Multiplex Polymerase Chain Reaction for Rapid Detection of Major Pathogens causing Gastroenteritis

Abstract

Infectious gastroenteritis (IG) is one of the most common diseases worldwide, killing millions of individuals each year. In industrialized countries, IG remains a major public health burden, although mortality is low. In developing countries, where sanitation is suboptimal, epidemics of bacterial gastroenteritis can develop and cause significant mortality. The etiology of IG includes viral, parasitic, and bacterial pathogens. Bacterial gastroenteritis is usually self-limited, but improper management of an acute infection can lead to a protracted course. The aim of this work is to evaluate multiplex PCR for the rapid detection of five major intestinal pathogens causing gastroenteritis (Salmonella enterica, Campylobacter jejuni, STEC, Shigella spp./ EIEC and Giardia lamblia) in stool specimens in comparison to the conventional culture methods. The study included fifty five patients suffering from gastroenteritis attending the outpatient clinic or admitted to Ain Shams University Hospitals. They were 32 females and 23 males with their age ranging from 3 months to 67 years (median 6 years). Forty nine (49/55, 89.1%) of patients suffered from watery diarrhea while 5/55 (9.1%) had bloody diarrhea. Twenty four patients (24/55, 43.6%) had associated abdominal cramps/colics, 14/55 (25.5%) had vomiting, 10/55 (18.2%) had dehydration, 4/55 (7.3%) had steatorrhea and 1/55 (1.8%) complained from weight loss. Only 12/55 (21.8%) of patients were on antibiotic treatment at time of sample collection. Fifty five diarrheal stool specimens were collected from patients and were subjected to macroscopic examination; microscopic examination using wet mount iodine preparation; routine culture on MacConkey agar, XLD agar and Skirrow’s agar medium; and Clostridium difficile toxin detection. Culture positive specimens were further identified by Gram stained films, oxidase test, API 10S test, E. coli O157 latex agglutination test for E. coli positive specimens to detect EHEC and serotyping for Salmonella and Shigella positive specimens. Moreover, a multiplex PCR was done on all samples using the Seeplex® Diarrhea ACE Detection kits (Seegene, Korea) for the detection of Salmonella spp., Shigella spp., Vibrio spp., Campylobacter spp., Clostridium difficile, Yersinia enterocolitica, Aeromonas spp., E.coli O157:H7, VTEC family, and Clostridium perfringens.