Preneoplastic proliferative changes induced by experimental Original blastocystosis Article Mazloum M Ahmed, Fayza SM Habib, Ghada A Saad, Heba M El Naggar

Department of Parasitology, Faculty of Medicine, Ain Shams University, Cairo, Egypt

ABSTRACT Background: Studying the pathogenic potential of spp. by in vitro and in vivo experimental studies led to suspicions concerning its role in development of cancer colon. Still, this hypothesis remains under investigations. Objective: To investigate the pathology induced in the gut of mice inoculated with Blastocystis spp. isolates derived from patients with and without colorectal carcinoma (CRC). Subjects and Methods: Seven Blastocystis spp. isolates were derived from patients with CRC, six from non-CRC patients with symptomatic blastocystosis and six from non-CRC asymptomatic Blastocystis spp. carriers. Isolates were used to induce experimental blastocystosis in three groups of three-weeks-old BALB-c mice: GI was inoculated by CRC isolates; GII by symptomatic non-CRC isolates; and GIII by non-CRC asymptomatic isolates. Always, one clinical isolate was used to infect one mouse. Each group contained one negative control mouse inoculated with parasite-free culture medium, kept under the same conditions. Results: Histopathological examination of sections of intestine of all inoculated mice in the three groups showed positive infection with parasites seen only in the cecum and colon, no parasites were seen in the .

Blastocystis Inflammatory cells infiltrations were detected in mice of the three groups with varying degrees. Vacuolar forms of observed being spp. more were severe seen infiltrating with polypoid the submucosaformation in in mice sections in GI from (4 mice) mice than in GI in (4 GII mice) (1 mouse). and GII (2 mice) but not Conclusion:in GIII. The significant Blastocystis difference spp. isolates in the pathologic associating changes CRC differinduced in in their the intestine proliferative of mice and in theinvasive three groupspathogenic was capabilities than symptomatic isolates. Asymptomatic Blastocystis spp. are non-invasive organisms causing only mildKeywords: inflammatory Blastocystis response spp.; colorectal in the large carcinoma; intestine histopathological of experimentally examination; infected animals.pathogenicity. Received: 18 April 2019, Accepted: 2 May, 2019. Corresponding Author: Ghada A Saad; Tel.: 0020 02 25217977, E-mail: [email protected] Print ISSN: 1687-7942, Online ISSN: 2090-2646, Vol. 12, No. 2, Ausgust, 2019. introduction of in cell lines[22,23] in contrast to the proliferation of human colonic cancer cell line, HCT116, Blastocystis spp. are the most common eukaryotic when incubated with Blastocystis spp. organisms[24,25]. protist encountered in the gut of both humans and In vivo studies demonstrated pre-neoplastic changes many animals[1]. The organism has been diagnosed with the formation of aberrant crypt foci in the colon of Blastocystis spp. experimentally infected rats[6]. symptoms such as nausea, vomiting, , These studies led to suspicions of Blastocystis spp. as a in both patients with non-specific gastrointestinal cause of cancer colon. However, until recently this view individuals[2-4]. Infection has been associated with a remains to be under investigation[20]. varietyabdominal of diseases pain, flatulence,such as irritable and inbowel asymptomatic syndrome (IBS)[5-9] The pathological lesions produced in animals by bowel disease (IBD) including Crohn’s disease and Blastocystis spp. isolates derived from asymptomatic ulcerous, non-specific[10,11], and colitis, with chronicCRC[12-14] . inflammatory It has been and symptomatic infected humans with gastrointestinal diagnosed in association with non-gastrointestinal manifestations[27-30], and from patients with IBS[7,31,32] disorders such as skin rash and chronic urticaria[15-18], have been studied and compared. The present study as well as Hashimoto’s thyroiditis[19]. Because of aims to investigate the pathology induced in the gut of these discrepancies in associations, and despite the mice inoculated with Blastocystis spp. isolates derived application of recent advances in immunological and from patients with and without CRC to discover the molecular methods to Blastocystis spp. research, the possible oncogenic potential of the parasite. pathogenicity of Blastocystis spp.is still controversial and inconclusive[11,20]. SUBJECTS and methods The pathogenic potential of Blastocystis spp. has been investigated by in vitro as well as in vivo experimental This descriptive experimental study was conducted at studies[21]. In vitro studies demonstrated the induction the Parasitological Research and Diagnostic Laboratory

Personal non-commercial use only. PUJ copyright © 2019. All rights reserved DOI: 10.21608/puj.2019.11959.1041

94 Blastocystosis: a precancerous infection? Ahmed et al., Unit of Parasitology Department, Faculty of Medicine, enteric parasites. All samples were cultured to exclude Ain-Shams University during the interval from Blastocystis infection. The animals were separately September, 2017 to February, 2018. maintained in polycarbonate cages with paper bedding, at 25°C, in the animal house, with a relative humidity Subjects: Individuals infected with Blastocystis of 40–60% and under a 12-hour light/dark cycle. They were fed a normal diet of commercial pellets and given stools were included in the present work. They were potable water ad libitum. The cages and paper bedding selectedspp. identified from attendants as the only of possiblethe outpatient parasite clinics in their of were changed at weekly intervals. El Demerdash Hospitals and from persons referred to the Parasitology Research and Diagnostic Laboratory Experimental infections and design: Blastocystis Unit of Medical Parasitology Department, Faculty of spp. organisms in 3–4 days-old cultures were Medicine, Ain Shams University. Participants in the study included patients with early diagnosed CRC who gradient centrifugation[35] to remove bacterial load had not received any chemotherapeutic drugs (GI); harvestedin each isolate, and and purified to exclude by Ficoll-Hypaque bacterial infection density as a symptomatic patients suffering symptoms related to possible cause of histopathological changes occurring gastrointestinal disorders, such as diarrhea, abdominal in experimental studies. After counting the number of pain and distension (GII); and asymptomatic carriers cells using a hemocytometer, number of cultured forms (GIII). Each participant was subjected to the was adjusted to 1 × 105/ml. Cells were immediately following: (i) questionnaire with a thorough medical intra-gastrically inoculated orally into the animals by history covering the personal data, the presence of 16-G ball-tipped feeding needle attached to one ml GIT symptoms related to Blastocystis spp. infection syringe[36]. current health status with special emphasis on diagnosis Mice were divided into three groups: GI, 7 mice of(diarrhea, colorectal abdominal carcinoma, pain, previous , parasitic flatulence),infections, inoculated by organisms of isolates derived from CRC and drug intake; (ii) complete clinical examination patients; GII, 6 mice inoculated by organisms of isolates including general and abdominal examination to derived from symptomatic non-CRC patients; and GIII, exclude other conditions. 6 mice inoculated by organisms of isolates derived from asymptomatic non-CRC Blastocystis carriers Stool samples: All participants were asked to (Table 1). In GI and GII one mouse was inoculated for provide three stool samples in clean, wide, disposable each clinical isolate. Each group of mice included one stool containers. Each sample was examined by additional mouse inoculated with one ml parasite-free direct smear examination, iodine, and trichrome culture medium, kept under the same conditions, and staining, and by formalin-ethyl acetate concentration considered as negative control for each group. These technique[33], to diagnose Blastocystis infection and were housed separately to avoid feco-oral transmission exclude other parasitic causes of GIT symptoms. of infection. Meanwhile, only one mouse was housed per cage to facilitate collection of stool samples and exclude , Cyclospora, Isospora, and diagnosis of infection. Stool samples from all mice were MicrosporidiumModified Ziehl-Neelsen-infected patients acid-fast[33] stain. In vitro was cultivation used to screened for the presence of infection daily for one was performed for each stool sample by inoculation of week post infection (PI) by wet mount examination fresh stool in Locke's egg serum medium[34]. The cultures and culture in Locke' egg slant serum medium[34]. The were incubated at 37°C and examination of 3–4 days-old culture was considered negative if the organism was Time absent until the 7th day. Two weeks PI, all mice On observing the typical vacuolar or granular forms of were anesthetized by ether and euthanized by disc Blastocystiscultures was spp. done organisms, under ×10 they and were ×40 sub-culturedmagnifications. in dislocation. (colon and cecum) was a new medium for several subcultures supplemented assessed by naked eye, removed, preserved in 10% with antibiotics to minimize bacterial contamination before being used for animal inoculation. formalin, and processed in paraffin blocks. Blastocystis isolates: Blastocystis Table 1. Date of diagnosis of infection in experimentally infected mice. isolates were employed in the present study. These included isolates derived from Nineteen patients with CRC Date of diagnosis of experimental Group/ blastocystosis in mice (n=7); from symptomatic blastocystosis patients No. of mice with gastrointestinal symptoms (n=6); and from D 4 PI D 5 PI D 6 PI asymptomatic Blastocystis spp. carriers (n = 6). G I (7) 4 3 0 G II (6) 6 0 0 Experimental animals: Three-weeks-old male G III (6) 0 3 3 BALB-c mice used in the present study, were obtained Group I: mice infected with isolates from CRC patients; Group from Ain-Shams University Research Institute Animal II: mice infected with isolates from non-CRC symptomatic House. Stool samples of mice were subjected to direct patients; Group III: mice infected with isolates from non-CRC asymptomatic individuals. PI: post infection. parasitological examination to confirm the absence of 95 PARASITOLOGISTS UNITED JOURNAL

Histopathology: redness and bloated cecum were observed in four were sectioned, stained with hematoxylin-eosin mice of GI (CRC group) and two mice in GII (non-CRC and examined microscopically Paraffin blocks using of tissue40x and samples 100x symptomatic group) compared to controls.

Histopathological examination: Histopathological describedobjectives. by Grading Riley et of al the.(37) .degree of inflammatory cell examination of the intestine of mice of the three groups, infiltration and tissue destruction was assessed as revealed Blastocystis vacuolar forms in the lumen Statistical analysis: Results are expressed as number of the large intestine, mainly in the cecum with no and percent. Statistical analyses were carried out using organisms detected in the small intestine. Blastocystis SPSS version 20. Pearson Chi-square (2 ) test was spp. parasites were detected at the edge of the mucosa, trapped in the epithelial lining near the surface (Figures considered at probability P<0.05. Data are presented 1 and 2). Different degrees of pathological changes were used to compare results. The significance level was detected among mice infected by isolates from CRC- (GI), non-CRC symptomatic- (GII) and asymptomatic- Ethicalby descriptive considerations: tables and appropriate All participants figures. were (GIII) patients (Table 2). Intense and severe degree acquainted with the study details and agreed to of pathological changes were seen in sections of the participate in it. The procedures employed in the intestine of all mice inoculated by CRC-isolates (GI) present experiments complied with the current ethics guidelines set out by Ain Shams University. General throughout the lamina propria namely lymphocytes, principles for care and use of animals in experiments eosinophils,with severe andand mixed sporadic inflammatory polymorphs cells infiltrationwith the appearance of lymphocytic aggregation throughout

Animalfor scientific Research. purposes The researchwere followed protocol as recommendedwas approved was moderate to severe in sections of the intestine of by the NationalEthics Committee, Advisory CommitteeFaculty of forMedicine, Laboratory Ain- micethe mucosal of GII and layer. mild The to moderate inflammatory in case cells of miceinfiltration of GIII Shams University. (Table 2; Figures 1, 2, and 3).

Collections of Blastocystis vacuolar forms were results observed in the submucosa and lamina propria in sections of the colon and cecum of 4 mice in GI. All isolates included in the study were infectious Precancerous proliferative lesions in the form of to mice with positive diagnosis starting from 4th to 6th with hyperplastic mucosa of the colon were noticed The infection was diagnosed on the 4th day PI in 4/7 inpolypoid some areas projections of the mucosa with fibrovascular of these 4 mice, core (Figure covered 1 mice day included PI with in no GI statisticallyinoculated with significant isolates differences. from CRC a-c). Only sporadic vacuolar forms were seen invading patients, and in all mice in GII inoculated with isolates the submucosa of 2 mice in GII (Figure 2). One of these from the non-CRC symptomatic patients. Infection was two mice showed one or two polypoid projections like diagnosed on the 5th day PI (3 mice) from GI, and on the Blastocystis 5th and 6th days PI (3 mice respectively) inoculated with organisms were seen invading the submucosa in isolates from the non-CRC asymptomatic GIII (Table 1). sectionsthose noticed of the in miceintestine of GI (Figureof mice 1d). of NoGIII. Also, no proliferative changes were seen in the mucosa of mice throughout the study. of GIII (Figure 3). There was no invasion detected in No mortality or lethargy was observed in the animals the muscularis mucosa by any of the Blastocystis stages Naked eye appearance of intestine: Gross morphology in all groups studied. The mucosal showed of the large intestine and cecum of all isolates of GIII mice, inoculated with asymptomatic isolates, showed differences existed on comparing the pathological no abnormalities compared to features seen in the lesionsno ulceration produced or in dysplasia.mice of the Statisticallythree groups significantin relation non-infected control animal. On the other hand, some to the clinical source of isolates (P<0.049).

Table 2. Blastocystis isolates derived from CRC patients (GI), non-CRC symptomatic patients (GII) and non-CRC asymptomatic carriers (GIII). Histopathologic findings in mice inoculated with GI GII GIII Group/Histopathological findings P value No. = 7 No. = 6 No. = 6 Degree of inflammation and inflammatory cells infiltration Moderate Mild to Severe - in mucosa to severe moderate Number (%) of mice with Blastocystis vacuolar forms 4/7 2/6 0/6 - mucosal invasion/total number of mice in the group (57.14%) (33.33%) (0%) Number (%) of mice with induced proliferative lesions 4/7 1/6 0/6 0.049* and polyp formation/ total number of mice in the group (57.14%) (16.6%) (0%) P value < 0.05

*Significant 96 Blastocystosis: a precancerous infection? Ahmed et al.,

Fig. 1. Tissue sections from large intestine of mice in GI (a, b and c) infected with CRC-Blastocystis spp., and a mouse in GII (d) (a) Blastocystis with the peripheral nuclei and the central vacuole. infected with non-CRC symptomatic isolate showing polypoid projections with fibrovascular cores. H & E, ×400. The box in shows magnified vacuolar forms of

Fig. 2. Tissue section from large intestine of a mouse in GII infected with an isolate of non-CRC-symptomatic Blastocystis spp. The lamina propria is packed with

eosinophils, lymphocytes, plasma cells, and vacuolar mixedforms of inflammatory Blastocystis cells (arrows) in the form of

infiltrating the submucosa (in circles) (H&E 400x).

Fig. 3. Section of the large intestine of a mouse infected with an isolate from GIII (non-CRC asymptomatic) showing the lamina propria with mild mixed inflammatory cells (black arrows) (H&E, 400 x).

97 PARASITOLOGISTS UNITED JOURNAL DISCUSSION bloating in contrast to those inoculated by asymptomatic isolates that showed normal gross morphology. This In the present study, experimental blastocystosis was similar to the results observed by Abaza et al.[6] was induced in mice to compare the pathogenic effects who noticed redness and bloating in cecum and colon of Blastocystis spp. isolates derived from CRC patients of rats infected by symptomatic isolates, but not in with those produced by isolates derived from non-CRC those inoculated by asymptomatic isolates and isolates and from other asymptomatic individuals. Blastocystis derived from IBS patients, suggesting differences in spp. isolates from CRC patients included in the study pathogenicity of human' isolates derived from persons were obtained from patients diagnosed with colorectal with different clinical presentations. carcinoma before beginning any chemotherapeutic therapy to exclude possible effects of the drugs on the The absence of parasites in the small intestine isolates. Also, persons infected with other parasitic in all infected mice is consistent with previous infections were excluded to ensure that the causative reports[6,28,29,31,43] that detected Blastocystis spp. organism of symptoms in symptomatic patients was organisms only in the cecum and colon. Only in immune- Blastocystis. compromised mice, were Blastocystis organisms found in the whole GIT[13]. Previously experimental blastocystosis was induced in many animal models including pigs, guinea pigs, Although the amoeboid form is the proposed pathogenic form of Blastocystis spp.[46-48], yet in pathogenic potential of Blastocystis[38]. Healthy pigs[20] studies describing the histopathology of experimental andmice, rats chickens[39] are recommendedand rats with conflictingfor use as animalresults models on the blastocystosis, only the vacuolar forms were reported for the pathogenicity of Blastocystis spp. However it to invade the wall of the intestine[6,20,28,31,32,45,49,50]. was recommended to screen these animals for current Similarly, in the present work in sections of the large infection before starting experimentation as both intestine of mice in GI (4/7 mice) and GII (2/6 mice), animals are natural hosts for Blastocystis spp. with only the lamina propria appeared to be invaded by the absence of signs of damage to their intestines[38,40,41]. vacuolar forms. Invasion of the lamina propria, sub- Although chickens could be inoculated with Blastocystis mucosa and reaching up to the muscular layer of the spp., authors observed that not all human isolates of intestine by vacuolar forms was seen only with high Blastocystis spp. can infect chickens and rats [42]. Mice infective doses[28,44], demonstrating the impact of the are more suitable for immunology studies than rats as Blastocystis the possibility of rats exhibiting previous immunity to spp. have been noticed in vitro studies to produce Blastocystis spp. can lead to errors[21]. It is important disruptionhigher dose of of intestinalinoculum. epithelialVacuolar formsmonolayer of and the to notice that older mice are refractory to Blastocystis spp. requiring induction of mild colitis using 2% DSS membrane permeability[51-53]. Proteases excreted (Dextran Sulfate Sodium) prior to infection[39]. Thus, occludingboth in and tight on the junctions cell surface (ZO-1), of asBlastocystis well as increased spp. are caution should be observed when choosing a suitable accused of being the virulence factor and their activity animal model to study blastocystosis. In our animal may determine the pathogenicity of an isolate. Cysteine experimental study, and as previously reported[28,29,43,44], proteases concentrated in the central vacuole of the 3 weeks-old mice were used successfully to produce vacuolar forms[54] are accused of causing apoptosis and blastocystosis. All Blastocystis spp. isolates were membrane permeability of host cells[51]. Therefore as infectious to animals used. observed, further investigation is needed to ascertain the role of the amoeboid and vacuolar forms in In the present work, experimental blastocystosis pathogenicity[20]. was successfully induced in all BALB-c mice by intra- gastric inoculation with 1.0×105 Blastocystis organisms Different degrees of pathological changes were reported among mice and rats experimentally similar inoculum doses produced infection in only 40% infected by symptomatic human isolates compared ofin100 the μlanimals saline. Inwhile the workin the of work El Wakil of Pavanelli and Hewedy[28], et al.[44], with asymptomatic isolates[6,31,45,49,50]. A mild degree of a smaller inoculum of 1.0 x 102 produced infection in all animals. These variations may be due to differences and aggregates of lymphocytes was previously reported in infectivity of Blastocystis spp. in different studies. ininflammation rats, with the with parasite few inflammatory seen at the cells luminal infiltration aspect Positive stools examinations of mice began from 4 of the colon when the animals were inoculated by to 6 days PI in the three groups. Earlier diagnosis of asymptomatic isolates[6,31]. Similarly, edematous lamina infection of 2 to 5 days[28,44] in mice, and 3 to 4 days[6] in rats were reported depending on the inoculum size. especially neutrophils, eosinophils and lymphocytes werepropria reported with mild in inflammatoryexperimentally cellular infected infiltration mice[29]. As regards the naked eye appearance of mice Researchers reported that Blastocystis spp. organisms intestine, cecum and colon of mice in GI inoculated were seen, only, on the luminal border of the large with CRC isolates and mice in GII inoculated with non- intestine[6,31] CRC symptomatic isolates, showed some redness and reaction was noticed in mice inoculated by isolates . A similar picture with mild inflammatory

98 Blastocystosis: a precancerous infection? Ahmed et al., derived from asymptomatic carriers included in our 2. study. On the other hand, isolates from symptomatic Blastocystis hominis patients have been observed, in many studies, to Taşovain Turkish Y, patients Sahin B, with Koltaş hematological S, Paydaş malignancy. S. Clinical cause more severe and intense pathological changes significanceActa Med Okayama and frequency 2000; 54 of (3): 133-136. 3. PA. Current views on the clinical relevance of inwith mice mucosal[28,30,44,49] sloughing, and rats[6,27,31,55] intense. In inflammatory addition, Hussein cells BlastocystisTan KSW, spp. Mirza Curr H, Infect Teo Dis JDW, Rep Wu 2010; B, 12: Macary 28– infiltration,et al.[31] and and Abaza parasite et al .infiltration[6] reported of the lamina induction propria of 35. a more severe degree of pathological change with 4. polypoid projections in colonic mucosa of rats infected et al. The microbial by symptomatic isolates, that the authors referred eukaryoteScanlan PD, Blastocystis Stensvold CR, is Rajilić-Stojanović a prevalent and M, diverse Heilig to as precancerous polyps. In the present study, memberHG, De Vos of the WM, healthy O’Toole human PW, gut microbiota. FEMS comparable results were recorded as the active colitis Microbiol Ecol 2014; 90: 326–330. produced in mice ranged from severe in mice in GI, to 5. In moderate in mice in GII, to mild in mice in GIII, with vitro susceptibility of Blastocystis hominis isolated Yakoob J, Jafri W, Jafri, N, Islam M, Beg MA. lymphocytic aggregation in mice in GI and GII. Polypoid Biomed Sci 2004; 61:75–77. intense inflammatory cells infiltration and formation of 6. from patients with . Br J noticed in our present study in 57.14% of mice in GI Mokhtar AB. Subtype analysis of Blastocystis spp. (projectionsP in colonic mucosa were also significantly isolatesAbaza SM,from Rayan symptomatic HZ, Soliman and RH,asymptomatic Nemr NA, GII, and were not found to occur in mice inoculated by patients in Suez Canal University Hospitals, asymptomatic = 0.049), and isolates insignificantly in GIII). in 16.66% of mice in 7. Recently, studies demonstrating phenotypic Ismailia, Egypt. PUJ 2014; 7: 56–67.et al. Blastocystis is differentiating characters between Blastocystis spp. Nourrissonassociated with C, Scanzi decrease J, Pereira of fecal B, NkoudMongo microbiota isolates derived from persons diagnosed with or C,protective Wawrzyniak bacteria: I, Ciancomparative A, analysis between without CRC have been published. Differences included patients with irritable bowel syndrome and control different in vitro growth pattern and susceptibility[56], and in the surface ultrastructure, 8. Azizian M, Basati G, Abangah G, Mahmoudi MR, [57] Mirzaeisubjects. A.PLoS Contribution ONE 2014; 9of (11): Blastocystis e111868. hominis the presence of differences in the pathogenic potential ofprotein different profiles Blastocystis and zymography spp. isolates. Our studywith confirmspossible in development of irritable bowel syndrome. oncogenic potential in some of them. Parasitolsubtypes Res and 2016; associated 115 (5): inflammatory2003-2009. factors 9. In conclusion, Blastocystis spp. isolates associating role of Blastocystis spp. as an etiology of irritable colorectal carcinoma differ in their proliferative and bowelLepczyńska syndrome. M, Dzika Pol AnnE, Kubiak Med 2016; K, KorycińskaJ. 23 (1): 57-60. The invasive pathogenic capabilities than symptomatic 10. isolates. Moreover, asymptomatic Blastocystis spp. are Blastocystis hominis: A controversial human pathogen.Basak S, RajurkarParasitol Res MN, 2014; Mallick 113(1): SK. Detection261–265. of response in the large intestine of experimentally 11. infectednon-invasive animals. organisms causing only mild inflammatory A. Blastocystis hominis: a mysterious and commonly disregardedTasić N, Milenković parasite. T, Med Bujić and V, BioZdravković 2016; 18 D, (2):39- Tasić Authors Contribution: Ahmed MM shared in designing 47. the plan of work and revising the manuscript; Habib 12. FSM conceived and designed the plan of work, analyzed S. Blastocystis spp. subtype 3 triggers higher the data, wrote and revised the manuscript; Saad proliferationKumarasamy V,of Kuppusamyhuman colorectal UR, Samudi cancer C, Kumar cells, GA shared in performing the culture, experimental HCT116. Parasitol Res 2013; 112: 3551–3555 13. HM performed the culture, shared in experimental DA, Al-Semany SA. Predominance of Blastocystis infectioninfections and and, histopathological histopathological analysis. analysis; ElNaggar hominisAhmed subtype MA, Mohamed I among AM, colorectal Ahmed cancer SAM, patients Zaglool

Conflict of Interest: Authors declare that there is no 7:5(Suppl). 14. Mohamedin Makkah, AM, Saudi Ahmed Arabia. MA, J Bacteriol Ahmed ParasitolSA, Al-Semany 2016; conflict of interest. association risk of Blastocystis hominis subtype I References inSA, colorectal Alghamdi cancer: SS Zaglool a case-control DA. Predominance study. Infect and Agents Cancer 2017; 12: 21. 1. 15. Blastocystis lesions in Blastocystis hominis infection. Acta Tanspp. Clin KS. Microbiol New Rev insights 2008; 21: on 639–665. classification, Valsecchi R, Leghissa P and Greco V. Cutaneous identification, and clinical relevance of DermVenereol 2004; 84: 322-323. 99 PARASITOLOGISTS UNITED JOURNAL 16. Stopsack K, Heinrich-Gräfe U, et al. Blastocystis 134. Vogelbergspp. subtype C, Stensvold2 detection CR, during Monecke recurrence S, Ditzen of A, 30. experimentally infected mice. PUJ 2012; 5(2): 127- gastrointestinal and urticarial symptoms. Parasitol et al. Int 2010; 59 (3): 469-471. ImmunopathologicalAbdel-Hafeez EH, Ahmad assessments AK, Abdelgelil of human NH, 17. BlastocystisAbdellatif MZ, spp. Kamal in AM,experimentally Hassanin KM, infected Farkas K, et al. Do not forget the stool examination, immune-competent and immune-suppressed mice. cutaneousBálint A, Dóczi and gastrointestinal I, Bereczki L, Gyulai manifestations R, Szűcs M,of Parasitol Res 2016; 115(5): 2061-2071. Blastocystis sp. infection. Parasitol Res 2014; 113 31. Hussein EM, Hussein AM, Eida MM, Atwa M. (4): 1585-1590. Pathophysiological variability of different 18. genotypes of human Blastocystis hominis Egyptian Blastocystis and urticaria: Examination of isolates in experimentally infected rats. Parasitol subtypesCasero RD, and Mongimorphotypes F, Sánchez in an A,unusual Ramí�rez clinical JD. Res 2008; 102: 853–860. manifestation. Acta Trop 2015;148: 156-161. 32. 19. Dosoky I, Azab MS. Genotyping of Blastocystis Blastocystis hominis hominisAbu El-Fetouh symptomatic NI, Abdel isolates Megeed andES, Attiakinetics RA, El-of preventsRajič B, Arapovićthe development J, Raguž K, Boškovićof symptomatic M, Babić SM, Maslać S. Eradication of 2015; 8: 115–122. Ctries 2015: 9 (7): 788-791. 33. Garciaassociated LS. localDiagnostic CD3 and Medical CD20 cellParasitology. infiltrate. PUJ6th 20. Hashimoto’s thyroiditis: a case report. J Infect Dev Blastocystis spp. research. Philip Sci Lett 2018; 34. 11(01):Adao DEV, 39-60. Rivera WL. Recent advances in Lamchuanedition,.2015 D. BoeckPress, andWashington. Drbohlav Locke egg serum 21. Ajjampur SS, Tan KS. Pathogenic mechanisms in mediumSaksirisampant for detection W, Nuchprayoon of Blastocystis S, hominis Pradniwat. Chula P, Blastocystis spp. - interpreting results from in vitro and in vivo studies. Parasitol Int 2016; 65 (6 Pt B): 35. 772-779. DescriptionMed J 2010; of54 an (6): improved 527-536. method for Blastocystis 22. Blastocystis ratti hominisLanuza culture MD, Carbajal and axenization. JA, VillarJ, Parasitol Borrás Res R. induces contact-independent apoptosis, F- 1997; 83: 60-63. Puthiarearrangement, MK, Sio SW, and Lu barrier J, Tan KSW.function disruption in 36. IEC-6 cells. Infect Immun 2006; 74 (7): 4114–4123. Iwatani S, Kimata M. Feco-oral transmission of the 23. cystYoshikawa form of H, Blastocystis Yoshida K, hominis Nakajima in rats. A, Yamanri Parasitol K, Res 2004; 94:361–366 enterocyteZhaona Wu, apoptosis Haris M, Joshuaby Blastocystis D, Teo W, Kevindisrupts S, 37. Tan W. Strain-dependent induction of human Dutt S, Turnberg LA. Comparison of delayed Caspase 3- and 9-dependent manner. BioMed Res releaseRiley SA,5-aminosalicylic Mani V, Goodman acid (mesalazine) MJ, Herd and ME, Internatepithelial 2014;209163 barrier and ZO-1 organization in a sulphasalazine in the treatment of mild to moderate 24. Chandramathi S, Suresh K, Kuppusamy UR. relapse. Gut 1988;29:669–674. Solubilized antigen of Blastocystis hominis facilitates 38. the growth of human colorectal cancer cells, HCT116. L, Owen H. Location and pathogenic potential of Parasitol Res 2010; 106 (4): 941-945. BlastocystisWang W, Bielefeldt-Ohmann H, Traub RJ, Cuttell 25. Chan KH, Chandramathi S, Suresh K, Chua KH, 2014a 9 (8): e103962. Kuppusamy UR. Effects of symptomatic and 39. in the porcine intestine. PLoS ONE asymptomatic isolates of Blastocystis hominis on Ex-vivo and in vivo mice models to study colorectal cancer cell line, HCT116. Parasitol Res BlastocystisAjjampur SSR, spp. Png , CW, Chia WN,colonization, Zhang, Y, Tanand 2012; 110: 2475–2480. pathology:KSW. closer to proving Koch's postulates. 26. 40. colonKumarasamy carcinogenesis V, Kuppusamy by Blastocystis UR, Jayalakshmi spp. PLoS One P, BlastocystisPLoS. ONE 2016; tropism 11(8). in the pig intestine. Parasitol 2017;Samudi 12(8): C, Ragavan e0183097. ND, Kumar S. Exacerbation of ResFayer 2014; R, Elsasser 113 (4): T, 1465-1472.R, Solano G, Urban Jr J, Santin M. 27. 41. T, Bielefeldt-Ohmann H. Molecular epidemiology Iguchi A, Yoshikawa H, Yamada M, Kimata I, ofWang Blastocystis W, Owen in H, pigs Traub and RJ,their Cuttell in-contact L, Inpankaew humans ratsArizono experimentally N. Expression infected of interferon with Blastocystis gamma andsp. in Southeast Queensland, Australia, and Cambodia. proinflammatory in the cecal mucosa of 140. 42. 28. strain RN94-9. Parasitol Res 2009; 105 (1): 135- IwataniVet Parasitol S, et 2014b;al. Infectivity 203: 264–269. of different genotypes Blastocystis hominis in laboratory mice. Parasitol ofIguchi human A, Ebisu Blastocystis A, Nagata hominis S, Saitou isolates Y, Yoshikawa in chickens H, El-WakilRes 2010; HS,Hewedi107: 685–689. IH. Pathogenic potential of and rats. Parasitol Int 2007; 56 (2): 107-112. 29. 43. Hegazy MM, Maklouf LM, El Hamshary EM, Dawoud pathology induced by human Blastocystis in El-Gebaly, NS, Zaki MM. Ultrastructural intestinal HA, Eida AM. Protein profile and morphometry of 100 Blastocystosis: a precancerous infection? Ahmed et al., cultured human Blastocystis hominis from children cysteine proteases. Cell Microbiol 2012; 14: 1474- 1484. Parasitol 2008; 38(2): 453-464. 52. 44. with and healthy ones. J Egypt Soc. in entero-adhesion accounts for differences in Ana L, Falavigna-guilherme AL, et al. Pathogenicity epithelialWu, Z, Mirza barrier H, disruption Tan KS. Intra-subtype and is associated variation with Pavanelliof Blastocystis MF, Kaneshimaspp. to the EN, gastrointestinal Uda CF, Colli tract CM, metronidazole resistance in Blastocystis subtype-7. of mice: relationship between inoculum size and period of infection. Rev Inst Med Trop Sao Paulo 53. 2015; 57(6): 467-472. inductionPLoSNegl Tropof humanDis 2014a enterocyte 8: e2885. apoptosis by 45. BlastocystisWu Z, Mirza H, Teo JD, Tan KS. Strain-dependent Experimental infection of mice with Blastocystis organization in a caspase 3- and 9-dependent hominisYao FR, Qiao JY, Zhao Y, Zhang X, Yang JH, Li XQ. manner. Biomed disrupts Res Int epithelial 2014; 209163. barrier and ZO-1 54. Blastocystis ratti contains 46. Tan T, Suresh, Zhongguo K. Predominance Ji Sheng ChongXue of amoeboid Yu Ji Shengforms cysteine proteases that mediate interleukin-8 ofChong Blastocystis Bing Za hominis Zhi 2005; in isolates23:444–448. from symptomatic Puthiaresponse MK, from Lu J, humanTan KSW. intestinal epithelial cells in patients. Parasitol Res 2006; 98 (3): 189-193. 47. Popruk S, Pintong A, Radomyos P. Diversity of 7: 435–443. Blastocystis 55. Chandramathian NF-kB-dependent S, Suresh manner. K,Eukaryot Sivanandam Cell 2008; S, Parasitol 2013; 36: 88-97. Kuppusamy UR. Stress exacerbates infectivity and 48. RajamanikamA, subtypes Govind in SK. humans. Amoebic J Tropforms Med of pathogenicity of Blastocystis hominis: in vitro and in Blastocystis spp.: evidence for a pathogenic role. vivo evidences. PLoS One 2014; 9: e94567. 56. 49. HM. Growth kinetics and metronidazole sensitivity etParasit al. Experimental Vectors 2013; Blastocystis 6: 295-303 hominis infection in ofHabib Blastocystis FSM, Abdel-Fattah spp. isolated NS, Saad from GA, Elcolorectal Naggar Moelaboratory KT, Singh mice. M, Parasitol Howe J, HoRes LC, 1997; Tan 83:SW, 319-325. Chen XQ, 50. 11(3): 175-180. histochemistry and infectivity of Blastocystis 57. carcinoma (CRC) and non-CRC patients. PUJ 2018; hominisAbou El-Naga IF, Negm A. Morphology, 635. zymographyAhmed MA, of Habib Blastocystis FSM, Saadspecies GA, isolated El Naggar from 51. cyst. J Egypt Soc Parasitol 2001; 31: 627– HM. Surface ultrastructure, protein profile and prevents rho kinase-mediated intestinal epithelial 2019; doi.org/10.1007/s12639-019-01092-9. 10 barrierMirza H, compromise Wu Z, Teo JD, induced Tan KS. Statinby Blastocystis pleiotropy pages.patients with colorectal carcinoma. J Parasit Dis

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