Association of Blastocystis hominis with colorectal cancer: A systematic review of in vitro and in vivo evidences

Table 1 Data extraction table

No.	Ref.	Country	Sampling	Setting	Method	Main results	Conclusion
					Please note: (1) Main outcomes assessed; (2) If the protocol is published; and (3) If risk of bias is reported
Prevalence studies
1	Esteghamati et al[35], 2019	Tehran, Iran	Study design: cross-sectional. Study duration: July 2016 and November of 2017. Sample size (190): 80 patients (Primary Immunodeficiency), 85 (cancer patients) and 25 (organ transplant recipients)	3 hospitals in in Tehran, Iran	The aim of this study to determine the prevalence of intestinal parasites in 3 different groups of patients referred to 3 hospitals. Method used for parasite identification: Conventional methods, nested PCR and amplification of the 18S rRNA gene	The prevalence of Blastocystis hominis among CRC patient was 13/39 (28.2%)	The prevalence of Blastocystis hominis was found high in cancer patients, especially CRC patients
2	Zhang et al[18], 2017	China	Sample size: 381 faecal specimens were collected from cancer patients including CRC. Study duration: 2016 to 2017	Tumor Hospitalof Harbin Medical University	The aim of this study to determine the prevalence and genotypes/subtypes-Blastocystis in CP and analysed for the Blastocystis by PCR amplifying and sequencing	Prevalence of Blastocystis was 4 (8.1%) among CRC patient	Blastocystis subtype 1 and 3 have been identified in humans and animals
3	Mohamed et al[19], 2017	Saudi Arabia	Total sample size: 218. Two groups of participants: (1) CP (138) of which 74 had CRC and 46 had cancers outside gastrointestinal tract; and (2) NCP (80). Exclusion criteria: (1) Patient started chemotherapy regime; and (2) Receiving any anti-parasitic medication. Study duration: 2013-2015	King Abdulla Medical city (KAMC), Makkah	Case control study design: Aim, to determine the prevalence of Blastocystis among CRC patients compared to patients who had cancers outside gastrointestinal tract and control group. Obtained Blastocystis isolates were grouped into 2 categories (A and C), then subtyped into 3 various subtypes; subtype-I, subtype-II and subtype-V	Prevalence of Blastocystis among CRC = 22 (29%). Blastocystis infection frequency was significantly different between CP group and NC group. There was a higher probability of Blastocystis sp. among CP. Subtype I was the common subtype among CRC patients (54.5%). Interestingly, an association risk between Blastocystis subtype 1 with a greater risk of association in CRC group	The study revealed a probable association between subtype 1 of Blastocystis and CRC
4	Toychiev et al[20], 2018	Uzbekistan	A total sample of 400 participants, two groups of participants: (1) 200 CRC patients; and (2) 200 of Tashkent residents (without any gastrointestinal tract complaints). Exclusion criteria: (1) patient had problems with stool sample collection; and (2) received any treatment 2–3 wk before the study. Study duration: 2015-2017	Research Institute of Epidemiology, Microbiology and Infectious Diseases and the Research Center of Oncology, Tashkent, Uzbekistan, during the period	Study design: Prospective cohort: Prevalence of some parasites including Blastocystis sp. in CRC patients before and after surgery and chemotherapy compared to control group. Methods: “3 stool samples for parasitological examination were taken at 2-d intervals during CRC diagnosis before and after surgery and chemotherapy”	A significantly higher prevalence of protozoa was found in CRC patients than in control population “the prevalence of Blastocystis in CRC patients is 4 times as high as in the control population. The overall prevalence of Blastocystis sp. was 2.8% and was higher than the other protozoa”	Data revealed a potential role for Blastocystis sp. in CRC pathogenesis
5	Kumarasamy et al[21], 2014	Malaysia	Sample size: 425 patients who go through diagnostic colonoscopy. faecal samples and colonic washouts were obtained from 221 control patients and 204 patients with CRC. Study duration: 2010 and 2012	University of Malaya Medical Centre	To determine the Blastocystis genotype present by comparing the prevalence using colonic washouts and faecal samples PCR and standard stool culture. Both techniques were used to detect Blastocystis from control and patients with CRC.	The prevalence of Blastocystis was 15.29% (65/425). “Colonic washouts and faecal samples showed 12.24% (n = 52) and 5.65% (n = 24) of Blastocystis infection respectively”. A total of 43 individuals were positive for Blastocystis in CRC patients and was significantly higher compared to control group. Subtype 3 was predominant compared to other subtypes. It was significantly higher in CRC group as compared with control group	Blastocystis sp. is common in CRC patients. Subtype 3 is the most common genotype in the infected individuals
6	Chandramathi et al[1], 2012	Malaysia	Stool samples were obtained from 46 and 15 breast cancer and CRC patients, respectively	Department of Parasitology, Faculty of Medicine, University of Malaya	Aim: To investigate whether intestinal parasites can be an opportunistic infection in breast cancer and CRC patients who are undergoing chemotherapy treatment. Molecular detection of microsporidia species was done using a PCR technique. The presence of Blastocystis hominis was further confirmed by culturing stool samples	This study found that 7 out of 15 CRC patients were positive Blastocystis in various chemotherapy cycles accounting for 46.7%	Blastocystis hominis and microsporidia could appear as opportunistic infections during chemotherapy treatment of CP. This infection may diminish the efficacy of chemotherapy treatments and consequently advance the progression of cancer
7	Majeed et al[22], 2019	Iraq	116 faecal specimens with Blastocystis and Helicobacter pylori infection, 15 biopsy specimens from CRC patients	Middle Technical University/Baghdad 1st Feb 2018-15th June 2018	Faecal specimens were screened for Blastocystis and Helicobacter pylori. Direct DNA sequencing was done to evaluate mutations in CRC-associated molecular pathways	Prevalence of Blastocystis infection statistically insignificant in various age groups. Prevalence of Blastocystis infection was more in females [females 29 (46.9 %), males 22(43.1%)]. Prevalence of mixed infection (Blastocystis and Helicobacter pylori) was 27 (23.32%)	Prevalence of Blastocystis infection was more in females. KRAS and TP53 gene mutation was observed in the CRC patients with mixed infection (Blastocystis and H. pylori)
In vitro studies
8	Chandramathi et al[7], 2010	Malaysia	In vitro study model. PBMCs were isolated from blood collected from healthy persons. Solubilised antigen of Blastocystis isolate was obtained from a human subject. Human colorectal carcinoma cell line, HCT116, was used	University of Malaya, Kuala Lumpur	Effect solubilised antigen of Blastocystis on the HCT116 proliferation quantified. Gene expressions of certain genes in HCT116 and PBMCs evaluated via real-time reverse transcription PCR. PBMCs were isolated from blood using Histopaque technique. Cell proliferations were measured using MTT assay	Increased number of PBMCs/ HCT116 cells observed with Blastocystis antigen. IFN-γ and TNF-α were downregulated and IL-6, IL-8 and NF-κB, p53 were upregulated in the PBMCs treated with the antigen. IFN-γ was downregulated and IL-6 and NF-κB was upregulated in HCT116 cells	Solubilised antigen of Blastocystis could facilitate increased number of PBMCs/ HCT116 cell and has the ability to downregulate immune cell responses
9	Chan et al[23], 2012	Malaysia	In vitro study model. Solubilised antigen of Blastocystis isolate was obtained from symptomatic and asymptomatic human subject. HCT116 was used	University of Malaya, Kuala Lumpur	Effects of solubilised antigen of Blastocystis isolate was obtained from symptomatic and asymptomatic human subject on HCT116. Gene expressions of certain genes in HCT116 and PBMCs evaluated via real-time reverse transcription PCR	Increased number of HCT116 cells observed with symptomatic Blastocystis antigen. Th2 cytokines/CTSB were upregulated in HCT116. NF-κB was observed upregulated in HCT116 exposed to symptomatic Blastocystis antigen	Solubilized antigen of Blastocystis from symptomatic individual was more virulent than that in asymptomatic. Higher inflammatory reaction and increased proliferation of cancer cells was observed
10	Kumarasamy et al[24], 2013	Malaysia,	In vitro study model using HCT116 treated with solubilised Blastocystis antigen from 5 Blastocystis subtypes	University Malaya research Lab	In vitro study. HCT116 treated with solubilised antigen from Blastocystis. Following Assays: Proliferation of the cell line, HCT116 on exposure to different Blastocystis subtypes; Gene expression profile of apoptotic genes like p53 and CTSB; Transcription factor gene expression profile	Blastocystis subtypes (5) increased the proliferation of HCT116, especially subtype 3. Blastocystis antigen caused the upregulation of Th2 and Th1 cytokines, and downregulation of IFN-γ and p53 in HCT116 cells. Blastocystis antigen caused a higher stimulation of gene expression of CTSB and TGF-β genes	Infection with Blastocystis caused exacerbation of existing colon cancer cells. The effect may be due to weakening of the cellular immune response and dysregulation of IFN-γ and p53 expression. Infection with Blastocystis subtype 3 has a higher pathogenic potential
11	Ahmed et al[25], 2019	Cairo, Egypt	Seven Blastocystis isolates were from stools specimen from patients with early diagnosed CRC (Oncology and Surgery and Colonoscopy unit) of a Hospital in Egypt. The different groups were: Group I (GI), 12 isolates from infected non-CRC; Group II (GII), 6 from infected symptomatic patients and Group III (GIII), 6 from infected non-symptomatic carriers	Department of Parasitology lab, Faculty of Medicine, Ain Shams University, Cairo, Egypt	Aim: To investigate some phenotypic characters like the surface ultrastructure, protein profiles and protease activity of Blastocystis from three different clinical groups. Techniques performed: Scanning electron microscopy to study morphology of the organism; SDS-PAGE to analyse the Blastocystis protein profiles and their protease activities	Observations: All CRC Blastocystis isolates showed a very rough intensely folded surface when compared to less rough and smooth surface of isolates from symptomatic and asymptomatic and non-CRC isolates; SDS-PAGE showed presence of 2 protein bands of 230 and 32 KDa in 42.9% of Blastocystis CRC isolates and these proteins were absent in Non-CRC isolates. When the protease activity of the parasite was tested, no significant difference existed between isolates of the three groups	There was significant difference in the surface structure and the protein profiles between different clinical isolates of Blastocystis. Differences indicate that it may be: (1) secondary to the altered gut environment in the presence of CRC or (2) indicators of a different pathogenic potential of the parasite in inducing malignancy
In Vivo studies
12	Kumarasamy et al[26], 2017	Malaysia	Different specimens collected: Blood, urine, faecal samples and gastrointestinal tract sections from 24 male Wistar rats. Age of the rats: 3 wk. Weight of each rat: Average of 65 g/rat	University Malaya research Lab	In vivo experimental study. Aim: To investigate the effect of infection with Blastocystis cyst on exacerbation of carcinogenesis. Twenty-four rats divided into different groups for the study (4 groups, 6 rats each): Control group, AOM group, group inoculated with Blastocystis cyst, the group inoculated with Blastocystis cyst and AOM injection. Body weights recorded once a week. Rat faecal samples screened for presence of Blastocystis post-inoculation. Histopathological assessment of the rat colon for aberrant crypts. Urine and blood samples assessed for oxidative stress	Observations: lower body weight showed by Blastocystis infected rats than rats infected with Blastocystis and injected with AOM (P < 0.05). Stools from AOM-rats with Blastocystis infection were softer and watery compared to the AOM-rats without Blastocystis infection. Blastocystis was present in the stool of all infected rats from Day 3 to 7 post-inoculation. All the rats injected with AOM developed numerous abnormal, hyperplastic colonic crypts. Co-administration of Blastocystis cyst showed a 1.6-fold increase in the number of crypts when compared with control rats treated with AOM only. Two of the co-Blastocystis infected AOM-rats were found to have adenomas. Major dysplasia and presence of hyperplastic aberrant crypts were observed in rats injected with AOM and co-infected with Blastocystis	Blastocystis infection considerably enhanced the AOM-induced carcinogenesis because of the oxidative damage of the intestinal epithelium

AOM: Azoxymethane; CP: Cancer patients; CRC: Colorectal cancer; CTSB: Cathepsin B; IFN: Interferon; IL: Interleukin; NCP: Non cancer patients; NF-kB: Nuclear factor kappa B; PBMCs: Peripheral blood mononuclear cells; PCR: Polymerase chain reaction; SDS-PAGE: Sodium dodecyl sulphate–polyacrylamide gel electrophoresis.