الخلاصـة: درس الباحثون درجة تكرار ظهور أنواع الإشريكية القولونية، والشيغيلة، والسلمونيلة في عيِّنات براز المرضى المصابين بالإسهال، مـمَّن يرتادون المراكز الصحية في مقاطعة همدان بجمهورية إيران الإسلامية. وقد تم استفراد ذراري الشيغيلات في 17 من أصل 144 عينة (11.8%): 10 منها شيغيلـة فلكسنرية، و3 شيغيلة سونيـة، و2 شيغيلة بويدية، وذريَّتان لم يحدَّد نمطهما. ولم يتم استفراد أي من ذراري السلمونيلات. وعندما استُخدمت طريقة التشخيص الجزيئي، تم اكتشاف وجود الإشريكية القولونية المسبِّبة للإسهال في 37 حالـة (25.7%)، معظمها مولِّدٌ للذيفان المعوي ETEC (22 حالة) ومنها (15 حالة) من ذراري الإشريكية القولونية المنتجة لذيفان شيغا. كما وجدت عدوى مشتـركة في 14 حالة (9.7%).

ABSTRACT: This study investigated the frequency of Escherichia coli, Shigella and Salmonella species in stool specimens from patients with diarrhoea presenting to health centres in Hamedan province, Islamic Republic of Iran. From 144 samples, Shigella strains were isolated in 17 cases (11.8%): 10 Sh. flexneri, 3 Sh. sonnei, 2 Sh. boydii and 2 untyped strains. No Salmonella strains were isolated. Using molecular diagnostic methods, diarrheogenic E. coli were detected in 37 cases (25.7%), the majority were enterotoxigenic (ETEC) (22 cases) and Shiga toxin-producing (STEC) strains (15 cases). In 14 cases (9.7%) there was co-infection.

La responsabilité d’Escherichia coli, des Shigella et des Salmonella dans la diarrhée aiguë dans la province d’Hamadan en République islamique d’Iran

RÉSUMÉ: Cette étude a évalué la fréquence d’Escherichia coli, des Shigella et des Salmonella dans des échantillons de selles de patients diarrhéiques consultant dans les centres de santé dans la province d’Hamadan en République islamique d’Iran. Sur 144 échantillons, des souches de Shigella ont été isolées dans 17 cas (11,8 %), à savoir 10 Sh. flexneri, 3 Sh. sonnei, 2 Sh. boydii et 2 souches de sérogroupe inconnu. Aucune souche de Salmonella n’a été isolée. Les techniques de diagnostic moléculaire ont permis de détecter des E. coli diarrhéigènes dans 37 cas (25,7 %), dont la majorité étaient des souches entérotoxinogènes (ETEC) (22 cas) et des souches productrices de Shiga‑ toxines (STEC) (15 cas). Quatorze cas (9,7 %) présentaient une co‑infection.

¹Research Centre of Gastroenterology and Liver Disease, Shaheed Beheshti University, Tehran, Islamic Republic of Iran (Correspondence to A.H.M. Alizadeh: ahmaliver@yahoo.com).

²Hamedan University of Medical Science and Health Services, Hamedan, Islamic Republic of Iran. Received: 12/04/05; accepted: 13/07/05

 

Introduction

Diarrhoeal diseases are among the most common causes of death in the world [1,2] with a greater medical and economic burden in developing countries. In spite of the lack of accurate investigations in developing countries, the problem of diarrhoeal diseases in these regions is much greater than it seems [3].

Escherichia. coli, Shigella and Salmonella spp. are among the most important agents causing diarrhoea worldwide [3]. At least 5 different categories of E. coli are responsible for diarrhoea: enterotoxigenic E. coli (ETEC), enterohaemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), and enteroaggregative E. coli (EAEC). EHEC excrete potent toxins called verotoxins or Shiga toxins and are referred to as Shiga toxin-producing E. coli (STEC).

The associated diseases comprise: childhood and traveller’s diarrhoea (ETEC), bloody diarrhoea and haemolytic uraemic syndrome (EHEC), infantile diarrhoea (EPEC), and bacillary dysentery-like diarrhoea (EIEC). EAEC has been associated with acute and persistent diarrhoea in children and adults in industrialized and developing countries in Europe, America, Asia and Africa [4,5].

Shigella is an important source of bacterial diarrhoea in developing countries [6–8]. The most important diarrheogenic Shigella strains are Sh. flexneri, Sh. sonnei, Sh. boydii and Sh. dysenteriae and untyped Shigella strains. Salmonella is another etiological factor in diarrhoea and the most common source of bacterial diarrhoea in developed countries [9]. It consists of serotypes A, B, C, D and S. typhi.

The pattern of major diarrhoea pathogens in the Islamic Republic of Iran seems to be different from that of many developed countries. E. coli species are the most frequently isolated pathogens, followed by Salmonella, Shigella, and Campylobacter [3,7,8], while in developed countries Campylobacter is more prevalent [10]. Developments in molecular analysis techniques have increased our ability to identify potential enteropathogens in stool specimens, and, in recent years, several new agents have been increasingly recognized in association with diarrhoeal disease [10]. There is little data about the molecular epidemiology of these in the Islamic Republic of Iran. Thus we decided to investigate the frequency of diarrheogenic E. coli, Shigella, Salmonella and their strains using both traditional and molecular diagnostic methods in patients with acute diarrhoea in Hamedan province.

Methods

Area and population

The provincial capital, also called Hamedan, is located 343 km south west of Tehran at the foot of Mount Alvand (3580 m) in the Zagros range, at an altitude of 1700 m, and dominates the wide, fertile plain of the upper Qareh Su River. It has long and severe winters from September to May with heavy snow and temperate summers.

Specimens

Between June 2003 and August 2003 all patients attending the health centres of the Hamedan province health network for acute diarrhoea were enrolled in the study. For every patient a fresh stool sample was taken and transported in less than 6 hours to the central laboratory in Cary-Blair transport medium and processed within 2 hours. All the specimens were examined for E. coli, Shigella spp. and Salmonella spp.

Bacteriologic methods

All samples were cultured on MacConkey agar, Shigella agar, Salmonella agar, xylose lysine decarboxylase (XLD), sorbitol MacConkey and bismuth sulfite MacConkey and incubated for 24 hours at 37 °C. The detection of Shigella and Salmonella spp. was conducted using traditional microbiological methods and with commercial anti-sera if necessary. Polymerase chain reaction (PCR) of mixed culture was used for the detection of diarrheogenic E. coli strains.

DNA was extracted from the primary mixed culture of faecal samples and was subjected to 6 different PCR reactions targeting STEC (stx1 and stx2 genes), ETEC (LT and ST toxin-producing genes), EPEC (eae gene), and EAEC (pCVD432 plasmid) respectively [10–14] (Table 1).

For PCR, a loopful of Gram-negative bacterial growth was taken from the first streaking area of the primary culture plate and was suspended in 0.5 mL of sterile distilled water and boiled for 20 minutes. Extracts of DNA from mixed culture were subjected to PCR by 6 pair of primers targeting stx1, stx2, eae, pCVD432 plasmid, LT and ST.

From the PCR positive primary faecal culture, distinct E. coli-like and other Gram-negative colonies were isolated and tested for the presence of sequences which had initially given a positive result. As many colonies as required for finding the isolate carrying these particular genes were assayed. The isolates were subsequently characterized biochemically according to standard methods. E. coli strains that carried eae and were negative for stx were interpreted as EPEC. The strains positive in PCR for pCVD432 were interpreted as EAEC, and those positive for stx genes as STEC. E. coli strains positive in PCR for LT, ST or both were considered ETEC. Positive controls were E. coli EDL 933, E. coli RH 4283, E. coli RH 4260, E. coli ATCC 35401, E. coli 43886 and E. coli ATCC 35401 and E. coli ATCC was a negative control.

Analysis

Descriptive statistics for patients were reported and continuous variables were summarized as mean and standard deviation (SD). Patients were analysed in 2 groups: ≤ 5 years and > 5 years. The chi-squared test was used to compare categorical variables between age groups and etiology of disease. The level of significance was P < 0.05.

Results

We collected a total of 144 samples from patients with acute diarrhoea: 3 samples could not be cultured. The male to female ratio of patients was 1.52:1. The mean age was 20.2 years (range 5 months to 90 years); 67 patients were in the age group ≤ 5 years.

Shigella strains were isolated in 17 (11.8%) cases: 10 Sh. flexneri, 3 Sh. sonnei, 2 Sh. boydii and 2 untyped Shigella strains. Shigella were found in 8 (11.9%) of the age group ≤ 5 years and 9 (11.7%) of the > 5 years age group.

No Salmonella strains were isolated.

In the molecular analysis, diarrheogenic E. coli were detected in 37 cases (25.7%): 2 (1.3%) EPEC, 10 (7.0%) EAEC, 22 (15.2%) ETEC and 15 (10.4%) (STEC).

In 14 patients (9.7%) there was co-infection: 9 patients (6.3%) had 2 or more types of diarrheogenic E. coli, 4 patients (3.0%) had 1 diarrheogenic E. coli strain and Shigella and 1 patient had 2 diarrheogenic E. coli strains and Shigella. E. coli were found in 18 (26.9%) patients aged ≤ 5 years and 24.7% of those aged > 5 years.

There was no relation between age and contamination with the bacterial agents. Furthermore such comparison was taken for different strains of diarrheogenic E. coli and similar results were seen for them. On the other hand there were no differences between these diarrheogenic E. coli in these 2 age groups. Table 2 shows the frequency of bacterial pathogens in the 2 age groups.

Discussion

Diarrhoeal diseases are a major problem in the Islamic Republic of Iran, as in other developing countries [11,12]. Clarification of the enteropathogens associated with diarrhoeal diseases in the country is an essential step towards the implementation of effective primary health care prevention activities against the diseases. The present etiological study is also meaningful in that it provides information on the prevalence of 3 important enteropathogens in the Hamedan area of the west of the Islamic Republic of Iran. The specimens analysed were taken from patients who had diarrhoea severe enough to bring them to a medical facility.

E. coli spp. played an important role in diarrhoea in all age groups. The frequency of isolation of E. coli spp. was 25.7% on average, which is markedly higher than that reported in other developing countries (11.0% and 16.9%) (Somalia and Thailand) [12,13] and previously in the Islamic Republic of Iran (16.2%, 6.8%) [14,15]. Almost all studies in this country have been performed on children and other age groups have been ignored. In this study all age groups were included and we did not detect any differences between the frequencies of this organism between the 2 age groups. So in our study, the frequency of E. coli in children (26.9%) was markedly higher than another study in the Islamic Republic of Iran from Islamshahr, south Tehran (6.8%) [15].

ETEC was detected in 22 patients (15.2%) and was the most common strain of diarrheogenic E. coli isolated, a finding that is consistent with previous studies which reported frequencies of ETEC of 22% [16]. This organism can be assumed to be one of the most important diarrheogenic E. coli strains.

The second most frequently detected diarrheogenic E. coli strain was STEC, detected in 15 cases (10.4%). In 2 studies the frequency of this strain was reported in the normal population in 2 provinces of the Islamic Republic of Iran (4.9% and 0.7% of people respectively) and with a greater frequency in the age group < 6 years [17,18]. Our data showed a higher frequency in comparison with Sweden (8%) where this strain is not a predominant agent among diarrheogenic E. coli [19]. STEC has an important role in fatal haemolytic uraemic syndrome, which is believed to be one of the most frequent causes of acute renal failure in children [20], and therefore more focus may be needed on the diagnosis of this organism using molecular methods such as PCR.

The EAEC strain is a new category of diarrheogenic E. coli and we detected this pathogen in 10 patients (7%), which is comparable with other studies conducted in Thailand and Brazil, where its frequency among children with acute diarrhoea was reported to be 12% and 11% respectively [11,12]. This contrasts with the low frequency of this organism in developed countries, for example in a study in western Europe its frequency was reported to be 2% in children with acute diarrhoea and 0% in individuals without diarrhoea [21].

We detected the EPEC strain in 2 patients (1.3%), and it was ranked fourth of the causes of diarrheogenic E. coli, whereas in another study in 1988 in the Islamic Republic of Iran this organism was the most frequently detected strain (31%) [16]. Despite differences between the methods used, this finding suggests a declining role of this organism in acute diarrhoea.

Shigella is a important pathogen in developing countries [13,22] and one of the most important causes of acute diarrhoea in the Islamic Republic of Iran in children and in adults [7,8]. The isolation frequency increases progressively with age [23,24]. In this study Shigella spp. were isolated in 17 (11.8%) of patients. We did not find any difference between children and adults (11.8% versus 11.7%). In other studies, Shigella spp. were associated with diarrhoea in children in a relatively older age group [3].

In general, Sh. sonnei is reported to cause the majority of cases of shigellosis in industrialized countries, and in developing countries where hygiene is poor. The incidence of other Shigella species such as Sh. dysenteriae, Sh. boydii and Sh. flexneri are relatively high [9]. In the present study, Sh. flexneri and Sh. sonnei accounted for 58.8% and 17.6% of all Shigella isolates, respectively. Considering these frequencies, the pattern of prevalence of each Shigella species in the Islamic Republic of Iran can be considered the same as that in a developing country. In studies in Kuwait and Nigeria Sh. flexneri was also the predominant isolated strain among Shigella spp. (46.0% and 51.6%) [25,26].

In our study we did not detect any cases of Salmonella infection, while in other studies in this country Salmonella was an important strain in bacterial diarrhoea (14.9%) with S. enteritidis, S. typhimurium, and S. typhi as the most common species [3–15, 27]. In a study in 1986 Salmonella was the most common diarrheogenic pathogen in our country [28] but later studies found this organism in second and third rank [15,27]. This finding is surprising as the reservoir of infection in animals constitutes the principal source of infection with Salmonella spp. [29] and in view of the presence of animals in people’s daily lives in the Islamic Republic of Iran, we expected a higher frequency of isolation of Salmonella spp.

Conclusion

We recommend that comprehensive surveys are needed in different parts of the Islamic Republic of Iran in order to identify the incidence of different diarrhoeal pathogens in the country.

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