{'fraction_non_alphanumeric': 0.03928881179531656, 'fraction_numerical': 0.02367736339982654, 'mean_word_length': 4.949948400412796, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Molecular Characterization of Diarrheagenic Escherichia Coli in Children Less Than 5 Years of Age with Diarrhea in Ouagadougou, Burkina Faso

Diarrheagenic Escherichia coli (DEC) is important bacteria of children’s endemic and epidemic diarrhea worldwide. The aim of this study was to determine the prevalence of DEC isolated from stool samples collected from children with acute diarrhea living in Ouagadougou, Burkina Faso. From August 2013 to October 2015, stool samples were collected from 315 children under 5 years of age suffering from diarrhea in the “Centre Médical avec Antenne Chirurgicale (CMA)” Paul VI and the CMA of Schiphra. E. coli were isolated and identified by standard microbiological methods, and the 16-plex PCR method was used to further characterize them. Four hundred and nineteen (419) E. coli strains were characterized, of which 31 (7.4%) DEC pathotypes were identified and classified in five E. coli pathotypes: 15 enteroaggregative E. coli (EAEC) (48.4%), 8 enteropathogenic E. coli (EPEC) (25.8%) with 4 typical EPEC and 4 atypical EPEC, 4 enteroinvasive E. coli (EIEC) (12.9%), 3 enterohemorrhagic E. coli (EHEC) 9.67%, and 1 enterotoxigenic E. coli (ETEC) 3.2%. The use of multiplex PCR as a routine in clinical laboratory for the detection of DEC would be a useful mean for a rapid management of an acute diarrhea in children.


Introduction
Diarrheal diseases remain a global health issue, particularly in children in developing countries. The number of annual deaths due to these diseases is estimated around 2.5 million [1,2]. Among the bacterial causes of diarrhea, diarrheagenic Escherichia coli (DEC) represents the most important etiologic agent of childhood diarrhea and constitutes a major public health concern in developing countries [3,4]. DEC is one of the predominant facultative anaerobes species present in the human gut and usually known as harmless to the human host [5,6]. However, a group of emerging pathogenic E. coli is responsible for several human diseases including diarrhea, urinary tract 221 infections, and meningitis [6,7]. E. coli strains associated with diarrhea were classifi ed into six different groups based on the clinical, epidemiological, and molecular criteria [8,9], namely, enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC), and diffusely adherent E. coli (DAEC) [10,11]. Each of the classes of DEC is defi ned based on the virulence characteristics [12]. Most of the E. coli isolated from stool are nonpathogenic. The DEC pathotypes represent a main cause of the bacterial infection of pediatric diarrhea in developing regions [13,14]. The prevalence and epidemiological features of these pathogens vary from different regions, even within the same country [15,16]. Several studies on the incidence of diarrheal caused by different classes of DEC have been conducted mainly in Latin-America, Africa, South and South East Asia, and the Middle East [17]. However, little is known about the prevalence of DEC categories and their importance in children with diarrhea in Burkina Faso, particularly in Ouagadougou. Ouagadougou has 30 districts, and Bonkoungou et al. [16] conducted a study in one district (CMA 30). Therefore, to defi ne the association of various categories of DEC with diarrhea, we undertook this study using the traditional culture technique and polymerase chain reaction (PCR) for the identifi cation of DEC specifi c virulence genes in two districts of Ouagadougou, CMA Paul VI and CMA of Schiphra both with an even higher population density.

Study area and target population
Ouagadougou (1,915,102 residents), the capital city of Burkina Faso, is composed of 30 districts. CMA Paul VI or CMA of Schiphra received most of residents with low and middle incomes seeking for health care services [18]. This study was conducted between August 2013 and October 2015 in CMA Paul VI and CMA of Schiphra located in peripheral areas and in the city center of Ouagadougou, respectively (Fig. 1). The choice of these health centers is justifi ed by the fact that they are located in different environments. The study population consisted of children less than 5 years of age with acute diarrhea and who were hospitalized or visited the health center as outpatient. Any child over the age of 5 years was excluded from the study.

Sample collection
Three hundred and fi fteen (315) stool samples were collected in sterile containers and transported to the dedi-

Multiplex polymerase chain reaction (16 plex PCR)
The 16-plex PCR was used to detect simultaneously 16 genes from the fi ve main pathogroups of E. coli (EHEC, EPEC, EAEC, EIEC, and ETEC) as described by Antikainen et al. [19]. The genes investigated and primers used are described in Table 1. DNA extraction was performed using heating method [20]. Briefl y, a lapful of bacterial growth of Mueller Hinton agar (Liofi lchem, Italy) plate was suspended in 1 ml of sterile water. The mixture was boiled for 10 min at 100 °C and centrifuged for 10 min at 12,000 rpm at +4 °C.  (Table 2).

Discussion
In this study, we investigated the occurrence of the major DEC pathogroups by 16-plex PCR in 315 stool samples from children with diarrhea in two health districts of Ouagadougou, Burkina Faso, namely, CMA Paul VI and CMA of Schiphra. Globally, the prevalence of DEC (7.4%) is quite comparable to the prevalence reported in Iran [21] but lower than results obtained previously in Burkina Faso [16], Mozambique [22], and Nigeria [12]. This lower prevalence may be due to the fact that nowadays, in developed and some developing countries, the improvement of the applied techniques in the recent years have greatly positively increased the level of detection of DEC infections. In fact, these improvements have consequently reduced the global death rate due to bacterial diarrhea diseases [17]. The present study concerned fi ve pathotypes of E. coli (EAEC, EPEC [typical EPEC and atypical EPEC], EIEC, EHEC, and ETEC) responsible for children diarrhea in Burkina Faso. The majority of the previous studies carried out also implicated E. coli as the predominant bacterial agent in diarrheal diseases [13,[23][24][25]. Moreover, in Burkina Faso and many developing countries, DEC continues to be one of the major causes of morbidity and mortality among infants and young children [14,26,27]. This situation could be explained by the migration of persons within the same region and from one country to another, which increases the risk of transmission. Thus, this requires a better understanding of the epidemiology of the DEC infection in order to provide to the health policy makers and health care providers the information on this peculiar disease in each region or country.
EAEC was the most frequently detected pathotype in the present study. Nevertheless, some studies reported that EAEC might not be the primary cause of diarrhea [16,28,29]. Other authors have found EAEC to be associated with diarrhea, which is in concordance with our present results [30,31]. EAEC has emerged as an important pathogen in several clinical scenarios including pediatric diarrhea   which is endemic among children in developing countries [28,29]. Thus, the high prevalence of the EAEC pathotype reported in our study could lead to public health concern. The second pathotype with high prevalence was EPEC (25.8%). The EPEC prevalence observed in the present study was higher than those reported in a previous study which revealed that atypical EPEC was more prevalent than typical EPEC in Burkina Faso [16]. Typical EPEC and atypical EPEC differ in genetic characteristics, serotypes, and virulence properties. Indeed, typical EPEC possess a virulence plasmid (bfpB), while atypical EPEC do not possess this plasmid. In the past, typical EPEC were more frequently isolated than atypical EPEC in developing countries [32]. However, some recent reports indicate an increased isolation of atypical EPEC in developed countries as well as in developing countries [33][34][35]. In the present study, atypical EPEC constituted half of the total EPEC isolates, suggesting an epidemiological situation of possible transmission in the study area.
In this study, EIEC (12.9%) were detected, which is in agreement with the prevalence reported recently in Iran [36]. However, our result is higher than those reported by several other studies respectively in Tanzania, Nicaragua, and Burkina Faso [16,20,37]. This result may be related to the diversity of this pathotype reported during the last decade in Africa where different EIEC prevalence has been reported [22,26,29,38,39]. Our study suggests that EIEC may be an important and unrecognized cause of diarrhea in children.
EHEC (9.7%) was also characterized in our study. Similar prevalence was reported in South Africa as far the serotype O 157 is concerned [40]. However, the abovementioned prevalence of EHEC is higher than the EHEC O157 prevalence (1.2%) reported in our previous study conducted in Burkina Faso [14]. One reason that could explain the differences might be that EHEC detection frequencies vary depending on countries. Moreover, in other African countries and in non-epidemic settings, EHEC appears to be more frequent in adults than children [26]. Furthermore, humans might acquire the pathogen from animal sources, given that EHEC can be transmitted primarily through the ingestion of fecal contaminated foods, particularly from the undercooked beef meat [41,42]. Previous studies showed contamination of slaughtered animals with DEC pathotypes in Burkina Faso [6,43]. However, in Iran, a large number of outbreaks of EHEC have also been associated with consumption of contaminated drinking water or contact with recreational water [44].
In the present study, ETEC was poorly detected (3.2%), while it is well-known as a causative pathogen of diarrhea in developing and developed countries [45]. Several countries in the world, including Bangladesh, Egypt, and Mexico, have reported ETEC as the most common cause of diarrhea among all E. coli intestinal pathotypes [11,[46][47][48][49].
Among the 8 isolated EPEC strains, 4 (50%) were typical EPEC (contained only bfp gene) and 4 (50%) were atypical EPEC (3 contained only escV gene and another 1 contained only eae gene). In contrast, a study reported in Iranian, typical EPEC containing both eae and bfp gene, atypical EPEC containing only eae gene, and another containing only bfp gene [50]. The estlb gene was harbored by the only one (1) ETEC detected in our study. All 4 EIEC strains harbored the ipaH gene. However, detection of the EIEC-specific gene ipaH could also indicate the presence of Shigella sp. in the sample. For both EIEC and Shigella, the reservoir is considered to be the gut of infected humans [51].

Conclusion
The data generated in the present study with the use of 16plex PCR assay, amplifying 16 virulence genes in a single reaction, is a practical and fast tool for DEC identification in routine clinical diagnostic and epidemiological studies. By using 16-plex PCR assay, DEC can be diagnosed in one PCR reaction that makes a conclusive diagnosis of diarrhea. Therefore, the use of 16-plex PCR as routine practice for detection of DEC in clinical laboratories would be an important diagnostic tool and thus resulting in better treatment of acute diarrhea in children although the cost would represent one major limitation for developing countries.