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

lNTRODUCTlON

(

)is one of the most commonly recovered microorganisms in faecal specimens,and its widespread presence is found in colorectal cancer(CRC)patients[1].Its distribution is known to be prevalent in both rural and urban areas[2].This microorganism has been in discussion since early 1900s[3,4];however,the taxonomic position of

remains unanswered.

treatment is often difficult due to its drug resistance and the failure of the host defenses to counter the infection[5].Previous studies suggest that

is a common and diverse element of microbiota in human host,as it has been highly prevalent in healthy individuals[6-8].

is commonly found in both patients with gastrointestinal symptoms and in healthy people widely across the world.More recently,researchers consider

as an emerging zoonotic disease,and its pathogenic potential in human is somewhat controversial[9].Although accumulating data suggest that

is a pathogen,the pathogenic role in humans is still a matter of debate.

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It is suggested that around 20% of cancer reported worldwide could have been due to infectious agents[10,11].Viruses such as hepatitis B virus,human papilloma virus and Epstein-Barr virus have been associated with carcinogenesis.Various other bacteria also have been described previously to exacerbate cancer[12].There are numerous epidemiological evidences that strongly support the fact that parasites can be a factor of various malignant tumours[13],but it is challenging to validate this relationship.Previously,a review article highlighted the correlation of various protozoan parasites including

with carcinogenesis[13].In addition,there was a case report in India that demonstrated a possible association of subtype 3

in the worsening of CRC[14].

There are a few other systematic reviews on the interventional studies done on

,but we did not find any systematic reviews on the association between

and CRC.Therefore,this systematic review aimed to(1)identify prevalence of

in CRC patients;(2)review

colorectal carcinoma cell line study models on the cytopathic and immunological effects of

antigens;and(3)review an

experimental animal model study to investigate the effect of infection with

on exacerbation of colorectal carcinogenesis.

MATERlALS AND METHODS

PRISMA guidelines were utilised in conducting this review[15].

Eligibility criteria

For

studies,the quality of the studies was assessed using Quasi experimental appraisal tool[16],which contained nine questions.For

studies,checklist of Systematic Review Center for Laboratory Animal Experimentation’s Risk of Bias tool for assessing risk of bias as quality assessment was used[17].The quality of all included studies was acceptable.

Exclusion

Articles that reported the association of

with cancer in general without specific findings on its association with CRC,reviews papers,conference proceedings and case reports were excluded from this review.

Search

PubMed,Science Direct,Scopus and Google scholar databases were searched.

If the newspapers, or even the whole world, said of a certain subject, It is so-and-so; and a little schoolboy declared he had learned quite differently, they would take his assertion as the only true one, because he belonged to the family

The various keywords used were:

infection and CRC and their MESH terms and synonyms.Manual searching through reference lists of included journal articles was done to find the missed studies during online search.

Study selection

We identified 872 papers in the initial screening(Figure 1).

The storms threwvessel after vessel on the fatal reefs; there were snow-storm andsand-storms; the sand flew up to the houses, blocking the entrances,so that people had to creep up through the chimneys; that wasnothing at all remarkable here

Identification

First,two authors developed a search strategy with different key words and their synonyms.All articles were moved to the Endnote X7 software(Clarivate Analytics,Philadelphia,PA,United States),and 146 duplicate papers were removed.Two authors independently went through the titles and abstracts of the remaining 726 papers.Subsequently,a total of 22 papers were retained for full text review.The eligibility of retained papers was evaluated by two other authors.Authors checked the reference lists of all included articles for any relevant studies that met this systematic review inclusion criteria and had not been found during the database searches.

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After full review of the 22 papers,12 were selected.All authors have completed data extraction and agreed eligibility of included papers(Figure 1).

Quality assessment

Original articles that reported the prevalence and association of

subtypes with CRC patients,

studies using

antigen and an

study using an animal model published before February 2,2020 were included.

Data extraction

The data extracted included author-year,country,sampling,setting,methods,results and study conclusion(Table 1).

RESULTS

Prevalence of Blastocystis in CRC patients

Based on the seven reviewed articles,the prevalence of

in CRC patients were found to be between 2.8%-46.7%,whereby subtype 1 and subtype 3 were predominantly isolated.

A study by Esteghamati

[35]evaluated the prevalence of

in 85 cancer patients,including 39 CRC and 46 cancers outside gastrointestinal tract(COGT).In this study,

was identified in 11/39(28.2%)among CRC group.Another study in China by Zhang

[18]showed the prevalence of

in 4 among 49(8.1%)patients with CRC.In another study conducted in Saudi Arabia,the prevalence of

among CRC patients was 29.7%[19].Subtype I was thepredominant(54.5%)among CRC patients,while subtype II was predominant(43.7%)among COGT patients[19].Higher prevalence of intestinal helminths and protozoa was observed in CRC patients than in the control population in a study conducted in Uzbekistan.The prevalence of

in CRC patients was four times higher than that in the control population.The overall prevalence of

(2.8%)was significantly higher than the other protozoa[20].In a study by Kumarasamy

[21]in Malaysia,among 221 control patients and 204 CRC patients with colorectal malignancies,the overall prevalence of

infection was 15.29%(65/425).A total of 43(21.08%)samples were positive for

infection in CRC patients and was significantly higher compared to normal individuals(

=22,9.95%,

＜ 0.01).Subtype 3 was present at higher levels compared to other subtypes detected in both groups and was significantly higher in CRC patients as compared with control patients[21].

Another study sought to investigate some phenotypic characteristics such as the surface ultrastructure,protein profiles and protease activity of

isolated from three different clinical groups:CRC patients,non-CRC symptomatic and asymptomatic infected persons.This study showed the presence of two protein bands of 230 and 32 KDa in 42.9% of

CRC isolates with their complete absence from non-CRC isolates.There was no significant difference in the protease activity of the protein among isolates of the three groups,CRC and Non-CRC

isolates[25].

In vitro colorectal carcinoma cell line studies on the cytopathic and immunological effects ofBlastocystis antigen

Intestinal colonisation of

(

)as a risk factor to the worsening of colorectal cancer(CRC).

The three

model studies used the human colorectal carcinoma cell line HCT116 and the

isolated from a human subject[7,23,24].One of the studies demonstrated the cytopathic effect of

antigen on peripheral blood mononuclear cells.This study findings showed increased cell proliferations in

antigen-stimulated HCT116 cell-lines,which suggested that the infection by

may facilitate the growth of colon cancer cells[7].Another

study showed that the five subtypes of

significantly increased the proliferation of HCT116,especially subtype 3.

antigen caused the upregulation of T helper(Th)2 and Th1 cytokine gene expressions,and downregulation of interferon gamma and

gene expressions in HCT116 cells.In addition,

antigen caused a significantly higher stimulation of Cathepsin B(CTSB)and Transforming growth factor beta(TGF-β)gene expression,which indicates the pathogenic potential of this protozoan[24].Another study showed an increase in cell proliferation in HCT116 cells inoculated with the symptomatic

antigen.Gene expression studies carried out in this research also showed a significant upregulation of Th2 cytokines,which indicates the parasites’ potential in weakening the cellular immune response[23].HCT116 cells exposed to symptomatic and asymptomatic

antigen caused a significant upregulation of

,which led to the postulation that the

antigen may enhance the invasive and metastasis properties of CRC[23].

Another study was designed to investigate the emergence of

and Microsporidia infections in breast and CRC patients undergoing chemotherapy treatment.This study found that 7 out of 15 CRC patients were positive

in various chemotherapy cycles,accounting for 46.7%.However,the researchers did not mention whether the isolate was from the same patients in different cycles[1].In a study carried out in Iraq,stool samples from 116 patients with

and

infections were investigated.Fifteen tissue samples of CRC were taken from 15 suspected patients out of 116 infected cases,and it was shown that the infection with

and

was associated with pathological gene mutation in the CRC patients[22].

In vivo experimental animal model to investigate the effects of infection with Blastocystis on exacerbation of colorectal carcinogenesis

is a commonly identified microorganisms in CRC patients.These studies have provided supportive data that

could exacerbate existing CRC

alteration in host immune response and increased oxidative damage.

DlSCUSSlON

is one of the most common gut microorganisms found in healthy individuals[27].Besides being associated with a healthy gut microbiota[27,28],

infection is also known to be opportunistic in immunocompromised patients[29].CRC is the third most common cancer diagnosed worldwide and one of the major causes of cancer-associated fatality[30].The reason for high mortality is due to the asymptomatic progression of the disease that usually results in late diagnosis[31,32].Certain gut microorganisms are known to be one of the important factors that had been associated with initiation and development of CRC[33].However,data on the roles of parasites are vague and restricted.Various findings have been reported regarding the association between

among CRC patients,whereby positive association was shown in all the studies[1,23,24].Therefore,the aim of this systematic review was to quantify the studies published so far that revealed the direct association of

and CRC.This paper outlines the results of a systematic review to evaluate the prevalence of

in CRC patients,

studies using

antigen and an

study using animal models.

Out of the data extracted from 12 studies relevant to this topic,all the studies showed positive association between

and CRC.Prevalence studies,

investigations and

studies were used to evaluate the pathogenicity of

with CRC.

The global prevalence of

infection ranged from 1.5%-20% in developed countries,which was much less than that in developing countries,which was 30%-50%[34].Based on our review,the prevalence of

infection in CRC patients ranged between 2.8%-46.7%.It has been widely reported in the world,in developing countries such as Iran,China,Saudi Arabia,Uzbekistan and Malaysia[18-20,24,35].The first demonstration of

infection in Iran was reported in 11 CRC patients in Tehran province[35].In Malaysia,a total of 43 samples were positive for

from 204 CRC patients[21].This study utilised colonic washout in addition to stool sample to recover the parasites.Subtype 3

was detected predominantly as compared to other subtypes[21].Some of these findings highlighted the high prevalence of certain subtypes of

among these patients[19,21].A previous study showed that subtype 1 was the most common genotype identified(54.5%)among CRC patients[19].DNA was extracted from

cultures

conventional method and subtyped using multiplex polymerase chain reaction(PCR)with restriction fragment length polymorphism and sequence-tagged site primers-based PCR.In another study,subtype 3 was predominant compared to other subtypes found in both CRC patients and healthy individuals[21].Subtype 3 is speculated as the most pathogenic subtype in symptomatic individuals[36].Some researchers have attributed subtype 3 to be more pathogenic compared to other subtypes[37,38].

was initially screened

culture and conventional PCR using stool samples and colonic washouts.The presence of

infection in CRC patients could be contributed by various reasons including health status.For instance,positive cases were more likely in patients with gastrointestinal symptoms compared to healthy individuals[18].Besides,

was also identified in higher frequency in immunosuppressed CRC patients who were undergoing chemotherapy treatment[1,18].

A total of three

studies were carried out using colorectal carcinoma cell line models to study cytopathic and immunological effects of

antigen[7,23,24].Some of the research studies suggested that solubilised antigen of

could facilitate the exacerbation of CRC cells,HCT116[7,23].In another study by Kumarasamy

[24],

subtype 3 stimulated significantly higher

and

gene expression in HCT116,which indicates the pathogenic potential of this protozoan.Result of

studies that were performed in Malaysia were similar[7,23,24].

is commonly found in both patients with gastrointestinal symptoms and in healthy people widely across the world.More recently,researchers consider

as an emerging zoonotic disease,and its pathogenic potential in human is unclear[9].The pathogenic potential of

was widely debated,as they are found in both symptomatic and asymptomatic patients.The significant expression of nuclear factor kappa light chain enhancer of activated B cells was observed in HCT116 exposed to

antigen isolated from individuals with gastrointestinal symptoms,but such observations were not found when the colon cells treated with

antigen isolated from asymptomatic individuals.This finding shows the potential pathogenicity of symptomatic

in CRC patients[23].Similarly,HCT116 cells exposed to symptomatic and asymptomatic

antigen caused a significant upregulation of

.These gene expression findings lead to a postulation that the

antigen may enhance the invasive and metastasis properties of CRC[23].Besides,proliferation of HCT116 when exposed with

antigen could be a result of higher levels of interleukin(IL)-6 and IL-8 expression[23].

Another study revealed that solubilised antigen isolated from subtype 2 and 3 isolates introduced to colon cancer cells showed significant IL-8 and IL-6 expression[24].The production of inflammatory cytokines such as IL-8 together with reactive oxygen species could contribute to the pathogenesis of cancer[38].In a few studies conducted previously,IL-6 expression was associated with proliferation of colon carcinoma[39,40].Besides that,subtype 3

also triggered positive expression of

in cancer cells.A previous study showed that

expression is significant in CRC patients[41].

Only one study was conducted to investigate the

effect of

in Wistar rats.In parallel with

studies,an

study showed similar findings on the possible role of

to exacerbate CRC.The results demonstrated that

may cause damage to the intestinal mucosal layer and result in increased crypts formation.Furthermore,an increased oxidative stress was also observed in these rats.There have been numerous animal models of human CRC and animal model of tumour carried out

quantification of aberrant crypt foci[42,43].This allows the study of gut microbiome and its role in pathogenesis.In humans,there are many potential pathogenic and nonpathogenic gut microbial infections,and various animal models have been used for such studies.Aberrant crypt foci are known as putative precancerous lesions of the colon in both animal models and humans[44,45].Even though various studies associating

and CRC were carried out

model using colon cancer cells,this study utilised animal model to bridge between

findings in the laboratory and studies in humans.As such,this extensive

study showed that

had a major impact on normal intestinal function in Wistar rats resulting in damage to the intestinal mucosal layer and inducing oxidative stress,which caused increase in crypts formation in AOM-treated rat models.The study establishes that

is a pathogen,and there is a need to screen cancer patients for harbouring this parasite.

This systematic review has some limitations.According to these investigations,a greater prevalence of

was found in CRC patients,but the question whether increased prevalence of

could be linked with increased high risk to CRC is unclear.The studies discussed in this review did not highlight the association of

according to cancer stages,and it was unclear if

itself could result in the initiation of malignancy as

acquisition alone is insufficient for cancer development.Even though strong association between

and CRC is apparent,some questions remain unanswered.Therefore,we propose future studies should focus on the pathogenicity of

in various stages of CRC by concentrating on the molecular pathways involved in tumorigenesis.

CONCLUSlON

In conclusion,according to various recent studies,

is one of the most commonly identified microorganisms in CRC patients,whereby subtype 1 and subtype 3 were predominantly isolated.It is apparent in most cases that the prevalence is higher in developing countries compared to developed countries.These studies have provided supportive data that

could exacerbate existing CRC

alteration in host immune response and increased oxidative damage.An

study wellestablished that

infections resulted in tissue damage from host inflammatory responses that may predispose the host towards neoplasm exacerbation.Upregulation of gene expression responsible for proinflammatory cytokines and downregulation of apoptotic genes was observed in

studies.Through continued research in

and CRC,we may discover new findings as well as develop new effective means of prevention.Future studies of CRC and

should attempt to determine the various stages of CRC that are most likely to be associated with

and its association with other intestinal bacteria.In addition,future

studies should evaluate exposure to various subtypes of

.

ARTlCLE HlGHLlGHTS

Research background

Three of the reviewed articles used

study models and observed considerable cytopathic and immunological effects induced by the solubilised antigen of

to the human colorectal carcinoma cell line[7,23,24].These findings speculated that

infection may enhance the proliferation,invasiveness and metastatic properties of CRC cells.One study investigated some phenotypic characteristics of

isolated from CRC patients[25].

Research motivation

There has been an increase in the prevalence of

in CRC patients.Besides,various

and in vivo studies have highlighted

as an important risk factor for the worsening of CRC.

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Research objectives

To perform a systematic review on all evidence on the association between CRC and

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Research methods

Future studies of CRC and

should attempt to determine the various stages of CRC that are most likely to be associated with

and its association with other intestinal diseases.

Research results

Out of 12 studies selected for this systematic review,seven studies have confirmed the prevalence of

.A total of four studies employing

human colorectal carcinoma cell line study models showed significant cytopathic and immunological effects of

.One

experimental animal model study showed that there was a significant effect of infection with

on exacerbation of colorectal carcinogenesis.

Research conclusions

An animal model study compared the effects of

infected rats and rats infected with

co-administered with Azoxymethane(AOM),a potent carcinogen.This finding showed that the co-administration of

cyst resulted in a 1.6-fold increase in the number of colonic crypts when compared with control rats treated with AOM only.Two of the co-

infected AOM rats were found to have adenomas.Major dysplasia and the presence of hyperplastic aberrant crypts were also observed in rats injected with AOM and co-infected with

[26].

Research perspectives

A systematic review of the literature was performed by searching PubMed,Science Direct,Scopus and Google scholar databases up to February 2020.

FOOTNOTES

Azzani M designed the research,performed the literature search and extracted the data;Kumarasamy V wrote the discussion and extracted the data;Atroosh WM wrote the methodology and extracted the data;Anbazhagan D wrote the results and extracted the data;Abdalla M wrote the introduction and extracted the data;All authors read and approved the final manuscript.

It doesn t matter when. I m sure the Addisons are nice people, but I m not going to waste an evening socializing with people who don t have any eligible3 daughters.

Dr.Azzani has nothing to disclose.

The authors have read the PRISMA 2009 Checklist,and the manuscript was prepared and revised according to the PRISMA 2009 Checklist.

This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers.It is distributed in accordance with the Creative Commons Attribution NonCommercial(CC BYNC 4.0)license,which permits others to distribute,remix,adapt,build upon this work non-commercially,and license their derivative works on different terms,provided the original work is properly cited and the use is noncommercial.See:https://creativecommons.org/Licenses/by-nc/4.0/

With arms outstretched and hands clutching the broomstick inserted through the sleeves of an old plaid shirt, I wore a felt hat and faded overalls stuffed with straw

Malaysia

Vinoth Kumarasamy 0000-0002-8895-1796;Wahib Mohammed Atroosh 0000-0003-3809-3460;Deepa Anbazhagan 0000-0002-2527-4126;Mona Mohamed Ibrahim Abdalla 0000-0002-4987-9517;Meram Azzani 0000-0002-7939-1539.

Chang KL

Filipodia

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Chang KL

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