Infectious diarrhea is endemic in most developing countries. We aimed to investigate the protozoan, viral, and bacterial causes of acute diarrhea in Taif, Saudi Arabia. A cross-sectional prospective 1-year study was conducted on 163 diarrheal patients of various ages. Stool samples were collected, 1 per patient, and tested for 3 protozoa, 3 viruses, and 9 bacteria with the Luminex Gastrointestinal Pathogen Panel. Overall, 53.4% (87/163) of samples were positives (20.8% protozoa, 19.6% viruses, 2.8% bacteria, and 9.8% mixed). Rotavirus (19.6%),
Infectious diarrhea continues to be a worldwide public health problem. According to an estimate, around 2 billion episodes globally occur per year [
Thus, prompt diagnosis of diarrhea should rely on laboratory tests that are subsequently performed based on a presumed etiology by the treating physicians. Nevertheless, the causative pathogen has been unrecognized in about 80% of cases [
A few years ago, several molecular assays were developed, properly validated, received FDA-clearance, and had a growing popularity for the detection of multiple GI pathogens in human feces [
The microbiological etiology of diarrhea has been received much concern among researchers in many parts of the world, including Saudi Arabia [
In a cross-sectional study, we investigated a range of relevant enteric protozoan, viral, and bacterial pathogens in a population with community-acquired acute diarrheal episodes from Taif Province, Saudi Arabia using the xTAG GPP assay. Our main objective was to estimate the frequency of acute infectious diarrhea caused either by 1 pathogen or by multiple pathogens in the setting. The distribution of the detected infections according to patients’ ages, genders, and seasons were also investigated.
A cross-sectional study was undertaken from March 2016 through February 2017 in Taif region, western Saudi Arabia (
Acute diarrheal episode was defined as passage of ≥3 loose bowels over a day for a period ≤2 weeks before interviewing. Those patients who presented with diarrhea of ≥2 weeks duration and those received anti-parasitic or antibiotic medications for their diarrhea episodes, 2 weeks prior to the meeting, were excluded from the study. Following these criteria, 163 patients were recruited in the current study.
Patients or their relatives were informed of the aim of the study and signed consent forms authorizing their voluntary participation. The regional ethics committee approved data collection, clinical samples collection, and analysis of the study results.
A total of 163 fresh stool samples, 1 sample per each patient, were collected in screw-capped appropriate containers and delivered within 2 hr to the microbiology laboratory at the College of Applied Medical Sciences, Taif University, Saudi Arabia. Upon arrival, each stool sample was inspected for consistency and for the presence of mucous or blood, carefully coded, and immediately stored at −20°C. On a monthly base, frozen samples were transported in ice packs to Al Borg central reference laboratory for molecular diagnosis with the xTAG™ GPP assay.
From a single fecal specimen, the GPP assay allows identification of 15 potential enteropathogens (types, genotypes, and toxin-production genes) frequently-associated with diarrhea. The sought-for GI pathogens included
Samples were initially prepared with the VERSANT Sample Preparation 1.0 Reagents kit (Siemens Healthcare Diagnostics, Erlangen, Germany) following the manufacturer’s protocol. The crude nucleic acids were directly retrieved from 700 μl of the fecal suspension using the automated platform VERSANT kPCR Molecular System. The purified nucleic acid samples were collected in 100 μl elusion buffer, following the manufacturer’s instructions.
The xTAG GPP assay was performed according to the manufacturer’s recommendations (Luminex Molecular Diagnostics, Austin, Texas, USA). Briefly, 10 μl of the nucleic acids extract together with the target-specific tagged primers and the biotin-labeled primers were initially used to set up the multiplex reverse transcription PCR (RT-PCR). The PCR reactions were carried out in Biometra T3 Thermocycler (Biometra GmbH, Göt-tingen, Germany). Amplification of the target DNA results in PCR amplicons with molecular weights ranging from 58 to 293 bp.
Aliquots of the xTAG bead mix, 20 μl each, were subjected to sonication, applied into a 96-well microtiter plate, and 5 μl of each PCR product was added into specified well. Subsequently, the xTAG 0.22 streptavidin, R-phycoerythrin conjugate (SAPE), was diluted with the xTAG reporter buffer, and 75 μl of the solution was added into each well.
Following hybridization, the median fluorescence intensity was generated for each xTAG bead population using the Luminex 100/200 instrument pre-heated to 45°C. The data were analyzed using the xTAG Data Analysis Software for the GPP assay.
The xTAG GPP assay results were tabulated for statistical analysis. The GI pathogen prevalence rate was calculated in Microsoft Excel and stated as a percentage of positive samples in relation to the total number of sample tested. The chi-square test was used to test associations between age groups, gender, and GI pathogens positivity variables. For<5 values, Fisher’s exact chi-square test was used, and the
Total 163 patients were allocated in the current study; 91 were males and 72 were females. The age range was 0.5–60 years, with a mean age of 29.9±18.9 with the median age of 19. GI pathogens, 15 kinds, were looked-for in 1 stool sample collected from each participant with the xTAG GPP test. At least 1 enteropathogen was detected in 87 samples, with an estimated detection rate of 53.4%. Mono-infections were found in 71 (43.5%) samples and co-infections were identified in 16 (9.8%) samples. Moreover, co-infections included 12 (7.4%) samples presented as double infections and 4 (2.4%) samples were existed as triple infections (
Among the 87 diarrheal episodes that were proved infectious, 34 (20.8%) were protozoan mono-infections, 32 (19.6%) were viral mono-infections, 5 (2.8%) were sole bacterial infections, and 16 (9.8%) were mixed infections (
All the detected pathogens were involved in co-infections except
In general, infections were more in younger patients and in males, but no statistically significant difference observed between any of the 2 variables and the total number of pathogens detected (
To the best of our knowledge, this is the first study, in Saudi Arabia, concerned with detection of multiple GI pathogens, commonly recognized to cause diarrhea in humans using the xTAG™ GPP assay. Also, this is the first report in the Taif region that studies, in an all-inclusive manner, the common viral, protozoan, and the bacterial etiologies of diarrhea.
One noteworthy finding in our study was the exceptionally high number of enteric pathogens detected in this setting. Pathogens were detected in 87 out of 163 patients, with an estimated prevalence rate of 53.4%, a prevalence has not been previously described in this setting. In other settings, variable estimates have been reported in the country. Fareid et al. [
Overall, young children were a most vulnerable group to infections in this setting, going with tremendous of reports in the literature. Moreover, infections were more in males than females, in the absolute numbers, consistent with a previous study [
Also in our study, we clearly demonstrated a variety of enteropathogens detected and displayed their proportions in the etiology of diarrhea in the population studied. Viral gastroenteritis was a leading cause of diarrhea, in line with the literature. Rotavirus was detected in 32 out of 163 (19.6%) patients studied. Again, little has been recently published regarding rotavirus infection in our setting. None the less, estimates of 4–46% have been reported for other populations [
Coincided with several reports, viral gastroenteritis was predominant in children under 4 years, with a statistical significance observed (
Regarding protozoa, all the 3 protozoan parasites targeted were detected in patients.
Regardless the type of protozoa, infection were most frequent in children 0–4 years, less in adult<40 years, and least frequent in patients >40 years. There was significant difference stated in risk of across age groups with
Among the 9 bacteria studied,
Also in this context, we clearly demonstrated the frequency of infections with multiple pathogens in patients with diarrhea. Co-infections were detected in 16 patients, with an estimated prevalence rate of 9.8%, comparable to estimates, 7.0–9.5%, documented, elsewhere [
Interestingly,
Lastly, our study could be bounded by a number of factors. Some interesting data could be statistically biased by the relatively small number of positive samples. Moreover, non-inclusion of a control group in conjunction with patients did not help to clear the clinical impact of some findings. Lastly, pathogens commonly reported to cause diarrhea in the country like astrovirus, intestinal helminths, and other coccidian parasites, are not covered by the xTAG GPP assay and therefore, these enteric pathogens were not investigated in this study.
To conclude, in this report, we clearly demonstrated the high burden of pathogens causing diarrhea in the study setting.
This research was funded by Taif University, Saudi Arabia (grant no. 1-437-4949). The authors would like to thank Dr. Rizk A, Dr. Henawy I, and their colleagues for their great help while recruiting patients and collecting fecal samples.
We have no conflict of interest related to this study.
Saudi Arabia map showing the location of Taif region and the nearby cities.
The results of xTAG GPP assay of 163 diarrheal stool samples.
Distribution of gastrointestinal pathogens (mono-infections and co-infections) relative to the patients’ age groups.
Monthly distribution of the detected gastrointestinal pathogens.
Distribution of GI pathogens (mono-infection and co-infections) relative to the patients’ age groups
GI Pathogens | Age (year) groups | Total | ||||||||||
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0–4 (n=36) | 5–19 (n=40) | 20–40 (n=48) | >40 (n=39) | |||||||||
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Mono- | Co- | Mono- | Co- | Mono- | Co- | Mono- | Co- | Mono- | Co- | Total | ||
9 | 3 | 5 | 3 | 2 | 2 | 2 | 1 | 18 | 9 | 27 | 0.006 sig | |
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6 | 1 | 4 | 0 | 1 | 1 | 1 | 0 | 12 | 2 | 14 | 0.036 sig | |
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0 | 0 | 1 | 0 | 2 | 0 | 1 | 0 | 4 | 0 | 4 | 0.463 | |
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0 | 0 | 0 | 1 | 2 | 0 | 2 | 0 | 4 | 1 | 5 | 0.673 | |
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0 | 0 | 0 | 1 | 0 | 1 | 0 | 0 | 0 | 2 | 2 | 0.273 | |
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0 | 0 | 0 | 0 | 0 | 2 | 0 | 1 | 0 | 3 | 3 | 0.173 | |
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STEC | 0 | 0 | 1 | 2 | 0 | 1 | 0 | 1 | 1 | 4 | 5 | 0.683 |
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ETEC | 0 | 0 | 0 | 1 | 0 | 3 | 0 | 0 | 0 | 4 | 4 | 0.463 |
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0 | 1 | 0 | 2 | 0 | 0 | 0 | 1 | 0 | 4 | 4 | 0.653 | |
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Rotavirus | 16 | 3 | 7 | 1 | 1 | 1 | 3 | 0 | 27 | 5 | 32 | <0.001 HS |
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Adenovirus | 0 | 2 | 3 | 0 | 1 | 0 | 1 | 0 | 5 | 2 | 7 | 0.573 |
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Total | 31 | 10 | 21 | 11 | 9 | 11 | 10 | 4 | 71 | 36 |
107 | 0.930 |
Different enteropathogens were concomitantly diagnosed in just 16 episodes (12 as double infections and 4 as multiple infections).
The monthly distribution of the detected GI pathogens
GI pathogen | Study duration | Total (163) | |||||||||||
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2016 | 2017 | ||||||||||||
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Mar (14) | Apr (13) | May (13) | Jun (14) | Jul (12) | Aug (14) | Sep (13) | Oct (11) | Nov (15) | Dec (14) | Jan (14) | Feb (16) | ||
5 | 4 | 3 | 1 | 1 | 1 | 3 | 2 | 2 | 1 | 2 | 2 | 27 | |
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3 | 2 | 1 | 1 | 2 | 1 | 1 | - | 1 | 1 | - | 1 | 14 | |
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- | 1 | - | 1 | - | 1 | - | - | - | 1 | - | - | 4 | |
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1 | - | 1 | - | 2 | - | - | 1 | - | - | - | - | 5 | |
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- | - | - | - | - | 2 | - | - | - | - | - | - | 2 | |
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- | 1 | - | 1 | - | - | - | - | - | - | 1 | - | 3 | |
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STEC | - | - | - | - | 2 | 2 | - | 1 | - | - | - | 5 | |
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ETEC | - | - | - | 1 | 2 | - | - | - | - | 1 | - | 4 | |
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- | - | - | - | 1 | - | 1 | - | 2 | - | - | - | 4 | |
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Rotavirus | 9 | 4 | 2 | 1 | 4 | 3 | 1 | 2 | 1 | 1 | 3 | 1 | 32 |
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Adenovirus | 1 | - | 3 | - | - | 1 | - | - | 1 | - | - | 1 | 7 |
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Total | 19 | 12 | 10 | 6 | 12 | 11 | 8 | 5 | 8 | 4 | 7 | 5 | 107 |
Including mixed infections that were displayed in 16 samples