enteric protozoa are a diverse group of unicellular microparasites inhabiting the intestinal tract of high vertebrate hosts including humans . infections usually occur through ingestion of cysts / oocysts contaminating raw food or drinking water [ 2 , 3 ] . diarrhea is relatively a frequent symptom for protozoan infections but asymptomatic colonization is also common . attributing diarrhea to an exact parasite identified in a patient 's feces is not certain for all protozoa . while a number of intestinal protozoa such as giardia duodenalis , entamoeba histolytica , cryptosporidium spp . , cyclospora cayetanensis , cystoisospora belli , and microsporidia spp . have been certainly recognized to cause diarrhea in humans , others like entamoeba coli and entamoeba dispar almost certainly have not . in addition , enteric protozoa like blastocystis spp . and dientamoeba fragilis have been recently identified in patients with diarrhea , but their causal role is still uncertain . regardless of the underlying protozoan parasite , diarrhea is usually mild and self - limited in healthy immunocompetent persons . nevertheless , severe and protracted diarrhea has been reported in immunosuppressed patients . by definition , patient with chronic renal failure ( crf ) is a patient with end stage kidney disease causing marked decline in the glomerular filtration rate as well as uremia and requires kidney replacement or scheduled dialysis to survive . it has been hypothesized that patients , at this stage , are more susceptible to infectious diarrhea than the normal population , secondary to immunosuppression [ 9 , 10 ] . this infectious diarrhea has been found associated with a number of intestinal viruses , bacteria , and protozoa . in saudi arabia , high prevalence of enteric protozoa - related diarrhea has been reported   and the number of patients with crf has been found rising . nevertheless , the frequency of these protozoa among patients with crf has been poorly studied . in a case - control study , we tested the above hypothesis using a matched population from taif , western saudi arabia . the burden of protozoan infections , associated symptoms , and potential risk factors were also sought in this study . two hundred and four patients comprising 139 with crf and 64 without crf from those patients attending the outpatient clinics at king abdulaziz specialized hospital , taif , saudi arabia , seeking treatment of their diarrheal episodes , were invited to participate in the study . an episode of diarrhea was defined as 3 loose bowel movements over a day prior to interviewing . individuals with history of diabetes mellitus , malignancy , autoimmune disease , or any other chronic diseases were excluded from the study . patients with history of taking antiparasitic , antidiarrheal , or immunosuppressive medications , 2 weeks prior to meeting , were also excluded from participation in the study . following these exclusion criteria , 50 patients with crf , described as cases , and 50 patients without crf , named as controls , all participants were informed of the purpose of our study and signed consent forms authorizing their voluntary participation . the regional ethics committee approved data collection , clinical samples collection , and analysis of the study . on enrollments , face - to - face interviews were conducted using a structured questionnaire for the basic demographic data in terms of age , sex , and residence . questions about drinking water resources , history of travel to foreign countries , and/or exposure to animals during the three - week period preceding diarrhea were also included . moreover , some clinical data concerning the number of bowels / day , duration of diarrhea , accompanying nausea / vomiting , mucous or blood in feces , abdominal pain , bloating / flatulence , general fatigue , and loss of weight were also sought for in the questionnaire . a total of 100 fecal samples , one sample from each participant , were collected between june and december 2014 . feces were transported to the microbiology laboratory at college of applied medical sciences , taif university , within 2 - 3 hours after collection . samples were appropriately coded and immediately stored at 4c till the time of parasitological examination . direct wet mount smears were microscopically examined for parasites ova , cysts , oocysts , and/or larvae , as previously described . formol - ether fecal concentrates of each sample were individually smeared and stained with native iodine , trichrome , modified trichrome , and modified ziehl - neelsen stains for identification of blastocystis spp . , microsporidium spp . , and intestinal coccidian protozoa , respectively . staining and examination techniques were carried out according to garcia , 2009 . fresh fecal specimens were also examined for g. duodenalis with rida quick giardia ( r - biopharm , darmstadt , germany ) , cryptosporidium spp . with rida quick cryptosporidium ( r - biopharm , darmstadt , germany ) , and for e. histolytica with e. histolytica ii test ( techlab , blacksburg , virginia , usa ) commercial kits . immunoassays were performed following the corresponding manufacturer 's protocol . statistical package for social science ( spss ) program , version 16 for windows , was used for data entry and data analysis . summaries with descriptive statistics were generated and the data was further statistically analyzed according to the objectives of the study . appropriate statistical tests ( parametric or nonparametric tests ) were used according to the type of data whether qualitative or quantitative . among 100 participants , 67 were males ( mean age of 54.4  12.8 ) and 33 were females ( mean age of 51.9  14.3 ) . eighty - three participants were urban , while the remaining 13 participants were from remote rural areas . forty - seven patients had travel history to one tropical country 13 months before their diarrheal episodes . twenty - six participants had contact with one or more domestic animal 3 - 4 weeks prior to their diarrheal episodes . treated bottled water was the main drinking water resource for 56 participants , followed by desalted sea water for 31 participants and the underground well water for 13 participants . intestinal protozoa were detected in 21 ( 42% ) cases and 14 ( 28% ) controls , with an overall prevalence rate of 35% . were the most frequently identified parasite ( 12/100 ; 12% ) followed by g. duodenalis ( 11/100 ; 11% ) and cryptosporidium spp . , g. duodenalis , c. cayetanensis , and e. histolytica were identified in cases at infection rates of 16% ( 8/50 ) , 10% ( 5/50 ) , 10% ( 5/50 ) , 4% ( 2/50 ) , and 2% ( 1/50 ) , respectively . in the control group , blastocystis spp . g. duodenalis , and e. histolytica were diagnosed with prevalence rates of 8% ( 4/50 ) , 6% ( 3/50 ) , 12% ( 6/50 ) , and 2% ( 1/50 ) , respectively ( figure 1 ) . concomitant parasitic infections were described in 5 cases ( 5/100 ; 5% ) and were absent in the control group . were concurrently identified with g. duodenalis in a case and with c. cayetanensis in another . were concomitantly detected with e. histolytica in a case and with g. duodenalis in two cases . no helminths infection was observed for either cases or the control group . neither c. belli nor microsporidia spp . importantly , 2 participants , one from each group , diagnosed as positives for e. histolytica / e . dispar by microscopy , were proved as e. histolytica positives by the elisa test . equally important , identification of protozoan cysts / oocysts in patients feces ( figure 2 ) was confirmed by the corresponding immunoassay . concerning the parasitized cases ( n = 21 ) , 14 ( 66.7% ) were males and 7 ( 33.3% ) were females . infections were more frequent in cases from urban areas ( 16/21 ; 76.2% ) than those residing rural areas ( 5/21 ; 23.8% ) . eleven cases ( 11/21 ; 52.4% ) had travel history and 10 ( 10/21 ; 47.6% ) cases had not . among the parasitized controls ( n = 14 ) , intestinal protozoa were identified more in males ( 9/14 ; 64.3% ) than in females ( 5/14 ; 35.7% ) , in persons from urban areas ( 9/14 ; 64.3% ) than those residing rural areas ( 5/14 ; 35.7% ) , and in participants with travel history ( 7/14 ; 50% ) than those without ( 7/14 ; 50% ) . no significant differences were statistically observed for any of these variables between the parasitized cases and controls ( table 1 ) . acute and transient diarrhea was reported in 71% ( 15/21 ) of the parasitized cases and by all parasitized controls ( 14/14 ; 100% ) , with no significant differences observed ( p = 0.06 ) between both groups . diarrhea continued for a duration of more than 2 weeks only in 28.6% ( 6/21 ) of protozoa - positive cases . positive cases , one blastocystis spp . positive case , and one c. cayetanensis positive case . mucous and blood were seen in feces of two cases ( 9.5% ; 2/21 ) and one control ( 7.1% ; 1/14 ) . the only patient in the control group who reported mucous and blood in his feces was e. histolytica positive . bloating / flatulence , abdominal pain , general fatigue , and nausea / vomiting were the common symptoms found associated with the diarrhea ( table 2 ) . diarrhea associated with abdominal pain and fatigue sensation was reported by all the parasitized cases . diarrhea associated with nausea / vomiting , bloating / flatulence sensation , and losing some weight was reported by 57% ( 12/21 ) , 76% ( 16/21 ) , and 9.5% ( 2/21 ) of the parasitized cases , respectively . among the parasitized controls ( n = 14 ) , diarrhea accompanied with symptoms of abdominal pain , nausea / vomiting , bloating / flatulence sensation , feeling fatigued , and losing weight was practiced by 79% ( 11/14 ) , 93% ( 13/14 ) , 71% ( 10/14 ) , and 100% ( 14/14 ) of patients , respectively . significant differences were found between parasitized cases and parasitized controls regarding the association of their diarrhea with abdominal pain ( p = 0.05 ) and with nausea / vomiting ( p = 0.02 ) . diarrhea was often mild ( 87.5% of cases versus 100% of controls ) and nondysenteric ( 87.5% of cases versus 100% of controls ) . the commonly associated symptoms were abdominal pain , fatigue ( 100% of cases and controls ) , nausea / vomiting ( 87.5% of cases versus 100% of controls ) , flatulence / bloating ( 75% of cases versus 25% of controls ) , and losing weight ( 12.5% of cases versus 0% of controls ) . no significant difference was found between the parasitized cases and controls ( table 3 ) . cryptosporidium species associated diarrhea was often mild ( 60% of cases versus 100% of controls ) and nondysenteric in all parasitized patients . the commonly reported associated symptoms were fatigue ( 100% of cases and controls ) , abdominal pain ( 100% of cases versus 33.3% of controls ) , nausea / vomiting ( 60% of cases versus 100% of controls ) , flatulence / bloating ( 80% of cases versus 100% of controls ) , and losing weight ( 12.5% of cases and controls ) . statistically , no significant difference was described for any of these symptoms between the parasitized patients in both groups ( table 4 ) . as described in table 5 , g. duodenalis associated diarrhea was frequently mild ( 60% of cases versus 100% of controls ) and nondysenteric ( 100% of cases and 83.3% of controls ) . the common associated symptoms were fatigue ( 100% of cases and controls ) , abdominal pain ( 100% of cases versus 83.3% of controls ) , nausea / vomiting ( 80% of cases versus 83.3% of controls ) , flatulence / bloating complaints ( 100% of cases versus 83.3% of controls ) , and losing weight ( 00% of cases versus 9% of controls ) . in the study 's population , protozoan infections associated with diarrhea were common ( 35% ) . intestinal protozoa were identified more in cases than in control group , reflecting the high association between the isolated protozoa and diarrhea in patient with crf . in agreement with this finding , in one case - control study carried out in turkey , protozoan infections have been described in 44% ( 62/142 ) of cases and 13% ( 19/150 ) of controls . in another study executed in brazil , kulik et al . have reported these infections in 45% ( 33/86 ) of cases and in 26% ( 36/146 ) of controls . have reported more protozoan infections ( 61% ) in controls than in cases from brazil ( 52% ) . it is far important to announce that the three aforementioned studies were community based . according to the literature , infections with intestinal protozoan infections are commonly asymptomatic . therefore , high proportions of asymptomatic infections are expected in these studies ; on the contrary , in hospital or physician - based study , like our study , where the control group was selected from those patients suffering from diarrhea , asymptomatic protozoan infection was misdiagnosed . this may put an explanation for the high frequency of protozoan infections in the above studies , especially in the control groups compared to our study . perhaps , variation of the geographical distribution of protozoa , the socioeconomic status of the target population , the demographic characters of participants , and the adopted methodology may be additional explanations for prevalence variations of these protozoan infections among studies . it has been argued that crf predispose patients to helminths   but this was not the case in our study where no helminths were detected in all participants . most of our study 's participants were urban , middle aged , and using treated bottled water for drinking . these demographic characters may give reasons for the absence of worms ' infections that have been broadly recognized by their intimate connection with poverty , remote rural areas , and young ages . another important reason to be considered is that patients with crf usually change their health behavior and receive much more health care attention than their peers in populations . these protozoa were detected more in cases than in controls , consistent with earlier studies [ 16 , 17 ] . higher prevalence rates ( 24% ; 34/142 in cases versus 10% ; 16/150 in the controls ) of blastocystis spp . have been identified more in controls ( 42% ; 36/86 ) than in cases ( 24.5% ; 27/110 ) , according to gil et al . . in a cross - sectional iranian study , blastocystis spp . , with prevalence rate of 13.6% ( 12/88 ) , have been reported in dialysis patients . lower prevalence rates ( 0.314% ) have been previously reported for this intestinal protozoan in saudi normal populations [ 2125 ] . considering the opportunistic enteric protozoa , neither c. belli nor microsporidia spp . were identified in our study . except for a recently published case report for c. belli infection overwhelming a patient with eosinophilic gastroenteritis , low frequency of microsporidium spp . infections ( 2% ; 3/142 ) has been reported in turkish patients with crf . high prevalence rate of cryptosporidium spp . was revealed in the present study . while higher rates ( 1135% ) have been described in earlier studies [ 2729 ] , lower estimates ( 28% ) infection has been stated with prevalence rate of 2.78.1% in saudi general populations [ 23 , 25 ] . to the best of our knowledge , this protozoan has never been reported in patients with crf . in saudi arabia , c. cayetanensis has been rarely detected even in immunocompromised patients . infections were identified more in the control group , compatible with an earlier study   and incompatible with another . moreover , e. histolytica , the tissue - invasive intestinal protozoan , was rarely determined among cases in this study , consistent with a previous study   and inconsistent with other . prevalence rates of 635% have been described in saudi arabia [ 13 , 2124 ] . interestingly , in a study carried out in saudi population , an infection rate of 60% has been described . in the aforementioned studies the low sensitivity of microscopy and its incompetence to determine the morphologically identical species may put an explanation for these exaggerated prevalence rates . acute dysenteric diarrhea was described in six cases , two of them were found parasitized with blastocystis spp . concomitantly with g. duodenalis in one patient and with e. histolytica in the other . in addition , acute dysenteric diarrhea was observed in three controls , one of them was found parasitized with e. histolytica . in the presence of coinfection , it is very challenging to attribute patients ' symptoms to one protozoan and neglect the other . perhaps , one may attribute dysentery to e. histolytica being a well - known tissue - invasive parasite . one else may relate the dysenteric diarrhea to blastocystis infection relying on a previous research [ 34 , 35 ] . any other dysenteric cases that have not been supported by the literature require further investigations . this is the first study comparing the frequency of intestinal protozoa - associated diarrhea in crf patients and nonrenal controls from taif , saudi arabia . in this study , special considerations were given to avoid well - known potential confounders while enrolling patients and subsequent selection bias . another strength of our study was the inclusion of a number of immunoassays in the study 's methodology to avoid the low sensitivity of microscopy - based methods in diagnosis of certain parasites such as e. histolytica . nevertheless , our study was not free from the limitations . low number of cases was enrolled in this study due to the strict selection criteria adopted . we could not rely on the recent immune status of participants based on doing laboratory tests because many participants were reluctant to give blood samples . lastly , we did not include tests for intestinal bacteria and viruses that could be concomitantly present with protozoan infection(s ) , due to time and cost limits of our study . all these limitations plus performing longitudinal study have to be considered in the near future research . in conclusion , further studies are required before attributing diarrhea in patients with crf to a protozoan detected in patients ' feces . until these studies , we advise screening all diarrheal patients for intestinal protozoa in the study 's population , with greater concern that should be given to patients with crf .
it has been hypothesized that chronic renal failure ( crf ) predisposes patients to infection with intestinal protozoa . 
 we tested this hypothesis with a matched case - control study to determine the prevalence of these protozoa and their diarrhea associated symptoms among 50 patients with crf ( cases ) from taif , western saudi arabia . 
 fifty diarrheal patients without crf were recruited in the study as controls . 
 participants were interviewed by a structured questionnaire and stool samples were collected . 
 samples were thoroughly examined with microscopy and three coproantigens detection kits . 
 enteric protozoa were detected in 21 cases and 14 controls . 
 blastocystis spp . 
 were the most predominant parasite ( 16% in cases versus 8% in controls ) , followed by giardia duodenalis ( 10% in cases versus 12% in controls ) and cryptosporidium spp . 
 ( 10% in cases versus 6% in controls ) . 
 cyclospora cayetanensis was identified in two cases , while entamoeba histolytica was described in one case and one control . 
 intestinal parasitism was positively associated with the male gender , urban residence , and travel history . 
 clinical symptoms of nausea / vomiting and abdominal pain were significantly varied between the parasitized cases and controls ( p value  0.05 ) . given the results , we recommend screening all diarrheal feces for intestinal protozoa in the study 's population , particularly those with crf .