The associated risk of Blastocystis infection in cancer: A case control study

Background Blastocystis is an anaerobic intestinal protozoan. Nine Blastocystis subtypes (STs) were detected in humans. A subtype-dependent association between Blastocystis and different cancer types has been debated in many studies. Thus, this study aims to assess the possible association between Blastocystis infection and cancer, especially colorectal cancer (CRC). We also screened the presence of gut fungi and their association with Blastocystis. Methods We used a case-control design; cancer patients and cancer-free (CF) participants. The cancer group was further sub-group into CRC group and cancers outside the gastrointestinal tract (COGT) group. Macroscopic and microscopic examinations were performed to identify intestinal parasites in participants’ stool samples. Molecular and phylogenetic analyses were conducted to identify and subtype Blastocystis. Furthermore, gut fungi were investigated molecularly. Results 104 stool samples were collected and matched between CF (n=52) and cancer patients (n=52); CRC (n=15) and COGT (n=37). As anticipated, Blastocystis prevalence was significantly higher among CRC patients (60%, P=0.002) and insignificant in COGT patients (32.4%, P=0.161) compared to CF group (17.3%). The most common subtypes were ST2 among cancer group and ST3 in the CF group. Conclusion Cancer patients have a higher risk of Blastocystis infection compared to CF individuals (OR=2.98, P=0.022). Increased risk of Blastocystis infection was associated with CRC patients (OR=5.66, P=0.009). Nevertheless, further studies are required to understand the underlying mechanisms of Blastocystis and cancer association.


Introduction
Blastocystis is an anaerobic intestinal protozoan found in humans and a wide range of animals. Morphological forms of Blastocystis include vacuolar, granular, amoeboid, and cyst forms, the vacuolar form is predominant in fresh stool samples and laboratory cultures (1). The Blastocystis prevalence rate is 60% in developing countries, due to poor hygiene and close contact with animals, compared to developed countries (5-20%) (2). More than 17 subtypes (STs) of Blastocystis spp. are known, and only nine subtypes are found in humans (3,4). Blastocystis was considered a commensal parasite and caused asymptomatic infections at most. However, there is an increasing number of studies investigating the role and pathogenicity of Blastocystis in the gut (3,4).
Some studies showed that Blastocystis infections contributed to the severity of multiple conditions, such as AIDs, cancer, IBD, and gut microbiota dysbiosis (5)(6)(7). Conversely, other studies have shown that the presence of Blastocystis promotes high diversity of gut microbiota in healthy individuals preventing intestinal disorders (8,9). Blastocystis culture filtrates did not affect the growth of cancer cell lines in one study (10). These conflicting findings are probably attributed to the genetic diversity of Blastocystis, inter and intra-subtype variations (7, 11,12).
Globally, cancer is regarded as one of the leading causes of death with an estimation of 10 million deaths in 2020 (13). The most common cancers in order are breast, lung, colorectal, prostate, skin, and stomach cancers according to WHO in 2020 (13). Some of risk factors include age, family history, obesity, diet, and infectious pathogens (13, 14). Since 30-50% of cancer is preventable by avoiding its risk factors and 30% of cancer are caused by infectious pathogens, it is important to identify all possible cancerpromoting infections to limit progression of existing cases and emergence of new cases (13). Colorectal cancer (CRC) is the third most common cancer, and the second leading cause of cancer-related deaths worldwide (2020) (15). CRC cases are detected in males more than females. The majority of CRC cases are diagnosed in the late stages of the disease, partially due to its non-specific symptoms (16)(17)(18)(19). One of the risk factors of CRC is the gut microbiota dysbiosis (17)(18)(19)(20). Several studies have investigated the role of microbiota in different cancers initiation or progression (21)(22)(23)(24)(25)(26)(27). However, previous studies focused on the bacterial content overlooking other micro-organisms such as protozoa and fungi (28).
Since Blastocystis spp. is considered a normal intestinal flora, investigating Blastocystis association with cancer, focusing on CRC, is essential to understanding the gut microbiota effect on tumor initiation and progression (8,29). Many studies investigated the interaction between Blastocystis spp. and gut microbiota, and one study reported an association between Blastocystis with increased levels of five gut fungi (Mycobiota) (7-9, 30). Other studies associated gut mycobiota with carcinogenesis, initiation, and development of different cancers (20,(31)(32)(33). Thus, this study aimed to assess the possible association between Blastocystis infection and fungi in cancer patients locally. Then, compare Blastocystis infection in different cancers' patients to cancer-free controls (CF).

Study design
In this observational study, a matched case-control study design was used. The case patients affected by cancer were gender and age-group-matched with a CF control group. The study participants were recruited from March 2020 to April 2022. This study followed the STROBE guidelines (34).

Study population and variables
All participants signed informed consent (n=104), and the study was performed per the Declaration of Helsinki. Participants of both genders were 18 years old and above. Age was categorized into three groups: Youth (18-24 years old), adults (25-59 years old), and elderly (≥60 years old). Nationalities were classified into six regions of origin: Africa, Americas, South-East Asia, Europe, Eastern Mediterranean, and Western Pacific. Patients who took any anti-parasitic drug in the last six months, did not provide informed consent, or were unwilling to give a stool sample were excluded from this study. The consumption of antibiotics, gastrointestinal surgery history in the past two years, hospitalization, cancer therapy, and the number of therapy cycles were extracted from patients' medical records.

Case patients
Patients referred to the Oncology Services at Tawam Hospital, United Arab Emirates, with confirmed cancer (n=52) cases histopathologically, in any stage, and undergoing any treatment were included in this study. The cases were further sub-categorized into two groups: CRC (n=15) and cancers outside the gastrointestinal tract (COGT) (n=37) patients.

Control participants
Community-based control participants (n=52) were gender-and age-group matched subjects recruited. Excluding subjects with intestinal disorders, immunological or neoplastic disorders, and antibiotics users in the last six months before recruitment.

Sample collection and processing
Stool samples were collected using a commercial stool collection kit (alpha laboratories, UK). All stool samples were transported for analysis to the Microbiology Laboratory, College of Medicine and Health Sciences (CMHS), United Arab Emirates University (UAEU). Stool samples were each split into two parts; one was stored at 4°C and processed within one to two days for macroscopy, and microscopy, while the second was put in Eppendorf tubes and stored at -20°C for molecular work.

Macroscopic and microscopic investigation
As mentioned previously (35), macroscopic and microscopic examinations were performed to define the types of organisms and check for blood and mucus. Stool samples were stained with Wheatley Trichrome for Blastocystis detection, following the manufacturer's instructions. Then, smears were examined microscopically under ×40 and ×100 magnification objectives. Two microbiologists examined all stool slides independently.

Molecular investigation
Genomic DNA was extracted from stool samples as previously published (35). Primers and PCR conditions used are listed in Table  S1. Blastocystis spp. and gut fungi DNAs were amplified by Polymerase Chain Reaction (PCR). A mix of 5µl of 5x Q-Solution, 2.5µl of 10x CoralLoad PCR buffer, 1µl of each primer (10mM), 0.5µl of QIAGEN Taq DNA Polymerase (250U),1µl of dNTP Blend (100mM) (Applied Biosystems, USA), and 2µl of stool genomic DNA diluted in free-nuclease water to reach final volume of 25µl. For the ITS reaction mixtures, an extra 0.5µl of MgCl 2 was added.

Sequencing and phylogenetic analysis
Gel and PCR Clean-Up System (Promega, Madison, Wisconsin, USA) was used following the manufacturer's instructions. Barcode region and ITS primers were used to sequence the purified products in both directions via capillary electrophoresis using a Big DyeTM Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) in ABI PRISM 3130xI Genetic Analyzer. The confirmed Blastocystis-positive sequences were assigned to the best-matched Blastocystis subtype by aligning them to reference sequences in the GenBank database using nBLAST program (36). Query cover and per identity of ≥97% were used to determine subtypes matches. The ClustalW algorithm of MEGA-X was used to align the sequences (37). Acquired sequences were submitted to PubMLST database to confirm subtypes and identify relevant alleles (38). The established fungipositive sequences were assigned to the best-match fungi species by aligning them to reference sequences in the GenBank database. Query cover of ≥80% and 97-100% per identity were used to determine the most probable match.
Blastocystis samples and reference sequences along with an outgroup species (Proteromonas lacertae, GenBank accession no. U37108) went through phylogenetic analysis. The best substitution models were determined via the Bayesian information criterion. The maximum Likelihood (ML) method and Bayesian Inference (BI) were used to construct the tree. ML tree was constructed using MEGA-X. Tamura 3-parameter with gamma distribution was used, and Bootstrapping analysis with 1000 replicates was performed. For the BI method, Jmodeltest v2.1.10 and MrBayes v3.2.7 were used (39,40). Hasegawa-Kishino-Yano model with gamma distribution was used, and four Markov chains were run for 5 million generations, with a sampling frequency of 100 and a 25% burn-in. Tree Graph 2 combined the two constructed trees (41).

Statistical analysis
Data were analyzed by IBM SPSS Statistics v26.0. Qualitative variables were expressed as numbers and percentages, while quantitative variables were expressed as means and medians. The chi-square, Fisher's Exact, and Fisher-Freeman-Halton tests for categorical variables. Logistic regression was used to predict factors associated with Blastocystis prevalence, and a p-value of ≤ 0.05 indicated statistical significance.

Ethical approval
The study was approved by the Tawam Human Research Ethics Committee (T-HREC) of Tawam Hospital, Al Ain, Abu Dhabi, UAE (THREC-678).

Results
In this case-control study, a total of 104 matched participants were recruited. The controls were CF participants (n=52), and the cases were cancer patients (n=52) (

Stool analysis
Stool samples macroscopic parameters had an insignificant association with Blastocystis infection. Thirteen samples were positive for Blastocystis via microscopy ( Figure 1). Moreover, eight other protozoans and three helminths were identified in participants' stool samples, and the majority of infections were identified in CF group. The most common protozoa found were Entamoeba, Cryptosporidium, and Retortamonas intestinalis (Table S4), and the most common helminths infection were Enterobius vermicularis and Ascaris lumbricoides (Table  S4). Blastocystis co-infection was studied; however, there was no significant association with other intestinal parasites.
The odds of Blastocystis infection were almost threefold higher in cancer group than in the CF group (OR=2.98, P=0.022) and more than fivefold higher in the CRC group (OR= 5.66, P=0.009). In contrast, the Blastocystis infection odds in COGT to CF group were insignificant (Table 1). Blastocystis fold change between CRC and COGT groups was insignificant (OR=2.35, P=0.209), even though the prevalence of Blastocystis spp. in CRC group alone was high (60%) ( Table 2).

Molecular investigation: Gut fungi
Via gel electrophoresis, amplicon size of~450-800bp using ITS primers was considered positive for gut fungi. Sixty individuals (57.7%) were tested positive for gut fungi, of which 30 were from CF group (57.7%), and 30 were from the cancer group (57.7%). Twenty-two individuals (59.5%) of the COGT subgroup had gut fungi, and 8 (53.3%) of the CRC subgroup.
Thirty-nine fungi-positive samples were sequenced. Eleven types of fungi were identified; eight were gut mycobiome, and three were environmental fungal species (Table 3). Saccharomyces cerevisiae was the most common gut fungi (n=25, 62.5%). S. cerevisiae was detected in 17 samples (77.3%) from CF group and 8 samples (44.4%) from the cancer group. However, S. cerevisiae prevalence was not significantly associated with cancer (P=0.193) nor Blastocystis spp.(P=0.478).
Fifteen Blastocystis-subtyped samples were assigned an accession number via GenBank. Allele sequence analyses showed allele 4 in all ST1-positive samples, allele 15 in three ST2-positive samples, and allele 12 in one ST2-positive sample (Table S6). In ST3-positive Blastocystis cyst (black arrow) in stool sample stained with Trichrome stain at 100x magnification objectives using light microscopy. OM478527-OM478529, OM976632, OM976635-OM976638, and ON185813-ON185815) (Figure 2). The topologies between the original reconstructed trees with ML ( Figure S1) and BI ( Figure S2) were broadly consistent. Figure S3 shows the branch lengths of the combined tree.

Discussion
Cancer is one of the most common causes of death worldwide (15,42). The majority of new cases in the UAE are among women compared to men. (42). Colorectal, skin, and prostate cancers are the most prevalent in men, whereas breast, thyroid, and colorectal cancers are the most common in women. (42). In our study, Blastocystis spp. was significantly higher in cancer patients (OR=2.98). Similarly, a regional study from the Kingdom of Saudi Arabia (KSA) reported a significant association between Blastocystis infection and patients with cancer (OR=2.15) (43).
In this study, patients' medical records were assessed to identify potential risk factors associated with Blastocystis in cancer patients. Blastocystis prevalence association with risk factors tested was found insignificant (Table S3), as reported in other studies (44, 52).
Except for two patients (breast and hematologic/blood malignancies), all cancer patients in our study had received cancer therapy. Twenty-four cancer patients received chemotherapy, of which 18 (75%) were COGT, and 6 (25%) were CRC. The potential association of the number of completed chemotherapy cycles with Blastocystis prevalence was insignificant (P=0.705). In contrast, other studies reported that patients receiving at least eight chemotherapy cycles had a significantly higher Blastocystis prevalence than patients receiving fewer cycles (47, 53). Generally, Blastocystis infections were detected consistently in patients receiving ≥8 chemotherapy cycles compared to earlier cycles, and compared to patients who did not start treatment (47, 53, 54). Interestingly, current study findings and previous studies suggest that CRC patients had a significantly higher Blastocystis prevalence than healthy participants, regardless of their treatment status (43,45,46). Also, COGT patients receiving multiple chemotherapy cycles presented a significantly higher Blastocystis infection than healthy participants (43,50). These findings may help in understanding the complex relationship between Blastocystis and cancer and the effects of chemotherapy on Blastocystis prevalence.
Reportedly, the gut fungi Aspergillus flavus, Debaryomyces hansenii, Mucor mucedo, Mucor racemosus, and Issatchenkia terricola were significantly higher in the presence of Blastocystis (30). Therefore, we investigated the relationship between Blastocystis and gut fungi in cancer patients. Fungal sequences were identified via nBLAST and confirmed as gut fungi via the human gut mycobiome database published previously (28,36,55,56). However, none of the aforementioned fungi were detected in our fungi-positive samples. We have detected S. cerevisiae in most gut fungi-positive samples 25 (62.5%). Various studies saw S. cerevisiae to inhibit CRC progression and metastasis and stimulate apoptosis of cancer cells (57,58). S. cerevisiae was more prevalent in CF group (77.3%) compared to cancer patients (44.4%) with an insignificant difference (P=0.193). The latter observation was consistent with previous studies, except the association was statistically significant (59,60).
The predominant Blastocystis subtype in this study was ST3 (n=5) in CF group and ST2 (n=5) in cancer patients. In accordance with studies from Egypt and the UAE, where ST3 was the most common subtype in control participants (44, 61). On the other hand, studies in KSA and France showed ST2 and ST4 are the most common subtypes in a healthy population (43,62). Moreover, ST2 was the most common sub-type in cancer group (n=5) and CRC patients(n=3), unlike other studies where ST3 and ST1 were the most predominant sub-type in cancer patients (43,44,46,47,51,53).
In our study, the predominant Blastocystis subtypes are ST2 and ST3 in COGT group, with the same percentage (40%). Similarly, in Mohamed et al. work, Blastocystis ST2 was predominantly seen in COGT patients (43.7%) (43). Also, we found ST7 in one breast cancer patient while Ali  Dendrogram representing combined (ML+BI) phylogenetic tree inferred using the Barcode region of SSU rRNA gene sequences. This tree includes 15 isolated samples, 19 reference sequences from the GenBank (isolates from Cancer-free participants and CRC patients), 5 sequences of different alleles from PubMLST (AB107968.1, KT438693.1, AB070987.1, AB091234.1, and AB107965.1), and an outgroup (Proteromonas lacertae). The best tree of ML analysis was with Tamura 3-parameter model, while Bayesian tree best model was Hasegawa-Kishino-Yano model. Bootstrapping Proportions of more than 50% are shown on the left side of the branch. Bayesian posterior probability was also performed (ngen=5 million, samplefreq=100 and Burn-in 25%) and values of more than 50% are shown on the right side of the branch. A solid triangle indicates an isolate from a CRC patient, a solid circle indicates an isolate from a COGT patient and a solid square indicates an isolate from a cancer-free patient.
We identified Blastocystis ST7 allele 137, a similar finding was reported in Rezaei et al. study (65). A study from Turkey reported alleles 2, 4, and 88 of ST1, and alleles 34 and 36 of ST3 in cancer patients (66).
In conclusion, Blastocystis infection was significantly associated with cancer, with ST2 being the most common subtype. Furthermore, CRC patients had a higher risk of Blastocystis infection than CF. Since Blastocystis infection is more common among cancer patients than CF individuals, further studies are needed to understand the association between Blastocystis infection and cancer in general, and CRC in particular. Thus, routine Blastocystis infection screening in cancer patients might be a useful tool to be added to the usual patient's care in the future.

Ethics statement
The studies involving human participants were reviewed and approved by Tawam Human Research Ethics Committee (T-HREC) of Tawam Hospital, Al Ain, Abu Dhabi, UAE (THREC-678). The patients/participants provided their written informed consent to participate in this study.

Author contributions
LL: Methodology, data curation, analysis, interpretation, visualization, writing, and editing. SZ: Conception and design, methodology, data analysis, interpretation, revision, and writing. SA: Methodology, interpretation, writing, revision, and editing. MO: Methodology, analysis, and revision. SS: Study design and methodology. ZR: Conception and design, supervision, funding acquisition, methodology, data analysis, interpretation, revision, and editing. All authors contributed to the article and approved the submitted version.

Funding
This work received a grant from the College of Medicine and Health Sciences, United Arab Emirates University (Fund number:12M081)