Intestinal protozoa and helminths in ulcerative colitis and the influence of anti-parasitic therapy on the course of the disease

Abdurakhim Toychiev, MDa, Behzod Navruzov, MD, PhD, DScib, Dinora Pazylova, MDc, Nikolay Davis, MDd, Najiya Badalova, BSce, Svetlana Osipova, MD, PhD, DSci*


• A. lumbricoides, E. vermicularis, H. nana, and G. lamblia do not affect the course of ulcerative colitis.
• The highest prevalence of Blastocystis sp. was found in ulcerative colitis patients.
• The combination of anti-Blastocystis and anti-inflammatory therapy shows better mucosal healing of ulcerative colitis patients than monotherapy.
• Further study of the role of intestinal protozoa in ulcerative colitis pathogenesis is required.


Purpose: The aim of this study is to determine the prevalence of intestinal helminths and protozoa in patients with ulcerative colitis (UC) and to estimate the influence of the anti-parasitic therapy on the course of the disease.
Methods: The study was conducted at the Research Institute of Epidemiology, Microbiology and Infectious Diseases and Coloproctology Department of the Republic Clinical Hospital №1 of the Ministry of Health of the Republic of Uzbekistan. One hundred UC patients and 200 healthy individuals were examined by triple coproscopy. Additionally, 20, 25 and 22 UC patients with Blastocystis infection were treated with nitazoxanide (1.0 g/day), mesalazine (1.5-2 g/day) or a combination of nitazoxanide (1.0 g/day) and mesalazine (≥1.5-2 g/day) for 14 consecutive days, respectively. Parasitological, clinical and endoscopic examinations were conducted before therapy, immediately after and 6 and 12 weeks after therapy completion.
Results: The overall prevalence of helminths in UC patients and control individuals was not significantly different: 14±3.4% and 8.5±1.9%, respectively (OR: 1.7524; 95% CI: 0.8258 to 3.7186; P=0.1). Giardia lamblia was the most prevalent parasite in both groups, but the difference compared to the control was insignificant (OR: 0.4565; 95% CI: 0.2020 to 1.0318; P=0.05). A significantly higher prevalence of Blastocystis sp., Chilomastix mesnili and Iodamoeba butschlii in UC patients compared to control individuals was found (P<0.0005): 65.0%, 14.0% and 22.0%, respectively. During all follow-up periods, the clinical response and clinical remission were not statistically different between the groups (P>0.05). Mucosal healing immediately and 6 weeks after therapy with a combination of nitazoxanide with mesalazine was significantly better than with a monotherapy of nitazoxanide, respectively (P<0.05). UC patients treated with a combination of nitazoxanide with mesalazine showed better mucosal healing than in patients treated with a monotherapy of mesalazine (P>0.05).
Conclusions: Diagnosis of Blastocystis sp. should be introduced in the complex examination of UC patients. Further clinical studies are necessary for assessment of the efficiency of anti-Blastocystis therapy in UC patients. Keywords Ulcerative colitis; Helminths; Intestinal protozoa; Blastocystis sp.; Nitazoxanide

1. Introduction

Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) with a chronic and recurrent course. Intestinal microbiota play a key role in IBD [1]. The most intensive studies have been of the bacterial microbiome [2-4], while information on protozoa and helminths is less extensive and controversial [5, 6].
According to the hygiene hypothesis, helminths or their products modulate immune pathology and suppress inflammation [7, 8]. There is information on the positive influence of helminths on the course of active UC in humans and experimental models [7-11]. However, conflicting opinions exist. Enterobius vermicularis does not reduce the risk for IBD [12]; there is insufficient evidence to allow any firm conclusions regarding the efficacy and safety of helminths used to treat patients with IBD [13]; and Trichuris suis ova therapy showed no statistical benefit for IBD patients [14]. There is no information on the influence of other helminths, especially those endemic for Uzbekistan, on UC pathophysiology.
Concerning protozoa, emerging evidence suggests that many common eukaryotic residents of the human gut are commensals or beneficial rather than parasitic, at least in some contexts [5]. Data on the prevalence of the commensals Entamoeba coli, Entamoeba dispar, Endolimax nana, Chilomastix mesnili, and Iodamoeba butschlii in UC patients are scant and do not include all species [6].
The most studied protozoan with debated pathogenicity is Blastocystis sp. It is a protozoan that commonly resides in the large bowel and caecum of human beings. According to Audebert et al. (2016) [15], Blastocystis sp. colonization is usually associated with a healthy gut microbiota, rather than with the gut dysbiosis typical of IBD. A high prevalence of Blastocystis sp. in healthy adults in comparison with IBD patients was reported [16, 17], as well as an association of a low prevalence of Blastocystis infection in patients with active UC [18-20].
On the other hand, data on Blastocystis sp. pathogenicity are accumulating. Blastocystis sp. antigens induced the expression of proinflammatory cytokines IL-1β, IL-6, and TNF-α in mouse intestinal explants, mouse colitis colon and macrophages [21]. Blastocystis subtype (ST) 7 significantly increased apoptosis in human colonic epithelial cell-line Caco-2 enterocytes and induced changes in epithelial resistance, permeability and tight junction localization, causing epithelial barrier compromise in the human intestine [22]. In an experimental mouse model, the most adhesive axenic isolate ST 7 preferentially binds to the colon tissue rather than the caecum. Blastocystis sp. also exerts a mucinolytic effect. Increased adhesion and colonization were associated with greater tissue damage. Reinfection of a second batch of mice with an ST7 isolate obtained from faecal culture demonstrated similar histopathological findings and tissue damage, proving Koch’s postulates for this parasite [23]. Peroxiredoxins are proteins that are essential for the survival and virulence of pathogenic eukaryotes and prokaryotes [24]. Peroxiredoxins (Prx1a and Prx1m) were identified in Blastocystis hominis [25]. A high prevalence of Blastocystis sp. was revealed in patients with gastrointestinal disorders, diarrhoea, irritable bowel syndrome [26, 27], and urticaria [28]. B. hominis dominated in the structure of intestinal protists in UC patients, and the authors believe that protozoa could be a contributing cause of persistent UC activity [6]. Cekin et al. (2012) [29] also acknowledged a possible role of blastocystosis in UC exacerbation. Treatment of UC patients refractory to anti-inflammatory therapy and concomitant Blastocystis infection with metronidazole resulted in positive clinical outcomes [30, 31]. Mylonaki et al. (2004) [32] emphasized the importance of routine microscopic stool analysis for Blastocystis sp. in IBD exacerbation.
Due to the controversy surrounding the potential pathogenicity of Blastocystis and the self-limited nature of symptoms, the treatment of this disease is equivocal. Metronidazole is the most frequently prescribed antibiotic for infections [33-35], but there have been reports of protozoan resistance to metronidazole [36, 37]. Nitazoxanide (Ntz), a broad-spectrum 5-nitrothiazole anti-parasitic agent, has also been reported to be effective against Blastocystis sp. [38, 39].
The objective of the present study is to determine the prevalence of intestinal helminths and protozoa among UC patients and estimate the influence of anti-parasitic therapy on the course of the disease.

2. Materials and methods

2.1. Study design and participants

The study was conducted at the Research Institute of Epidemiology, Microbiology and Infectious Diseases and Coloproctology Department of the Republic Clinical Hospital №1 of the Ministry of Health of the Republic of Uzbekistan from January 2015 to January 2018.
The study was approved by the Medical Ethics Committee of the Ministry of Health of the Republic of Uzbekistan in accordance with the Declaration of Helsinki. Both informed and written consent were obtained from the patients and the controls. The trial is registered at the US National Institutes of Health ( #NCT03441893. Diagnosis of UC was confirmed using standard clinical, endoscopic, radiographic and pathological criteria according to the Montreal classification of the extent and severity of UC [40]. UC categories include proctitis, left- sided colitis and extensive or pancolitis. The activity of the disease was measured using the Mayo Clinic score consisting of 4 items: stool frequency, rectal bleeding, findings of flexible proctosigmoidoscopy, and patient’s functional assessment [41]. The disease duration was measured in years from the first time of symptoms onset.
In the first stage, a case-control study was performed. One hundred UC patients hospitalized at the Coloproctology Department of the Republic Clinical Hospital №1 underwent standard clinical, microbiological, instrumental and parasitological examination before receiving medication or surgery. The control group included 200 residents of the Tashkent region without any complaints of gastrointestinal tract disorders who applied to the clinic for planned medical examinations.
In the second stage, a randomized controlled double-blinded clinical study was performed. For assessment of the role of Blastocystis sp. in the UC course, an additional 67 UC patients with Blastocystis infection were randomized into 3 groups and examined before therapy, immediately after therapy, and 6 and 12 weeks after therapy. Twenty UC patients received anti-parasitic therapy with Ntz (Ntz group), 22 UC patients received a combination of anti- parasitic and anti-inflammatory therapy with Ntz and Ms (Ntz+Ms group) and 25 UC patients received anti- inflammatory therapy with mesalazine (Ms) (Ms group). All subjects were evaluated and screened for study eligibility by the first author prior to study entry.
Inclusion criteria for patients were 17 to 69 years of age with active UC according to the Mayo Clinic score and exacerbation of the disease after remission. UC patients included in the study were not treated with any preparation for the last 60 days. Patients with a diagnosis of Crohn’s disease were excluded from the analysis. Other exclusion criteria were toxic megacolon, abdominal abscess, symptomatic colonic stricture, stoma, a history of colectomy, an increased risk of infectious complications (e.g., as a result of recent pyogenic infection, enteric pathogens detected on stool analysis, active or latent tuberculosis, HIV-infection, hepatitis B or C) or recent inoculation with live vaccine, clinically meaningful laboratory abnormalities, pregnancy or lactation, an unstable or uncontrolled medical disorder, an anticipated requirement for major surgery, colonic dysplasia or adenomas and malignancy. For the groups undergoing anti-parasitic and anti-inflammatory therapy, in addition to the abovementioned exclusion criteria, patients who had ever used immunodepressants or biological therapy and the presence of pathogenic bacteria in gut microbiota (Salmonella spp., Shigella spp., Clostridium difficile, Campylobacter spp., Yersinia spp.) were excluded. For exclusion of UC patients with pathogenic bacteria, the following microbiological methods were used: C. difficile was determined by detecting C. difficile toxin A + B in stool samples by the ELISA technique (Microwell ELISA Kit, IVD Research Inc., Carlsbad, USA). For detection of Salmonella and Shigella, stool samples were plated on MacConkey medium (BD, Erembodegem, Belgium). Yersinia and Campylobacter were detected using cefsulodin-irgasan-novobiocin agar plates (BD, Erembodegem, Belgium) and campylosel agar plates (BioMérieux, Brussels, Belgium), respectively.

2.2. Parasitological examination

Parasitological examination was provided for the detection of endemic Uzbekistan helminths (E. vermicularis, Ascaris lumbricoides, Hymenolepis nana, etc.), intestinal pathogenic protozoa (Giardia lamblia, Cryptosporidium parvum and Entamoeba histolytica), protozoans with debated pathogenicity (Blastocystis sp.) and commensals (E. coli, E. nana, E. dispar, C. mesnili, I. butschlii).
Collection of stool samples. Three stool samples for parasitological examination were taken from both control subjects and UC patients at 2-day intervals before therapy (all participants), immediately after therapy and at 6 and 12 weeks after completing the course of therapy (only additional groups). Stool samples (1-2g) were collected in individual containers containing 5 ml of Turdiev’s preservative, providing for conservation and staining of protozoan cysts and eggs of worms for a year. Turdiev’s preservative includes the following: 80 ml of 0.2% aqueous solution of sodium nitrite, 10 ml of formaldehyde, 2 ml of glycerine, 8 ml of Lugol’s solution, and 250 ml of distilled water. Stool samples (1-2g) for the detection of C. parvum and E. histolytica were collected in individual empty containers immediately before parasitological examination.
Microscopy. Parasitological diagnosis was performed by triple coproscopy using the formalin – ethyl acetate concentration technique [42] and iodine stained smears [43]. To stain the preparations, Lugol’s solution was used. The intensity of protozoa was estimated by the number of protozoa in the field of view (ocular x10, objective x40) in iodine stained smears taken before treatment with the formalin – ethyl acetate concentration technique. The number of protozoa was calculated in at least 10 fields of view. Then, 1-2, 3-4 and ≥5-6 microorganisms in the field of view were considered an infection of low, medium and high intensity, respectively.
Detection of C. parvum. A modified Ziehl – Neelsen method [44] was used for staining the preparations. The stained smears were scanned using the ×100 oil immersion lens for the presence of C. parvum.
Detection of E. histolytica. Microscopic examination of direct smears for the detection of trophozoites of E. histolytica was performed. The presence of haematophagy in direct smears was the criterion to distinguish E. histolytica and E. dispar species in direct stool smears [45].

2.3. Trial protocol

After informed consent had been obtained, an additional 67 UC patients with Blastocystis sp. infection were randomized to 1 of 3 groups to receive one of the following treatments:
1 – Ntz group: 20 UC patients with Blastocystis sp. infection were treated with Ntz, 1.0 g/day (two pills) two times a day orally for 14 consecutive days.
2 – Ntz+Ms group: 22 UC patients with Blastocystis sp. infection were treated with Ntz, 1.0 g/day (one pill – 500 mg) two times a day orally, and Ms, 1.5 g/day (one pill – 500 mg) three times a day orally for 14 consecutive days. The dose of Ms for UC patients with high body weight was increased up to 2 g/day.
3 – Ms group: 25 UC patients with Blastocystis sp. infection were treated with Ms, 1.5 g/day (one pill – 500 mg) three times a day orally for 14 consecutive days. The dose of Ms for UC patients with high body weight was increased up to 2 g/day.
Other pathogenic protozoa and helminths revealed in the samples were treated with a standard dosage of conventional anti-parasitic drugs. Giardiasis, ascariasis, enterobiasis and hymenolepiasis were treated with metronidazole (dose and duration of drug intake were 750-1000 mg/daily for 10 days), albendazole (a single dose of 400 mg), mebendazole (a single dose 100 mg repeated in a fortnight) and praziquantel (a single dose of 25 mg/kg, repeated in 4 days), respectively. All patients were examined by an investigator before the regimen was assigned, and during all periods of the study, participants were contacted by telephone if necessary.

2.4. Randomization

UC patients were randomized using simple randomization method. Randomization was performed with the use of computer-generated randomization schedules. Sequence was generated by a person not directly involved in execution of the study. The randomization list was sealed and maintained by the Ministry of Public Health (study sponsor) in its study files until the study was completed. The patients, principal investigators and their staffs and laboratory personnel and study monitors were blinded as to treatment assignment.

2.5. Follow-up

The purpose of follow-up was to monitor and record the response to induction therapy, adverse events, and recurrence of symptoms. Parasitological, clinical and endoscopic examination was conducted before therapy, immediately after completion of the therapy course and at 6 and 12 weeks after therapy. In each examination of UC patients, a Mayo Clinic score was calculated and the intensity and the presence/absence of Blastocystis sp. was detected.

2.6. Outcome measures

The outcome measures were eradication/persistence of intestinal parasites, including Blastocystis sp.; reduction/persistence/increase of the infection intensity; a clinical response to therapy according to the Mayo Clinic score; and adverse effects of medicines in UC patients immediately and at 6 and 12 weeks after therapy completion. A positive clinical response was defined as a reduction according to the Mayo Clinic score of at least 3 points and a decrease of at least 30% from the baseline score, with a decrease of at least 1 point on the rectal bleeding subscale or an absolute rectal bleeding score of 0 or 1. A clinical remission was defined as a Mayo Clinic score of 2 or lower and no subscore higher than 1. Mucosal healing was defined as a Mayo Clinic scale for endoscopic subscore of 0 or 1.

2.7. Statistical Analysis

The results are expressed as the mean±standard deviation (SD) for categorical data. All data followed the normal Gaussian distribution. An independent Student’s t-test as well as matched paired t-tests were used for the comparison of numerical variables among two independent groups and two linked data, respectively. Categorical data were analysed by odds ratios (OR) with 95% confidence intervals (CI) of the mean. One-way analysis of variance (ANOVA) was used to assess differences between the means of three independent groups. A value of P<0.05 was considered statistically significant. Data were analysed using Origin 6.1 software (OriginLab, Northampton, MA). 3. Results 3.1. Baseline Characteristics of the participants In total, 514 participants were evaluated for eligibility; 147 of them did not meet one or more inclusion criteria, and 367 participants were included in the study. The demographic and clinical characteristics of the participants are presented in Table 1. 3.2. Structure of intestinal macro- and microbiota in UC patients The prevalence of pathogenic intestinal helminths in UC patients and control individuals is shown in Fig. 1. The overall prevalence of helminths in UC patients was not significantly different from that of control individuals, respectively, 14±3.4% and 8.5±1.9% (OR: 1.7524; 95% CI: 0.8258 to 3.7186; P=0.1). Mixed pathogenic intestinal parasitic infections were not found in UC patients or the controls. The clinical assessment of disease activity by Mayo Clinic score among UC patients with and without intestinal parasites was not significantly different (data not shown). Treatment of UC patients with helminths and giardiasis in all cases led to parasite elimination. Any influence of parasite elimination on the course of UC was not noticed. A significantly higher prevalence of Blastocystis sp. in UC patients (65.0±4.7%) in comparison with control individuals (18.0±2.7%) (OR: 8.4603; 95% CI: 4.8968 to 14.6171; P<0.0001) was observed. Moreover, infection of a high intensity was observed only in patients with UC (28.0±4.4%). The frequency of Blastocystis sp. infection of medium intensity in UC patients (24.0±4.2%) was higher than in the control group (4.0±1.3%) (P<0.0001). C. mesnili infection rates in UC patients (14±3.4%) and the controls (3.0±1.2%) were significantly different (OR: 5.2636; 95% CI: 1.9568 to 14.1586; P=0.0003). The medium intensity of the infection was found only in UC patients (7.0±2.5%). I. butschlii were found in UC patients 3.5 times as frequently as in the controls: 22±4.1% and 6.0±1.6%, respectively (OR: 4.4188; 95% CI: 2.0846 to 9.3667; P<0.0001). A medium and high intensity of infection was observed only in UC patients (Fig. 2A). The prevalence of E. nana in UC patients (7±2.5%) was higher than in control subjects (3.0±1.2%) (OR: 2.4337; 95% CI: 0.7956 to 7.4448; P=0.1), but in both groups, the intensity of the infection was low. The prevalence of E. dispar was also low in both groups: 3.0±1.7% and 0.5±0.4%, respectively (OR: 6.1546; 95% CI: 0.6319 to 59.9431; P=0.1). The percentage of individuals with E. coli among UC patients (11±3.1%) was lower than that in the control group (19.0±2.7%) (OR: 0.5269; 95% CI: 0.2567 to 1.0816; P=0.07). G. lamblia was the most prevalent parasite in both groups, but the difference between UC patients and the control group did not reach statistical significance (OR: 0.4565; 95% CI: 0.2020 to 1.0318; P=0.05). The prevalence of these protozoa in UC patients did not differ significantly from control individuals (Fig. 2B). Thus, we found a higher prevalence of Blastocystis sp., C. mesnili and I. butschlii in UC patients than in the control group. A high intensity of infection was revealed only in UC patients for Blastocystis sp. and I. butschlii, respectively. 3.3. Anti-Blastocystis and anti-inflammatory therapy and their influence on the course of UC After completion of anti-parasitic therapy (Ntz group) or the combination of anti-parasitic and anti-inflammatory therapy (Ntz+Ms group), elimination of Blastocystis sp. among UC patients was observed in 100% of cases. However, at 6 weeks after the course of anti-parasitic therapy (Ntz group), a low intensity of Blastocystis sp. was detected in 5.0±4.8% of UC patients. At 12 weeks after the course of therapy, a low intensity of Blastocystis sp. was detected in 10±6.7% and 18.2±8.2% of UC patients in the Ntz and Ntz+Ms groups, respectively. Medium and high intensity of Blastocystis infection was not observed 6 and 12 weeks after the therapy in Ntz and Ntz+Ms groups (Fig. 3A, B). Any significant changes in the prevalence and intensity of Blastocystis sp. were not found in UC patients in the Ms group after anti-inflammatory therapy completion and 6 and 12 weeks after the course of therapy (P>0.05) (Fig. 3C). One-way ANOVA test showed that the clinical response and clinical remission were not statistically different between the groups (Fig. 4A, B). Mucosal healing immediately and 6 weeks after therapy in Ntz+Ms group was significantly better than in Ntz group, respectively (P<0.05). Mucosal healing in Ntz+Ms group showed the tendency to a better result as compared to the Ms group, but it did not reach statistical significance (Fig. 4C). 4. Discussion To date, there are no satisfactory therapies for IBD, and progress in this direction is connected with target therapy. The dysbiosis in UC is characterized by alterations of bacterial composition, including decreases in Bacteroidetes, along with Firmicutes (in particular Clostridium IXa and IV groups, Bifidobacteria, Lactobacillus, Faecalibacterium prausnitzii), and an increase in Proteobacteria and Actinobacteria [1, 46-48]. While the exact aetiology of UC remains unclear, successful faecal microbiota transplantation may be associated with an increase in microbiota diversity and richness and certain changes in bacterial composition [49]. This procedure can substitute the patient’s microflora, supposedly containing microorganisms triggering intestinal inflammation, with microorganisms with probiotic properties. Another direction focuses on blocking the inflammatory cascade (anti-TNF-α, anti-integrin), but these preparations possess serious side effects and in some cases limited efficiency [50]. In our study we evaluated a possible influence of intestinal eukaryotes on the UC course. The prevalence of pathogenic parasites (A. lumbricoides, E. vermicularis, H. nana and G. lamblia) in UC patients did not differ from the control group. All patients infected with pathogenic parasites were admitted to the hospital at the exacerbation period and after anti-parasitic treatment, any changes in the course of the disease were not observed. Therefore, inhibition of the inflammation process by the species of parasites mentioned above seems to be not very intensive, if it takes place at all. Our results are in accordance with those suggesting that enterobiasis does not reduce risk for IBD [10] and helminth infections remain widespread in populations at risk of IBD [51]. To our knowledge, studies on A. lumbricoides and H. nana in UC patients are absent, as well as the influence of concomitant giardiasis on the UC course. Most data on the inhibition of intestinal inflammation in UC patients are concentrated on application of Trichuris trichiura and Necator americanus [9, 10, 52] and amelioration of colonic disease in experimental models by helminths (Heligmosomoides polygyrus, Hymenolepis diminuta, Schistosoma mansoni, Schistosoma japonicum, Trichinella spiralius) [11]. Currently, an opinion is being formed that helminth therapy has no statistical benefit for IBD patients [13, 14, 21]. Our data showed that the overall prevalence of Blastocystis sp., C. mesnili, I. butschlii and E. nana in UC patients (65.0±4.7, 14±3.4, 22±4.1 and 7±2.5%, respectively) was higher than in the control group (18.0±2.7, 3.0±1.2, 6.0±1.6 and 3.0±1.2%, respectively). We did not find studies characterizing the prevalence of C. mesnili, I. butschlii, E. dispar and E. coli in UC patients. According to Yamamoto-Furusho et al. (2012) [53], the prevalence of E. nana in Mexican UC patients corresponds to 9%, which does not differ significantly from our data (7%). The prevalence of eukaryotes in the control group are not comparable with data obtained from various regions (Iran, Mexico, China, Southeast Asian countries), because the prevalence of intestinal protozoa and helminths in populations vary to a great extent depending on the region. In particular, the prevalence of Blastocystis sp. was in the range from 7.2% to 49%, similar to that of other parasites. E. nana and C. mesnili were significantly more prevalent in people with loose stool specimens in combination with Blastocystis sp. [54-56]. Our results are opposite those of Audebert et al. (2016) [15], Petersenet al. (2013) [18], Rossen et al. (2015) [19], Coskun et al. (2016) [20], Rossen et al. (2015) [57] and others, who showed that Blastocystis sp. is usually associated with healthy gut microbiota, but are in accordance with the data of Lim et al. (2014) [21], Wu et al. (2014) [22], and Ajjampur et al. (2016) [58], who interpreted Blastocystis sp. properties as representing their pathogenicity as well as the association of Blastocystis sp. with gastrointestinal disorders and irritable bowel syndrome [26, 27]. One of the plausible explanations of these contradictions could be regional peculiarities, including possible variations in the structure of Blastocystis subtypes. The methods used (PCR or single, double or triple coproscopy with concentration method) undoubtedly affect the results of examination, but the tendency should remain as a low or high prevalence of Blastocystis sp. in healthy individuals and UC patients. Blastocystis sp. is a diverse genus comprising 17 characterized subtypes (ST1-ST17) [59]. Distribution of Blastocystis subtypes depends on geographic area. In general, ST3 is the most common in France, followed by ST1, ST4, ST2, ST6 and ST7, but the pattern of ST distribution varies in different centres. ST4 is commonly found in Europe but is much less frequently detected or is absent in Africa and Asia [60]. Subtypes differ in pathogenicity, so Blastocystis ST7, but not ST4, significantly increased apoptosis in enterocytes and activated Caco-2 caspases 3 and 9, but not caspase 8. ST7 induced changes in epithelial resistance, permeability and tight junction (ZO-1) localization [61]. Phenotypic variations within a particular subtype were established; in particular, phenotypic variations in Blastocystis sp. ST3 were found [62]. Blastocystis genomes demonstrated increased intrasubtype variability of ST1 and ST2 in comparison with ST3 and ST4 [63]. The high prevalence of Blastocystis sp. in UC patients was the basis for the evaluation of the efficiency of Ntz monotherapy and the combination of Ntz with Ms compared to the results with Ms monotherapy. During all follow- up periods, the clinical response and clinical remission were not statistically different between the groups (P>0.05). Mucosal healing immediately and 6 weeks after therapy with a combination of Ntz with Ms was significantly better than with a monotherapy of Ntz (P<0.05). UC patients treated with the combination of Ntz with Ms showed better mucosal healing than patients treated with monotherapy of Ms. After completion of anti-parasitic therapy or the combination of anti-parasitic and anti-inflammatory therapy, elimination of Blastocystis sp. among UC patients was observed in 100% of cases. The same picture was observed at 6 weeks after therapy completion when the number of UC patients with mucosal healing in the group obtained the combination therapy was significantly higher than in the patients treated with Ntz or Ms. However, at 12 weeks after the courses of therapy, Blastocystis sp. was detected in UC patients in the Ntz and Ntz+Ms groups. The reason for the appearance of Blastocystis can be explained by re-infection and a possible high susceptibility of the patients to these protozoa. So the mucosal healing effect in patients treated with the Ntz+Ms is associated with inhibition of Blastocystis sp. growth or their elimination as removal of one of the factors initiating or maintaining inflammation. Assessment of anti-Blastocystis therapy efficiency with the estimation of clinical response, clinical remission and mucosal healing has been carried out for the first time. The advantage of Ntz+Ms combination over monotherapy (Nz or Ms) indicates the expediency of further investigations in this direction. It would be of great importance to identify Blastocystis subtype depending property to induce the most intensive inflammation as well as susceptibility to anti-Blastocystis drugs. It is obvious that Blastocystis sp. is not the only factor determining the development of inflammation, but nevertheless, the protozoa are involved in the inflammatory process. The effect appears to be connected with Blastocystis sp. elimination/low prevalence and intensity of the infection and consequently cessation of properties, resulting in disorders of the epithelial barrier (mucinolytic activity, changes in epithelial resistance, permeability and tight junction localization, expression of proinflammatory cytokines IL-1β, IL-6 and TNF-α [21, 22]). This is confirmed by the results of combination therapy including Ntz and Ms. Possibly, the study of larger groups could confirm more clearly the influence of Blastocystis sp. on the course of UC. In total, our results are in accordance with Tai et al. (2011) [30] and Jeddy & Farrington (1991) [31], who obtained good clinical outcomes in UC patients refractory to anti-inflammatory therapy and concomitant Blastocystis infection after treatment with metronidazole. Information on the relationship between Blastocystis sp. and gut bacteria are accumulating. Andersen et al. (2015) [64] used a metagenomic approach to find that individuals with Bacteroides dominating in intestinal microbiota were less prone to having Blastocystis sp. positive stools than individuals with Ruminococcus- and Prevotella-driven enterotypes. The prevalence of C. difficile in UC patients varied from 0.013% [19] to 3.4% [65, 66]. It was found that C. difficile is not a common trigger for exacerbations of IBD in clinical practice in the Netherlands [66]. Thus, information on the relationship of Blastocystis sp. and representatives of the bacterial part of intestinal microbiota is rather scarce. Our study has several limitations. First, the number of participants who gave consent was lower than anticipated. Second, for estimation of anti-parasitic therapy efficiency, only parasitological, clinical and endoscopic examinations were used. Additionally, detection of faecal calprotectin, proinflammatory cytokines, C-reactive protein, and histological examination of the colon in UC patients before and after therapy could give a more complete estimation. Third, observation of UC patients with intestinal parasites after anti-parasitic therapy continued only for 12 weeks due to the impossibility of follow –up in patients from different regions of the country. It is possible that longer observation of parasitological, clinical and endoscopic changes in UC patients could be more informative. 5. Conclusion This study characterizes intestinal eukaryotes in UC patients. The similar prevalence of A. lumbricoides, E. vermicularis, H. nana and G. lamblia in UC patients at the stage of exacerbation and control individuals, as well as the absence of any noticeable clinical manifestations after parasitic elimination, suggest the absence/weak inhibition of the inflammatory process in UC patients. 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