Molecular Diagnosis of Shigella, Salmonella and Campylobacter by Multiplex Real-Time PCR in Stool Culture Samples in Ouagadougou (Burkina Faso)

Background: Bacteriological diagnosis of Campylobacter spp, Salmonella spp and Shigella spp could be necessary in case of infectious gastroenteritis syndrome. The objective of this study was to diagnose concomitantly the three enteropathogenic bacteria by multiplex Real-Time PCR in stool culture samples in Ouagadougou (Burkina Faso). Materials and Methods: The study was conducted from February 5th to March 9th, 2013. Two hundred stool samples were received during the study period. The bacteria were identified by bacterial culture following by multiplex Real-Time PCR. Results: Shigella spp and Campylobacter spp were sought by culture in all 200 samples. Enteropathogenic E. coli was sought only in 37 samples from all children under 2 years old. The bacterial culture was positive in 12 stool samples. Shigella spp and Salmonella spp. were isolated respectively in 5 (2.5%) and 3 samples (1.5%). Enteropathogenic E. coli was isolated in 10.8% (4/37) of the samples tested. The multiplex real-time PCR identified bacteria in 20 patients, including 17 cases of Shigella spp., 1 case of Salmonella spp. and 2 cases of Campylobacter spp. Conclusions: This study has highlighted the low frequency of 3 sought bacterial genera in stool samples. It has also demonstrated a significant difference between the culture and the multiplex Real-Time PCR method in the diagnosis of Shigella.


Introduction
Each year, there are about two billion cases of diarrheal diseases worldwide causing the death of more than 2 million people, including 1.9 million children under the age of 5 mainly in developing countries [1].In Burkina Faso, diarrhea is responsible for 12% of deaths in children under 5 years old [2].Among the pathogens involved in these infections, enteric bacteria such as Campylobacter, Salmonella and Shigella are important because of the frequency and severity of the symptoms they could cause.Thus, Campylobacter is currently considered the leading cause of intestinal bacterial infections in humans worldwide with an increasing incidence in developed countries [3][4][5].
Although, Campylobacter infection is usually mild, the increase in cases of campylobacteriosis, the existence of rare but serious complications such as abortions, peritonitis, meningitis, Guillain-Barre syndrome and the alarming increase of Campylobacter antibiotic resistance explain the renewed interest in this bacterial genus [3].Regarding Salmonella, the World Health Organization (WHO) estimates about 17 million cases of typhoid fever in the world with more than 500,000 deaths and 1.3 billion annual cases of gastroenteritis due to non-typhoid Salmonella with 3 million deaths annually [6].
As for Shigella, it is the leading cause of malnutrition in developing countries where shigellosis remains endemic [7].The cases of antibiotics' multi-resistant bacteria at high levels are also reported for Salmonella and Shigella [8][9][10][11].The direct diagnosis of these three bacteria can be done by stool culture.Campylobacter diagnosis is generally not performed in bacteriology laboratories in Burkina Faso.In this country, no study has yet reported the molecular diagnosis of Shigella, Salmonella and Campylobacter in human stool samples.The objective of this study was to diagnose by multiplex realtime PCR, Shigella, Salmonella and Campylobacterin routine stool culture samples in Ouagadougou.

Sample Collection
A total of 200 stool culture samples were collected, including 101 samples from El Fateh Suka clinic and 99 at Saint Camille Medical Centre in Ouagadougou between February 5th and March 9th, 2013.For each stool sample, a portion of the sample was transferred into a sterile tube and stored at -80 ∘ C for molecular diagnostics.The rest was used for stool culture process within 2 h of receipt.
Patients or parents and/or guardians of under-age children answered a questionnaire including their socio-demographic and clinical characteristics.

Stool Culture
Salmonella, Shigella and enteropathogenic E. coli (EPEC) were sought on the basis of existing repositories [12].For all samples, the Hektoen agar Petri dish and Salmonella enrichment broth (Selenite broth) were inoculated and incubated in an oven at 37 ∘ C.After 5 to 6 hours, selenite broth was sub-cultured on a second Hektoen agar plate.
Both Hektoen boxes were then incubated in an oven at 37 ∘ C for 18 to 24 hours.The suspected colonies were biochemically identified by API 20E (Biomérieux ® , Marcy-Etoile, France) and also sub-cultured on Kligler-Hajna medium.Salmonella and Shigella colonies identified on Kligler-Hajna medium were agglutinated with Salmonella and Shigella antisera (Bio-Rad ® , Marnes-la-Coquette, France) for confirmation and species or serotype identification.The detection of EPEC (Enteropathogenic E. coli) was carried out on the samples of the children under 2 years old.Its identification was performed on EMB agar.Typical colonies were agglutinated with an Enteropathogenic E. coli antisera (Bio-Rad ® , Marnes-la-Coquette, France).

Sample Preparation, Extraction and Amplification of Bacterial Genomes
After thawing the samples, 1 mL (for liquid stool) or 1 gram (for solid faeces) was used for the extraction of bacterial DNA using the DNA-Sorb-B extraction kit (Sacace Biotechnologies ® , Como, Italy) according to the manufacturer's instructions.
The amplification reaction was performed in a SaCycler-96 Real-Time PCR (Sacace Biotechnologies ® , Como, Italy) according to the manufacturer's recommendations.
The target genes were respectively IpaH, TtrB, and 23SrRNA for Shigella, Salmonella and Campylobacter.

Statistical Analysis
The results were analyzed using SPSS 17.0 Software and Epi Info version 3.5.1.The chisquare test was used for comparisons and the difference was considered significant for p-value < 0.05.

Epidemiological and Clinical Characteristics of Patients
The stools were predominantly from female patients (60.5%), outpatients (94.5%) and subjects over 15 years (57.5%).The major clinical features of patients were respectively, fever (44.5%), diarrhea (21%) and vomiting (13%).The presence of blood and leucocytes in stool was observed in 2.5% and 5% of patients, respectively.Some of the patients (14.5%) had started antibiotic therapy before sample collection.The epidemiological and clinical characteristics of the patients are shown in Table 1.

Identification of Bacteria from Stool Specimens by Culture and Multiplex Real Time PCR
The number and percentage of germs diagnosed by the two diagnostic methods are shown in Table 2. Enteropathogenic Escherichia coli was sought only in samples of children aged less than 2 years was isolated 4 times by culture (4/37, 10.8%) The prevalence of the three bacteria detected by multiplex real-time PCR was higher than that detected by stool culture; but the observed difference was not statistically DOI 10.18502/sjms.v12i3.931significant (10% vs 6%, p = 0.140).Bacteria prevalence detected by multiplex real time PCR and by stool culture were respectively, 8.5% vs 2.5% for Shigella and 0.5% vs 1.5% for Salmonella.Shigella prevalence detected by PCR was significantly higher compared to that detected by culture (p = 0.006).The distribution of Shigella species identified in the 5 patients was as follows: 3 cases of S. flexneri, 1 case of S. boydii and 1 case of S. dysenteriae.
The 3 cases of Salmonella identified were represented by three different serotypes: Salmonella Typhi, Salmonella Paratyphi B and Salmonella sp. of the 3 positive samples by culture, two were negative by PCR.Campylobacter spp. was identified by PCR only in 1% of patients (Table 2).The distribution of positive samples for the three bacteria by PCR based on epidemiological and clinical features of the patients is summarized in Table 3.
The distribution of Shigella spp.among patients was not associated with age, neither with sex or fever.The prevalence of Shigella was higher among patients with diarrheal compared to non-diarrheal patients (23.8% vs 4.4%; p < 0.001).

Discussion
This study aimed the concomitant search for Shigella, Salmonella, and Campylobacterby multiplex real-time PCR in stool samples from patients in Ouagadougou.To our knowledge, this is the first study on the molecular diagnosis of these three bacteria in Burkina Faso.However, the diagnosis of these pathogens by traditional methods has been reported in previous studies in children with diarrhea [13][14][15][16][17].The prevalence of Shigella, Salmonella and Campylobacter by multiplex real-time PCR was 8.5%; 0.5% and 1%, respectively.This reduced prevalence of enteropathogenic bacteria could be explained the weak frequency of diarrhoeal patients among the stool specimens tested.Diarrhea is the major cause of stool specimens' culture [12,18].This suggests that in Burkina Faso, stool culture is done as well as parasites stool examination in the systematic search for certain gastrointestinal disorders in primary care.Diarrheas were more common in infants than adults (68.6% vs 12.2%).Similar results have been reported in Burkina Faso [13,14] and Brazil [20].Despite this high rate of diarrhea, bacteria were isolated only in 28.6% of infants.This could be explained by the fact that, viruses are the leading cause of diarrhea in children [12,20].In addition, the period of the study coincided with the cold dry season where the frequency of virus is highest among children with diarrhea in Burkina Faso [16,17].Bloody stool samples having a positive leukocyte microscopy examination, were all positive by culture and by PCR, which is in agreement with the entero-invasive nature of these bacteria [12].Shigella was the most identified bacteria by stool culture as well as multiplex realtime PCR.Culture has identified 5 cases of shigellosis.Shigella flexneri was the most frequently isolated (3 of 5 cases), which is consistent with previous studies which have shown that it is the most common species in developing countries [16,17,21,22].
The frequency of Shigella spp. was higher by PCR than stool culture.This difference should be interpreted with caution.Indeed, the PCR target gene IpaH which allowed the detection of Shigella is also present in the entero-invasive E. coli (EIEC).Thus, the highest frequency of Shigella by PCR could be due to the presence of EIEC in stool of DOI 10.18502/sjms.v12i3.931patients.To our knowledge, there is no molecular technique to differentiate Shigella from EIEC [23].
Thus, 5 samples were found positive for Shigella in culture and by the PCR technique.
For 12 samples negative in culture but positive by PCR, the presence of the EIEC could not be excluded; although a recent study in Burkina Faso showed a very low frequency of EIEC (0.3%) compared to enteroaggregative E. coli (12%) and enteropathogenic E. coli (8%) [17].
The prevalence of Shigella spp.showing a discrepancy between the results of PCR and culture have been reported in Vietnam [24].The present report and previous studies in Vietnam [24], India [25] and Thailand [26] suggest that stool culture has a lower sensitivity than PCR in the diagnosis of Shigella spp.
Regarding Salmonella, of the 3 positive samples in stool culture, two were PCR negative.The absence of Salmonella in negative PCR samples suggests either a lower specificity of culture (biochemical and antigenic similarity to other bacteria) or a lower sensitivity of PCR in the diagnosis of Salmonella compared to stool culture.A low sensitivity of PCR compared to culture in the detection of Salmonella was reported by Cunningham et al, [27].However, because of the reduced size of our sample, additional data are needed to confirm one or the other of the two hypotheses.
The frequency of Campylobacter (1%) found in this study is similar to those reported in Burkina Faso [17] and France [28].A study combining enzyme immunoassay methods, molecular and culture had reported a 9.5% rate in hospitalized patients with gastrointestinal problems [29].The detection of Campylobacter by stool culture has been recommended by some studies [3,28] and repositories [12].It may be necessary for the diagnosis of certain enteritis and diarrhea.

Conclusion
This study has highlighted the low prevalence of the three bacteria diagnosed in routine stool culture samples.It also demonstrated a significant difference between multiplex real-time PCR and stool culture in the diagnosis of Shigella.