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| RESEARCH ARTICLE |
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| Year : 2014 | Volume
: 7
| Issue : 1 | Page : 56-67 |
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Subtype analysis of Blastocystis spp. isolates from symptomatic and asymptomatic patients in Suez Canal University Hospitals, Ismailia, Egypt
Sherif M Abaza1, Hanan Z Rayan1, Rasha H Soliman1, Nader A Nemr2, Amira B Mokhtar1
1 Department of Medical Parasitology, Faculty of Medicine, Suez Canal University, Ismailia, Egypt
2 Department of Endemic and Infectious Diseases, Faculty of Medicine, Suez Canal University, Ismailia, Egypt
| Date of Submission | 27-Nov-2013 |
| Date of Acceptance | 27-Jan-2014 |
| Date of Web Publication | 25-Sep-2014 |
Correspondence Address:
Sherif M Abaza
PhD, Department of Medical Parasitology, Faculty of Medicine, Suez Canal University, Ismailia
Egypt
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/1687-7942.139691
Background
Major symptoms associated with blastocystosis include diarrhea, abdominal pain, fatigue, constipation, flatulence, urticaria, and skin rash. It may play a significant role in several chronic gastrointestinal illnesses such as irritable bowel syndrome (IBS).
Objective
The main objective was to identify Blastocystis spp. subtypes (STs) of clinical isolates obtained from three different groups of patients: IBS and non-IBS with and without gastrointestinal tract symptoms. The secondary objective was to evaluate the infectivity and pathogenicity of detected STs from each group in experimental rats.
Patients and methods
This study was designed as a case-control study. Stool samples were collected from patients attending Suez Canal University Hospitals. Only positive samples for Blastocystis spp. were included in the study and the three groups were identified (19 patients each). Blastocystis spp. STs were identified using seven pairs of primers (SB83, SB155, SB227, SB332, SB340, SB336, and SB337) to explore the relationship of different STs with different clinical presentations of each group. Detected STs, from each group, were then used to evaluate the infectivity and pathogenicity in experimentally infected rats monitored by parasitological and histopathological parameters.
Results
STs using seven different sequence-tagged site primers revealed 54 isolates with single infection and three isolates with mixed infection. ST3 was the most common one in the present sample of Egyptian population (56.1%) followed by ST1 (35.1%), then ST2 (3.5%), whereas 5.3% were mixed infection (ST1 and ST3).
Conclusion
Our results showed that the clinical outcome of blastocystosis is not likely associated with a specific ST, although some STs are predominant in other epidemiologic studies. Keywords: Blastocystis spp., Egypt, epidemiology, genotyping, molecular, polymerase chain reaction
How to cite this article:
Abaza SM, Rayan HZ, Soliman RH, Nemr NA, Mokhtar AB. Subtype analysis of Blastocystis spp. isolates from symptomatic and asymptomatic patients in Suez Canal University Hospitals, Ismailia, Egypt. Parasitol United J 2014;7:56-67 |
How to cite this URL:
Abaza SM, Rayan HZ, Soliman RH, Nemr NA, Mokhtar AB. Subtype analysis of Blastocystis spp. isolates from symptomatic and asymptomatic patients in Suez Canal University Hospitals, Ismailia, Egypt. Parasitol United J [serial online] 2014 [cited 2023 Dec 3];7:56-67. Available from: http://www.new.puj.eg.net/text.asp?2014/7/1/56/139691 |
| Introduction | |  |
Blastocystis spp. are anaerobic parasites that inhabit human intestinal tract, besides a wide range of animals [1] . Blastocystis spp. are reported as the most common eukaryotic organism reported in human fecal samples, as infection is common in tropical, subtropical, and developing countries, with a prevalence rate ranging from 30 to 50% in some developing countries compared with 1.5-10% in some developed countries [2],[3],[4] . A higher risk for blastocystosis has been found in humans with close animal contact reinforcing its zoonotic potential [5] . Recently, findings concerning taxonomy, biology, and virulence of Blastocystis spp. were reviewed [6] .
Regarding the pathogenic potential of Blastocystis spp., it was widely debated in the literature during the last two decades because it may be found in both symptomatic and asymptomatic patients [7] . However, major symptoms associated with blastocystosis include diarrhea, abdominal pain, fatigue, constipation, flatulence, and urticaria and skin rash. In addition, it may play a significant role in several chronic gastrointestinal tract (GIT) illnesses such as irritable bowel syndrome (IBS) [8],[9],[10] . Researchers have classified IBS on the basis of stool consistency into IBS with constipation (IBS-C), IBS with diarrhea (IBS-D), IBS mixed type (IBS-M), and IBS unsubtyped (IBS-U). Patients with IBS-M have both hard and loose stools over periods of hours or days, whereas IBS-A patients have alternating bowel habits change over periods of weeks and months [11] . The group of IBS-M is the most prevalent group in primary care, identified in about 50% of referred patients [12],[13] .
At the morphological level, Blastocystis spp. is a polymorphic protozoon with four main forms described in stools and/or in-vitro cultures: vacuolar, granular, amoeboid, and cyst forms [14] . The water-resistant infective cyst represents its transmissible form [15] , whereas the irregular amoeboid form has been suggested as the pathogenic form [16] . Besides the heterogeneity in morphology, Blastocystis spp. is genetically and antigenically diverse, both within and among geographical regions, suggesting that several genotypes exist. Although several studies used rats [17],[18] and mice [19],[20] as suitable animal models to study the potential pathophysiological changes in clinical blastocystosis, no definite correlation between different subtypes (STs) and pathogenicity was determined.
The extensive genetic diversity of Blastocystis spp. was demonstrated using various molecular approaches including, in most cases, random amplified polymorphic DNA analysis, restriction fragment length polymorphism analysis, PCR using sequenced-tagged site primer, and dideoxy sequencing [7,21-23]. Recently, the potential application of the inexpensive matrix-assisted laser desorption/ionization time-of-flight mass spectrometry technique was evaluated to discriminate Blastocystis spp. STs [24] . The investigators found the method to be an effective technology for efficiently discriminating Blastocystis spp. STs in axenic cultures better than PCR. These studies showed that sequence divergence among Blastocystis spp. isolates of humans and animals reflected the existence of 17 Blastocystis spp. STs, with at least nine STs (1-9) found in humans [25],[26],[27],[28] . In Colombia, the investigators subtyped 446 Blastocystis spp.-positive samples from humans, birds, and mammals and observed the occurrence of STs 1, 2, and 3 in 34, 23, and 11.4%, respectively. STs 6 (19.8%) and 8 (10.5%) were detected only in birds and mammals. A lower proportion (0.8%) of ST4 was also found in the whole sample [29] . As the human Blastocystis spp. population is genetically highly polymorphic, it has been postulated that certain genotypes may contribute to its pathogenic potential [18],[30] . In addition, a recent study showed that ST3 Blastocystis spp. antigen caused a significant higher upregulation of cathepsin B, which may enhance the exacerbation of existing colon cancer cells by weakening the cellular immune response [31] . Therefore, the aim of the present study was to identify the influence of different STs of Blastocystis spp. clinical isolates from symptomatic and asymptomatic patients in Ismailia, Egypt in an attempt to determine the pathogenic potential of this parasite. In addition, we evaluated the infectivity and pathogenicity of the detected STs in experimental rats.
| Patients and methods | | |
Type of the study: case-control study.
The study was conducted in Medical Parasitology and Endemic and Infectious Diseases Departments, Faculty of Medicine, Suez Canal University, Ismailia, Egypt, during the period from October 2011 to May 2012.
Patients
The target population included patients attending Gastroenterology and Endemic Diseases Clinics in Suez Canal University Hospital in three groups. Group I included IBS patients diagnosed according to the Rome III criteria [11] . Group II included non-IBS GIT symptomatic patients, whereas group III included patients without any GIT symptoms. All patients proved to harbor Blastocystis spp. The inclusion criteria included Blastocystis hominis-infected patients and eligible patients from 15 to 60 years of age and of both sexes. Exclusion criteria included B. hominis-infected patients with associated pathogens (parasite or bacteria) that may be responsible for GIT symptoms and/or IBS. The sample size was calculated according to the following equation [32] : n = [(p1q1 + p2q2 )/(p2 -p1 ) 2 ] × f, where n is the number of patients per group, p1 is the prevalence of B. hominis in IBS patients (46%), p2 is the prevalence of B. hominis in control (7%) [8] , q1 = 1-p1 and q2 = 1-p2 , and f (a, power) for a two-tailed test was 7.9 at significant level (a) of 0.05 with a power of 0.80. The number in each group was calculated as 17 patients per group, and by adding 10% drop out, which is equal to 1.7, the number was 19 per group. A total of 305 stool samples were examined to collect 57 Blastocystis spp.-positive patients. Each patient was subjected to the following: (i) questionnaire with a thorough medical history covering the personal data, source of drinking water, special habits of food handling, contact with animals, presence of GIT symptoms related to B. hominis infection (diarrhea, abdominal pain, constipation, flatulence) or dermatological symptoms, current health status, previous parasitic infections, and drug intake and (ii) complete clinical examination including general and abdominal examination to exclude other conditions.
Stool samples
All patients were asked to provide three stool samples in clean, wide, disposable stool containers, and then each sample was examined by direct smear examination and iodine staining and by formalin ethyl acetate concentration technique [33] . Only positive samples for B. hominis were included in the study. The stool samples were also examined using trichrome and acid fast trichrome stains [33] to exclude other parasitic causes of GIT symptoms. In addition, stool samples were subjected to stool culture using Salmonella-Shigella and MacConkey agar [34] to exclude common pathogenic bacteria causing GIT symptoms. Blastocystis spp.-positive samples were cultivated in Jones' medium [35] . Consecutive subculturing every 3-4 days in fresh medium was performed. After 1-2 subcultures, Blastocystis spp. isolates were collected by centrifugation and packed organisms were frozen at -20°C until DNA extraction.
DNA extraction [22]
Genomic DNA was extracted from positive stool cultures with a DNA extraction kit, according to manufacturer's directions (QIAamp; Qiagen Inc., Hilden, Germany).
Polymerase chain reaction
To identify the STs of Blastocystis spp., a PCR was carried out using seven pairs of primers (SB83, SB155, SB227, SB332, SB340, SB336, and SB337) [Table 1] [22] . Two microliters of extracted DNA was used as template for amplification in a 25-ml reaction mixture containing 25 pmol/l of each primer pair (of one ST at a time), 12.5 μl Quick-Load Taq 2× Master Mix, (New England Biolabs Inc, Ipswich, MA, USA) and 9.5 μl nuclease-free water. It was followed by 40 cycles, including denaturing at 94°C for 30 second, annealing at 58°C for 30 second, and extending at 72°C for 1 minute, and an additional cycle with a 5 minute chain elongation at 72°C (Thermocycler; Eppendorf, Hamburg, Germany). The amplification products were electrophoresed in 1.5% agarose gels (Promega, Madison, WI, USA) and Tris-borate-EDTA buffer. Gels were stained with ethidium bromide and photographed using an ultraviolet gel documentation system (Uvitec, Cambridge, UK). The PCR amplification for each primer pair was performed twice for each isolate [Table 1]. | Table 1: Sequenced-tagged sites primers used in polymerase chain reaction-based Blastocystis spp. subtyping [22]
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Assessment of infectivity and pathogenicity of Blastocystis subtypes
Experimental animals
Four-week-old SPF Wistar male rats obtained from Theodor Bilharz Research Institute, Egypt, were used in this study. They were housed in polycarbonate cages with paper bedding and were fed a normal diet of commercial pellets and given potable water ad libitum. The animals were maintained in an animal house at 25°C, with a relative humidity of 40-60% and under a 12 hour light/dark cycle. The cages and paper bedding were changed at weekly intervals. Three groups of rats were used for each group of patients. The rats were divided into subgroups; each subgroup included four rats infected with one isolate of each of the detected STs in the corresponding group of patients. As we detected two STs in the IBS and non-IBS group without symptoms and three STs in the non-IBS group with GIT symptoms, the total number of rats in the present study was seven subgroups×four rats = 28 rats. Only isolates with parasite number equal to 5 × 10 6 /ml were used in animal experimental studies.
Animal experimental infection
Blastocystis spp. organisms in 3-4 day-old cultures were purified by the subtraction method of centrifugation [36] . Blastocystis spp. cells were first isolated by centrifugation at 500 g for 10 minute. The supernatant culture media was discarded and the pellet was washed with ringer solution and centrifuged at 350 g for 5 minute at room temperature. This was repeated at least five times to reduce the bacterial load in each isolate and to exclude bacteria as a possible cause of histopathology occurring in experimental studies. After counting the number of cells using a hemacytometer, the in-vitro culture forms were adjusted to 5 × 10 6 /ml. Cells were immediately inoculated orally into the animals by 16-G ball-tipped feeding needle attached to 1 ml syringe. One rat, for each subgroup, was used as a negative control by inoculation of 1 ml parasite-free culture medium in each. Control rat for each subgroup was housed alone in a separate place because of fear of feco-oral transmission of infection. Meanwhile, only one rat was put in the cage for easy collection of stool samples for the diagnosis of infection. Care was taken while cleaning and feeding of rats in each group and subgroup to avoid cross infection. All rats were screened for the presence of infection daily for 1 week postinoculation (PI). Stool samples from all rats were screened for the presence of infection daily for 1 week PI by wet mount preparation or culture in Jones' medium supplemented with 10% horse serum in case of negative specimen. Culture was considered negative if the organism was absent until the 7 th day. All rats were monitored for weight loss, presence of loose stool or mucus in stool, lethargy, and fur loss.
Two weeks PI, all rats were anesthetized and killed. Large intestine (colon and cecum) was assessed by naked eye, removed, preserved in 10% formalin, and processed in paraffin blocks. Intestinal sections were stained with H&E for histopathology examination [37] , which was graded as mild, moderate, or severe according to the degree of inflammatory cell infiltrate with or without tissue destruction [38] .
Statistical analysis
Collected data were entered into Microsoft Excel software file and SPSS (version 16.0; SPSS Inc., Chicago, Illinois, USA) software for analysis. Data were presented by descriptive tables and appropriate figures. Significance of statistical difference was measured by the χ2 and Fisher's exact tests. The significance level was 0.05.
Ethical considerations
Written consent was obtained from patients before the study, and they were later given the results of their stool examinations. Treatment by suitable antiparasitic drugs was prescribed for infected patients, followed by stool re-examination to ensure parasite elimination. General principles for the care and use of animals for scientific purposes were followed as recommended by the National Advisory Committee for Laboratory Animal Research [39] .
| Results | | |
Slightly more than half (52.6%) of IBS patients lived in rural areas, whereas the majority of acute GIT symptomatic patients (63.2%) and asymptomatic patients (78.9%) were from urban areas. Group II included non-IBS acute GIT symptomatic patients suffering from one or more of the following symptoms for less than 14 days: diarrhea, abdominal pain or discomfort, constipation, flatulence, nausea, vomiting, and loss of appetite. Most of the IBS patients (63.2%) had a history of contact with animals, compared with 52.6% of acute GIT symptomatic patients and 26.3% of asymptomatic patients [Table 2].
Abdominal pain was the main symptom (89.5%), followed by diarrhea (84.2%) and flatulence (15.8%), whereas vomiting and fever were the least frequent symptoms. Semiformed stool was detected more frequently than watery stool (52.6 vs. 31.6% of the patients) [Table 3]. | Table 3: Distribution of symptoms in the acute gastrointestinal tract symptomatic group
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The majority of patients (45/57, 78.9%) were found to have infection with less than five Blastocystis spp. parasites/high power field (HPF). Only four, three, and five patients (21.1, 15.8, and 26.3%) had at least five organisms/HPF in the IBS, acute GIT symptomatic, and asymptomatic groups, respectively, with no statistically significant differences (P = 0.918) (data not shown).
On examining the 3-4-day-old culture media, Blastocystis spp. vacuolar form was the most frequently detected form, followed by the granular form. The multivacuolar form was found to predominate in four patients in the asymptomatic group. The cyst form was detected in the asymptomatic and IBS groups more frequently than the acute GIT symptomatic group [Figure 1] and [Figure 2]. With respect to the presence of Blastocystis spp. amoeboid form in 3-4-day-old cultures, it was detected in most of the acute GIT symptomatic group (84.2%) compared with 15.8% in the IBS group and 0% in the asymptomatic group. This difference was statistically significant (P < 0.001) [Figure 3] (data not shown). | Figure 1: Different morphological forms of Blastocystis spp. Vacuolar forms: (a) wet mount, (b) iodine stain, (c) iodine stain with the vacuole darkly stained, (d) trichrome stain, (e) trichrome stain with the vacuole stained red. Granular forms: (f) wet mount, (g) iodine stain, (h) trichrome stain. Amoeboid forms: (i, j) wet mount showing pseudopodia (arrowheads). Multivacuolar forms: (k) wet mount, (l) iodine stain. ×1000.
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 | Figure 2: Blastocystis spp. fecal cyst forms with loose outer coat: (a) wet mount, (b– d) iodine stain, (e) trichrome stain, (f, g) fecal cysts stained with iodine, and (h) trichrome stain. It is noted that (a)– (e) have loose outer coat and (f)– (h) are cysts with no loose outer coat. ×1000.
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 | Figure 3: Amoeboid form in 3– 4-day-old cultures among the study groups. GIT, gastrointestinal tract; IBS; irritable bowel syndrome.
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Of the seven primers used, 20 (35.1%) isolates were amplified with SB83 primer (˜351 bp), which was determined as ST1. Only two (3.5%) isolates were amplified with SB340 primer (˜704 bp), which was determined as ST2, whereas 32 (56.1%) isolates were amplified with SB227 primer (˜526 bp), which was determined as ST3. Three (5.3%) isolates showed positive amplification with both SB83 and SB227 primers; therefore, these isolates were judged to have been a mixed infection with STs 1 and 3. Regarding the relationship of Blastocystis spp. STs with different groups, no statistically significant differences in distribution of the STs among the groups were observed (P > 0.05) [Figure 4] and [Table 4]. | Table 4: Detection rate of Blastocystis spp. genotypes among the studied groups
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 | Figure 4: Example of Blastocystis spp. subtypes (STs) from patients in the asymptomatic group. M is the ladder DNA at 100 bp. Lane 1 is the negative control sample. (a) ST1 (351 bp) in lanes 2, 4, 6, 7, 9, and
10. (b) ST3 (526 bp) in lanes 3, 5, 6, 8, and 11. From (a) and (b) the isolate in lane 6 shows coinfection with STs 1 and 3.
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STs 1 and 3 were more commonly identified (65 and 62.5%, respectively) among the residents of urban areas, whereas ST2 infections were detected only in residents of rural areas. The difference was not statistically significant (P > 0.05). The majority of patients with STs 1 and 3 had no history of contact with animals (55 and 50%, respectively), whereas two patients with ST2 infection were living in rural areas and were in contact with fowls and cattle, with no significant statistical difference (P > 0.05). Distribution of the detected STs in clinical IBS types also did not show statistically significant difference (P > 0.05). However, diarrhea-predominant IBS (IBS-D) was the most frequent IBS type detected in patients infected with ST1 (50%), whereas for ST3 infection, IBS-D and IBS-M were the predominant IBS clinical types with 30.8% each [Table 5]. | Table 5: Distribution of Blastocystis spp. subtypes according to residence, animal contact, and clinical irritable bowel syndrome types
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Assessment of infectivity and pathogenicity of different Blastocystis spp. subtypes in experimentally infected rats
All isolates that were identified as representatives of the detected STs (1, 2, and 3) were infectious to rats. All rats showed positive infection 3-4 days PI. Rats infected with STs isolated from patients of the IBS and asymptomatic groups (STs 1 and 3) showed normal stool consistency. Gross examination of the large intestine showed normal features and no bloating of cecum or colon [similar to features detected in noninfected rats [Figure 5]a. Rats infected with STs isolated from patients of the acute GIT symptomatic group (all detected STs) showed bloated cecum and colon and the distal colon showed semiformed or diarrheic stool [Figure 5]b and c. Only ST1 induced mortality in one (25%) rat, which was preceded by lethargy for 2 days. | Figure 5: (a) Gross appearance of large intestine of normal non-infected rat (arrow), (b) Gross appearance of large intestine of rat infected by Blastocystis spp. isolated from patients of acute GIT symptomatic group (all detected STs) showing bloated caecum and colon with distal colon containing semi-formed stool (arrow), (c) From the same group with the distal colon containing diarrheic stool (arrow).
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Tissue sections from rats infected with STs isolated from patients of the IBS group (STs 1 and 3) showed mild to moderate inflammatory reaction in the cecum and mild inflammatory reaction in the colon. Both STs showed similar histopathological features. Tissue sections from rats infected with STs isolated from patients of the acute GIT symptomatic group (all detected STs) showed intense inflammatory reaction throughout the mucosal layer. Some areas in the mucosa showed polypoid projections with fibrovascular cores covered by hyperplastic mucosa (mainly in cecum) [Figure 6]. Blastocystis spp. parasites were detected at the edge of the mucosal epithelium, trapped in the epithelial lining of the crypts near the surface or deep in mucosa, and sometimes invading the stroma [Figure 7]. | Figure 6: Tissue section from a rat infected with Blastocystis spp. isolated from patients of the acute gastrointestinal tract symptomatic group showing polypoid projections with fibrovascular cores. H&E, ×400.
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 | Figure 7: Tissue section showing (a) Blastocystis spp. vacuolar form (arrow) trapped in the epithelial lining of the crypts deep in the colon mucosa, (b) another vacuolar form (arrow) invading the cecal stroma, which appears intensely infi ltrated by infl ammatory cells. H&E, ×1000.
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| Discussion | | |
Blastocystis spp. is commonly found in human stool; yet, its role as a pathogen is still unclear [40] . The pathogenic potential of Blastocystis spp. remains controversial with numerous conflicting reports regarding its ability to cause disease. This is because it is frequently found not only in patients with GIT symptoms, but also in asymptomatic patients [41] . Moreover, the association of Blastocystis spp. with IBS was reported to be frequently encountered in daily practice, suggesting a possible role for the parasite in IBS etiology [42] .
In the present study, abdominal pain was the most frequent symptom among acute GIT symptomatic patients (89.5%), followed by diarrhea (84.2%) and flatulence (15.8%). These symptoms were often reported in association with symptomatic Blastocystis spp. infection by several studies [43],[44],[45] . Semiformed diarrhea was the most common form reported in the present study (52.6%) compared with watery diarrhea (31.6%). Anorexia, nausea, vomiting, and fever were also reported in the present study. These symptoms were accounted by other studies [46],[47] , where diarrhea-predominant IBS (IBS-D) was the most frequent IBS clinical ST detected among IBS patients with blastocystosis (36.8%), followed by IBS-C (26.3%) and IBS-M (21.1%). In agreement with our results, Blastocystis spp. was detected in a high percentage of patients with IBS-D (71%) [42],[48] . However, in another study, IBS-C was the most frequent clinical ST detected among IBS patients with blastocystosis (64%) followed by IBS-D (24%) [49] . The predominance of IBS-D among IBS patients with blastocystosis in the present study could be explained by the fact that the washing effect of diarrhea to a large number of parasites may lead to easy detection of the parasite in liquid than in formed stools [50] .
In the present study, no correlation was found between parasite abundance and clinical presentation of different groups. This result is in agreement with another report, stating that only two of the 36 (6%) symptomatic patients and six of the 63 (10%) asymptomatic patients had five or more Blastocystis spp. parasite/HPF [46] . Moreover, it was reported that only few IBS patients had large quantities of Blastocystis spp. parasites in their stool samples [51] . More recently, a highly sensitive real-time qPCR assay was developed to detect Blastocystis spp. parasites in stool samples, and the investigators found no correlation between parasite count and clinical expression of symptoms in patients with blood malignancy compared with healthy controls. They suggested that parasite shedding in stools does not reflect interactions with the host in the digestive tract [52] . In contrast, there was an old report on patients with IBS who were significantly more likely to have at least five organisms/HPF (7.4%) compared with 2% of non-IBS GIT symptomatic patients, suggesting that an abnormal intestinal condition in IBS patients may provide an environment suitable for Blastocystis spp. proliferation [53] . The reason for this discrepancy is presently unclear but may be due to genotype differences among Blastocystis spp. isolates or host factors such as age and genetic background variations in the populations studied [54] .
In the present study, the amoeboid form was detected in culture from most of the acute GIT symptomatic group (84.2%) and only in 15.8% of the IBS group, whereas none of the asymptomatic group showed the presence of the amoeboid form during the 3-4 days cultivation period. This result was statistically significant (P < 0.001). These forms were detected in cultures of all symptomatic isolates (10 patients), whereas none were detected in cultures of asymptomatic isolates (10 patients) throughout the 10-days period of in-vitro growth. As postulated by Tan and Suresh [16] , the presence of these amoeboid forms could be a contributing factor to pathogenicity. In addition, it was reported that the most predominant stages detected in stool and culture samples of symptomatic patients were (in a descending order) the vacuolar, granular, and amoeboid forms, whereas other forms (multivacuolar and cyst forms) were detected less frequently [55] . In contrast, amoeboid forms were detected in culture of two isolates from asymptomatic patients, suggesting that the presence of this form may be poorly correlated with patient symptoms [7] . However, Vassalos et al. [56] reported an asymptomatic Blastocystis spp. carrier in whom symptoms developed within a short period after the initial testing for Blastocystis spp. Hence, it is postulated that the amoeboid form could be the life cycle stage that contributes to the pathogenicity of Blastocystis spp. [16] .
The genetic diversity among human Blastocystis spp. population has supported the hypothesis that certain STs may contribute to the clinical outcome of infections. Therefore, the availability of data on Blastocystis spp. STs found in different patient groups would be of interest in understanding the pathogenic potential of the parasite [57] . A wide varieties of STs (1-17) were identified [27] , and Blastocystis spp.-rDNA sequence data were able to detect 15 STs from captive and free-living nonhuman primates [58] . In our study, the identity of Blastocystis spp. STs was investigated by PCR with seven different primers. It was anticipated that this study would provide further knowledge on the pathogenicity of the different Blastocystis spp. STs, as well as a link to human GIT diseases such as acute diarrheal illnesses and IBS. Three Blastocystis spp. STs variants were identified in our study (1, 2, and 3); ST3 was the most dominant (56.1%) followed by ST1 (35.1%), whereas ST2 was the least detected (3.5%). The other STs (4-7) were not detected among the study population. Epidemiological studies from other locations around the world have also identified ST3 as the predominant variant in humans: in Turkey 75.9% [59] , Thailand 57.1% [60] , France 53.5% [7] , Iran 53% [61] , Sweden 47.6% [62] , Italy 47.1% [63] , Australia 44% [64] , and the Netherlands 42% [65] . In Egypt, ST3 also predominated in 61.9% [66] , in 54.5% [18] , and in 49% [67] of Blastocystis spp. isolates. In contrast, few studies have identified other STs as predominant variant. In three studies, ST1 predominated in humans in Thailand, China, and Brazil with a prevalence of 94.8, 51.4, and 41%, respectively [68],[69],[70] . ST4 was detected in 84.1% of isolates from two rural communities in Nepal, with none belonging to ST3 [71] .
In the present study, ST1 was the second dominant variant detected (35.1%). ST1 also came second after ST3 in Iran (48%) [61] , Australia (31%) [64] , France (25.6%) [7] , China (24.5%) [72] , and the Netherlands (22%) [65] . In Egypt, as detected by Hussein et al. [18] and Souppart et al. [66] , ST1 shared the second position with both ST6 (18.2% each) and ST2 (19% each), respectively. The present ST distribution in the Lebanese population was reported as ST3 (33.3%), ST2 (33.3%), ST1 (30.6%), and ST4 (2.8%), with ST1 significantly more prevalent among symptomatic patients of this population [73] .
In our series, ST2 was the least detected (3.5%). This was in agreement with other studies reporting ST2 at minor percentages. It was reported in 3.2, 4.7, 5.0, and 9.3% of isolates from Pakistan [42] , Australia [64] , China [69] , and France [7] , respectively. In the Netherlands, however, ST2 was detected in 22.0% of their Blastocystis spp. isolates [65] . Globally, other genotypes (5-8) were identified sporadically and at lower frequencies [65],[74] . Such variations in the number and relative proportions of STs between countries as well as within the same country could reflect true differences between communities and might indicate divergent epidemiological contexts including reservoirs and ways of transmission [72] . It was postulated that these differences are likely due to prevailing local living conditions and customs rather than differences in the susceptibility to infection [66] .
In the current study, no significant difference was observed in the distribution of STs 1 or 3 among the study groups. Blastocystis spp. ST2 was detected only in the acute GIT symptomatic group. These results were in agreement with other studies denying any association between the presence of symptoms and Blastocystis spp. STs. Yoshikawa et al. [22] detected STs 1 and 3 in both asymptomatic patients and GIT symptomatic patients (15 and 11 isolates, respectively) in Bangladesh, concluding no correlation between STs and Blastocystis spp. pathogenic potential. In Turkey, STs 1, 2, and 3 were detected in isolates from asymptomatic patients (18 isolates) and STs 1, 2, 3, and 4 in isolates from symptomatic patients (69 isolates) with no association between Blastocystis spp. STs and the presence of symptoms [59] . In France, the investigators detected STs 1, 3, 4, and 7 in isolates from asymptomatic patients (12 isolates) and STs 1, 2, 3, and 4 in isolates from symptomatic patients (19 isolates) [7] . Interestingly, all ST2 isolates were associated with symptoms; however, no significant differences in distribution of the STs between the symptomatic and asymptomatic groups were observed. In addition, other Egyptian investigators found no definite correlation between genetically distinct STs and pathogenicity [75] . In the same year, Turkish investigators also identified STs 2 and 3 in patients with IBS but with no correlation between symptoms and STs, as the same STs were detected in their asymptomatic group [57] . Moreover, a recent study found that STs 1 and 3 were the most frequent variants in both the IBS and non-IBS control groups, concluding lack of correlation between the presence of IBS and any particular ST [49] .
In contrast, Yan et al. [69] detected only ST1 in a group of symptomatic patients, whereas ST3 was isolated predominantly from asymptomatic individuals. Similar results were obtained in Egypt [18] and Turkey [76] . The Egyptian investigators found STs 1, 3, and 6 in isolates from GIT symptomatic patients, whereas STs 3, 6, and 7 were isolated from asymptomatic patients, and they concluded that ST1 was relevant to the blastocystosis pathogenicity, whereas ST7 was irrelevant and suggested the presence of pathogenic and nonpathogenic strains among STs 3 and 6. Moreover, ST1 was the predominant variant among IBS patients with diarrhea, whereas ST3 predominated among healthy controls [42] . The same result was obtained by another Egyptian study conducted 1 year later, which reported that ST1 was the most pathogenic variant of B. hominis isolates in patients with IBS, whereas ST2 was not detected among those patients [67] .
In our study no statistically significant differences were observed between the detected Blastocystis STs, residence of patients (urban or rural) and contact with specific animals, although both cases with ST2 were living in rural areas and in contact with fowls and cattle. In contrast, other studies linked the predominance of ST3 to urbanized areas where there is limited opportunity for zoonotic transmission, such as in Singapore [23] and in Sydney, Australia [64] . Indeed, further studies to identify specific risk factors for infection with different Blastocystis spp. STs have to be performed in Egypt to explore the potential association of STs with specific environmental compartments and reservoirs [66] .
In our animal experimental study, all isolated Blastocystis spp. STs (1, 2, and 3) were infectious to rats, which shows that rats are suitable animal models for studying the pathogenicity of blastocystosis. This result is in agreement with several studies [18, 77, 78]. In addition, the infectivity of various Blastocystis spp. STs from humans (2, 3, 4, and 7) was studied in rats and chickens using two different isolates for each ST. There was variability in the infectivity of the isolates, as STs 2, 4, and 7 were capable of infecting chickens, whereas two isolates of ST2 and one isolate of each of STs 4 and 7 were capable of infecting rats. However, in another study, ST3 could not infect chickens or rats [17] .
In our study, different degrees of histopathological changes were reported from infected rats. Tissue sections of the large intestine from rats infected with STs isolated from patients in both the IBS and asymptomatic groups (I and III) were roughly similar, with mild inflammatory reaction detected in the colonic and cecal mucosa (sometimes moderate inflammatory reaction was detected in the cecum of rats in the IBS group). This histopathological reaction was similar to that recorded in another Egyptian study [18] , which reported mild pathological changes in rats inoculated by isolates from asymptomatic individuals, irrespective of the ST to which these isolates belonged (STs 3, 6, and 7). However, Eida [19] found that mice infected by isolates from asymptomatic individuals showed normal histological appearance of their large intestine and only one showed scanty inflammatory cells in the mucosa of large intestine.
Our histopathological findings in rats infected with isolates from IBS patients were similar to those detected in a study conducted in 2004, which showed that examination of the rectal and sigmoid colonic tissues of IBS patients with blastocystosis revealed nonspecific inflammation with mild increase in inflammatory cells [8] . More recently, another study found normal colonoscopic picture in all patients with IBS infected with STs 1 and 3 [49] . In contrast, on examination of sigmoidoscopic biopsies from IBS patients, Fouad et al. [67] found that severe inflammation was present mainly in patients harboring ST1 isolates (26.66%) and concluded that it is the most pathogenic ST.
Low-grade inflammation is a proposed mechanism of IBS through persistent antigenic exposure as in persistent carriage/infection with Blastocystis spp. parasite [79] . Moreover, it was postulated that the clinical outcomes of blastocystosis in humans are not only determined by the presence of the organism itself, but also are probably associated with host genetics, immune status, and/or intrinsic factors [80] . It was suggested that some IL-8 and IL-10 single-nucleotide polymorphisms could change individual susceptibility increasing the relative risk in the development of IBS in Blastocystis spp. carriers [81] .
In the present study, tissue sections of the large intestinal mucosa from rats infected with STs 1, 2, and 3 isolated from patients in the acute GIT symptomatic group showed severe inflammatory reactions, and Blastocystis spp. vacuolar forms were detected at the edge of the mucosal epithelium and invading the mucosa. Only ST1 induced mortality in 25% of infected rats in this group. In agreement with our results, other Egyptian investigators [82] reported intense inflammatory reactions in the large intestines of mice infected by isolates from symptomatic patients. They reported detection of the parasite at the mucosal epithelium but with no tissue invasion. The ultrastructural changes of intestinal mucosa in Blastocystis spp.-infected mice studied by SEM [83] revealed individual parasites invading the ileocecal mucosa and partially destroying the mucosal microvilli, with lymphocyte infiltration and eosinophilia in intercellular stroma. In addition, Hussein et al. [18] found that all ST1 and the majority of ST3 symptomatic isolates induced a severe degree of pathological changes in experimentally infected rats with polypoid projection in colonic mucosa of rats infected by ST1 isolates, which was referred to as a precancerous polyp. They also reported mortality in 25% of rats infected by ST1 isolates. A recent study showed that Blastocystis spp. isolates from GIT symptomatic patients caused active colitis with infiltration of mixed inflammatory cells in experimentally infected mice, and the parasite was found invading the lamina propria, submucosa, and muscle layers in the form of collections of vacuolar forms [84] .
The findings from experimentally infected rats in our study indicated that the severity of pathological changes is not dependent on the inoculated Blastocystis spp. ST but rather on the clinical presentation of patients from whom the Blastocystis spp. parasites were isolated, suggesting that a variation of virulence exists between isolates of the same ST from symptomatic and asymptomatic individuals.
| Conclusion | | |
It seems that ST3 could be the only ST of human origin with high host specificity, being the most predominant. Virulence or pathogenic potential of Blastocystis spp. could be because of intra-ST variation or because pathogenic and nonpathogenic strains may exist in different isolates of the same ST, which would explain the detection of some STs in both the disease and control groups.
| Acknowledgements | | |
The authors thank Dr Rehab Ibrahim Aly, Pathology Department, Faculty of Medicine, Suez Canal University for her help in reading all the histopathological slides conducted in the present study.
| Authors contribution | | |
S.M. Abaza supervised the study and revised the manuscript. H.Z. Rayan proposed the research idea and helped R.H. Soliman in the study design and writing the manuscript. R.H. Soliman helped A.B. Mokhtar in PCR subtyping and animal studies. N.A. Nemr helped A.B. Mokhtar in cases selection and shared in writing the manuscript. A.B. Mokhtar collected the samples, performed the parasitological, PCR, and animal studies, and conducted the statistical analysis.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]
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