of eltB (gene of labile toxin of ETEC), primer ST for estA (gene of stable toxin of ETEC), primer SHIG of ial (invasion-associated locus of the invasion plasmid found in EIEC) and primer EA of pCVD (the nucleotide sequence of the EcoRIPstI DNA fragment of pCVD432 of EAEC). The study revealed that diarrheagenic E.coli strains were significantly isolated from patients more than control using multiplex PCR. Out of 70 E.coli strains isolated from patients, 17(24.3%) isolates were proved to be diarrheagenic by multiplex PCR where 53 (75.7%) isolates were non diarrheagenic. Out of 30 E.coli isolates recovered from control group, 1 (3.3%) isolate was proved to be diarrheagenic by multiplex PCR where 29 (96.7%) isolates were non diarrheagenic(Chi-square=18.5 & p ≤ 0.001) as shown in table (1). As regard to serology of isolated E.coli strains, serologically typable strains were insignificantly isolated from both patients and controls. Out of 70 E.coli isolates recovered from patients, 23(32.9%) isolates were serologically typable and 47 (67.1%) isolates were serologically non-typable. Out of 30 E.coli isolates recovered from control group, 7(23.3%) isolates were serologically typable and 23(76.7%) isolates were serologically non-typable (Chi-square = 2.28 & p ≤ 0.20) as shown in table (2). Matching results of multiplex PCR and results of serology revealed that multiplex PCR was significant in differentiating diarrheagenic E.coli strains in both patients and control. Out of the tybable 23 E.coli strains isolated from patients, 12(52.2%) strains were proved to be diarrheagenic by multiplex PCR where 11(47.8%) strains were non diarrheagenic. Out of the nontybable 47 E.coli strains isolated from patients, 5 (10.6) strains were proved to be diarrheagenic by multiplex PCR where 42 (89.4) strains were non diarrheagenic(Chi-square = 40.17 & p ≤0.001) as shown in table (3). Non of the typable 7 E.coli strains isolated from control was proved to be diarrheagenic by multiplex PCR. Out of the nontybable 23 E.coli strains isolated from control, 1(4.3%) strain was proved to be diarrheagenic by multiplex PCR where 22 (95.7%)strains were nondiarrheagenic(Chi-square = 4.39 & p ≤0.05) as shown in table(4). Out of the diarrheagenic E.coli isolated, 9(52.9%) isolates were ETEC; 5(29.4%) isolates were EPEC; and 3 (17.6%)isolates were EAEC. 4(23.5%), 3(17.6%), and 2(11.8%) isolates of ETEC showed the eltB, estA, eltB+ estA genotypes respectively. 4 (23.5%)& 1(5.9%) isolates of EPEC showed eae and eae+bfp genotypes respectively. The 3(17.6%) EAEC isolates showed pCVD genotype table (5). This study concluded that multiplex PCR could be used as a rapid method for rapid diagnosis and characterization of diarrheagenic E.coli in children and recommended that further studies must be done for the application of multiplex PCR for the rapid diagnosis of diarrheagenic E.coli directly from stools.