Colorectal cancer and Blastocystis sp. infection

Blastocystis sp. is a common intestinal protozoan found worldwide. Based on gene analysis, 17 subtypes (STs, ST1–ST17) have been identified, 9 of which have been isolated from humans. Differences in clinical consequences may depend on differences among the STs. Here, we evaluated the prevalence of Blastocystis sp. in patients with colorectal cancer (CRC) compared to a control group and assessed the relationships between Blastocystis sp. infection and sex; age; and CRC grade, stage, and location. The study included 107 CRC patients (41 women and 66 men, median age 65 years); 124 subjects without colorectal cancer or a history of oncological disease comprised the control group (55 women and 69 men, median age 63). Stool samples were collected from patients before oncological treatment and examined using light microscopy (iodine-stained smear). Additionally, PCR-based identification of Blastocystis sp. was performed in 95 stool samples from CRC patients and 76 stool samples from the control group. Light microscopy showed that the prevalence of Blastocystis sp. was significantly higher in CRC patients than in the control group (12.15% and 2.42%, respectively; p = 0.0041). Multivariate analysis showed that the odds of Blastocystis sp. infection were fivefold higher in the CRC group than in the control group. PCR-based molecular examinations demonstrated that the proportion of patients infected with Blastocystis sp. was significantly higher in the CRC group than in the control group (12.63% and 2.63%, respectively; p = 0.023). The predominant ST in the CRC group was ST3, detected in nine patients (75%), followed by ST1 (2 patients, 16.7%) and ST2 (1 patient, 8.3%). No association was found between Blastocystis sp. infection and age, sex, or CRC stage, grade, or location. The results showed that CRC was associated with an increased risk of opportunistic Blastocystis sp. infection, even before oncological treatment. To the best of our knowledge, this is the first report estimating the prevalence of Blastocystis sp. infection in CRC patients before oncological treatment in Europe.


Background
The International Agency for Research on Cancer has estimated that 16% of cancer worldwide is caused by infectious factors, including parasites [1]. Colorectal cancer (CRC) is one of the most common neoplasms in humans. Most CRCs are sporadic, and the contributions of environmental risk factors have been widely investigated [2]. Microbes colonizing the gut are also considered potential cancer risk factors [3,4].
Blastocystis sp. is a common parasitic protozoan with a worldwide distribution that is found in the gastrointestinal (GI) tract of humans and a wide range of animal hosts [5,6]. Its prevalence in humans is estimated to be as high as 10% in developed countries and 50-60% in developing countries [7]. Blastocystis sp. is transmitted through the faecal-oral route as well as through contaminated water and food [8,9]. The pathogenicity of this protozoan is controversial, as it causes non-specific digestive tract symptoms, such as abdominal pain, nausea, vomiting, anorexia, acute or chronic diarrhoea, and weight loss. Blastocystis sp. infection is usually associated with alternating episodes of diarrhoea, normal defecation, and even constipation [10]. Poirier et al. suggested an association between Blastocystis sp. infection and irritable bowel syndrome (IBS) [11]. On the other hand, a higher rate of Blastocystis sp. infection in asymptomatic patients than in those with IBS symptoms was detected in Denmark [12].
The clinical significance of Blastocystis sp. infection remains uncertain, mainly because of its common occurrence in both dyspeptic patients and healthy individuals [9,13]. Some studies have maintained that Blastocystis sp. is part of a healthy gut microbiome [13,14]. However, it has also been reported that Blastocystis sp. infection can have features of opportunistic infection, as has been observed in patients with CRC treated with chemotherapy [15].
The identification of this organism at the species level is difficult. Blastocystis was originally named B. hominis, but subsequent phylogenetic studies limited the name to "Blastocystis species" because of the genetic diversity among members within the genus [16]. It was discovered that host specificity and the pathogenic potential of different isolates are correlated with sequence variations in the small subunit ribosomal RNA (SSU-rRNA) gene [17]. Based on these variations, members of the genus have been ordered into several subtypes (STs) [18]. Based on SSU-rRNA gene analysis, 17 STs (ST1-ST17) have been identified, 9 of which have been isolated from humans [19]. Differences in clinical consequences may depend on differences among STs [20]. Because Blastocystis sp. are found in both symptomatic and asymptomatic patients, the pathogenicity of this organism remains unclear [21][22][23]. Some studies have shown an association between Blastocystis sp. ST variation and pathogenicity. Dogruman-Al et al. [24] suggested that ST2 is a non-pathogenic genotype of Blastocystis sp.
The predominance of Blastocystis sp. ST3 among patients with chronic GI illness has been shown in Malaysia [25], Singapore [26], Egypt [27], Turkey [28], the United States [29], and Iran [30]. Khademvatan et al. [20] showed that in southern Iran, the most common ST of Blastocystis sp. was ST3, which correlated with the presence of GI symptoms in 44.83% of cases.
Studies evaluating the prevalence of Blastocystis sp. in the French population (inhomogeneous population of 788 patients from 11 hospitals) showed that the frequency of Blastocystis sp. infection in patients with symptoms of GI disorders was not significantly higher than that in patients without symptoms, and the most common ST was ST3 [31].
The aim of this study was to evaluate the prevalence of Blastocystis sp. in patients with CRC compared to that in a control group without colorectal cancer or a history of oncological disease and to assess the relationship of Blastocystis sp. infection with the sex and age of the subjects as well as CRC stage, grade, and location. To the best of our knowledge, this is the first report estimating the prevalence of Blastocystis sp. infection in CRC patients before oncological treatment in Europe.

Patients
This study included 107 consecutive patients with CRC treated between 2009 and 2014 in the Department of General and Oncological Surgery at Pomeranian Medical University (Szczecin, Poland). There were 41 women (38.3%) and 66 men (61.7%) in the study group, with a median age of 65 years. The control group comprised 124 individuals without CRC or other neoplasms in their medical history, including 55 women (44.4%) and 69 men (55.6%), with a median age of 63 years. Detailed patient characteristics are presented in Table 1. CRC was diagnosed based on colonoscopy examinations conducted in the Department of Gastroenterology at Pomeranian Medical University. Histopathological confirmation of cancer was obtained for all CRC patients. Patients with concomitant neoplasms or with a history of another cancer were excluded from the study. No patient included in the study had previously undergone chemotherapy. Stool samples were taken from CRC patients on the day of admission to the Department of General and Oncological Surgery (before surgery) and were delivered to the Department of Biology and Medical Parasitology at Pomeranian Medical University. The study did not require input from the Bioethics Committee, but the study was approved by the Bioethics Committee of Pomeranian Medical University (No. KB-0012/238/06/18). According to the Bioethics Committee instructions, verbal consent was obtained from patients because of the non-invasive nature of the study. Attestation statements of verbal consent of all participants in the study signed by the physicians were documented in medical records.

Light microscopy (LM)
Parasitological diagnosis was performed by coproscopy. For the detection of Blastocystis sp., the stool samples were examined using iodine-stained smears (×40 magnification).

Polymerase chain reaction (PCR) analysis
Among the patients with CRC (n = 107), PCR examinations of the stool samples for the presence of Blastocystis DNA were performed in 95 patients (37 women and 58 men) with a median age of 66 years. Among the control group (n = 124), PCR examinations were conducted in 76 patients (26 women and 56 men) with a median age of 64 years. The detailed characteristics of patients with CRC who were PCR-tested for the presence of Blastocystis sp. (n = 95) are presented in Table 1. DNA extraction from 200 mg of each stool sample was performed using the QIAamp DNA Stool Kit (Qiagen, Hilden, Germany). Before DNA extraction, three cycles of liquid nitrogen/ water bath (100 °C) incubation, each for 2 min, were performed to destroy the cyst walls of the protozoans present in the samples. Further DNA extraction was conducted according to the manufacturer's instructions. For molecular identification of Blastocystis sp., nested PCR was performed to amplify a 1.1-kb region of the SSU-rRNA gene using two pairs of primers: SR1F and SR1R for the first reaction [26,32] and the Forward B and Reverse B primer set for the second reaction (Böhm-Gloning et al. [33], modified by Wong et al. [26]). Each amplification reaction was conducted in a total volume of 10 μL with the following components: 1 μL of DNA, 1× reaction buffer, 2.5 mM of MgCl 2 , 3 pM of each primer, 0.75 nM of each nucleotide, and 0.5 U of polymerase (Allegro Taq Polymerase, Novazym, Poland). For the first reaction, amplifications were conducted as follows: 3 min of initial denaturation at 94 °C; followed by 35 cycles of denaturation at 94 °C for 45 s, annealing at 57 °C for 45 s, and extension at 72 °C for 1 min. The PCRs ended with a final extension at 72 °C for 5 min. For the second reaction, the following conditions were set: initial denaturation at 94 °C for 3 min; 35 cycles of denaturation at 94 °C for 30 s, annealing at 59 °C for 30 s, and extension at 72 °C for 45 s; followed by final extension at 72 °C for 4 min. All PCR amplifications were performed in two replicates in Mastercycler Pro thermal cyclers (Eppendorf, Hamburg, Germany). DNA isolates for positive controls were obtained from stool samples examined previously for the presence of Blastocystis sp. using LM. The PCR products were visualized in 1.5% agarose gels stained with ethidium bromide. All samples that were positive for the Blastocystis sp. SSU rRNA gene were sequenced (Macrogen, Seoul, Korea) with the Forward B and Reverse B primer sets [26,33]. The obtained sequences were initially aligned with homologous sequences published in GenBank using BLAST (www. ncbi. nlm. nih. gov) and then using MEGA 6.06 software with ClustalW [34]. Subtype confirmation was performed using the sequence query facility in the Blastocystis 18S database (https:// pubml st. org/ organ isms/ blast ocyst is-spp).

Results
Statistical analysis did not show differences in sex or age between the CRC and control groups ( Table 2).
Giardia lamblia was detected in one examined stool sample, and Entamoeba coli was detected in one stool sample. Both cases of parasitic infections were detected in patients with CRC without coexisting Blastocystis sp. infection.

Light microscopy Univariate analysis
The presence of Blastocystis sp. was detected in 13 patients with CRC (12.15%) and in 3 individuals in the control group (2.42%). Blastocystis sp. infection occurred significantly more often in patients with CRC than in individuals in the control group (p = 0.00409; Table 2).

Multivariate analysis
Multivariate logistic regression analysis including Blastocystis sp. infection, CRC diagnosis, age and sex of all participants (CRC and control groups, together n = 107 + 124) as independent variables showed that the odds of Blastocystis sp. infection were fivefold higher in the CRC patients than in the control group (odds ratio [OR] 5.41, 95% confidence interval [CI] 1.48-19.74, p = 0.010; Table 3). There was no association between Blastocystis sp. infection and age or sex (Table 3).

PCR analysis Univariate analysis
Blastocystis sp. infection was detected in 12 patients with CRC (12.63%) and in two individuals from the control group (2.63%). Blastocystis sp. detection by PCR occurred significantly more often in patients with CRC than in the control group (p = 0.023).

Multivariate analysis
Multivariate logistic regression analysis including Blastocystis sp. infection, CRC diagnosis, and age and sex of all participants (CRC and control groups) as independent   Table 3). There was no association between Blastocystis sp. infection and age or sex ( Table 3). The predominant ST among patients with CRC was ST3, which was detected in nine patients (75%). ST1 was detected in two patients, and ST2 in one patient. ST1 and ST3 were detected in one patient each in the control group (

Comparison of LM and PCR analysis results
Among the patients with CRC for whom both LM and PCR were performed for diagnosis of Blastocystis sp. infection (n = 95), Blastocystis sp. was detected by LM in 12 patients (12.6%), and 9 of those samples (9.4%) were confirmed by PCR examination (Table 5). In those samples, sequencing identified ST3 (Table 4). In the remaining three samples, Blastocystis sp. infection detected by LM was not confirmed by PCR. In 3 of 83 stool samples, the presence of Blastocystis sp. was not detected by LM but was detected by PCR (2 samples with ST1 and 1 sample with ST2) ( Table 4). In all nine samples positive for ST3 from patients with CRC, Blastocystis sp. was also detected using LM. There was a strong positive correlation between the LM and PCR results in the CRC group (phi coefficient = +0.71).
In the subgroup of controls in which both LM and PCR analysis were performed (n = 76), the presence of Blastocystis sp. was detected by LM in one sample (1.3%), but this was not confirmed by PCR. In two stool samples, Blastocystis sp. presence was detected by PCR (1 sample with ST1 and 1 sample with ST3), but it was not detected by LM in either case. Therefore, in the control group, there was no correlation between LM and PCR, as no sample was Blastocystis-positive by both methods (phi = −0.02). There were also no associations between Blastocystis sp. infection and age, gender, grade, Astler-Coller and tumour-node-metastasis (TNM) stages, location of tumour in the rectum compared to the colon, or left-or right-side location of the tumour according to splenic flexion in CRC patients.

Discussion
Microbiota alterations, referred to as dysbiosis, are often associated with CRC. Both human studies and studies conducted in animals showed that the gut microbiota related to CRC was distinct from that in subjects without CRC [35][36][37]. In addition, two meta-analyses of faecal metagenome changes specific to CRC were published in 2019 [38,39]. The human gut microbiota comprises bacteria, viruses, and eukaryotes (e.g., protozoa, helminths, and fungi). In human CRC samples, cytomegalovirus (CMV), John Cunningham (JC) virus, and human papillomavirus (HPV) have been identified, although the data are conflicting [37,40]. Additionally, changes in the mycobiome associated with human CRC have been reported [41].
The mechanism of the impact of dysbiosis on CRC carcinogenesis encompasses inflammation, immune regulation, metabolism of dietary components, and genotoxin production [42]. The gut microbiota interacting with the host immune system can affect the inflammatory process in the GI tract [43]. The microbiota produce numerous metabolites significant for human physiology [44]; on the other hand, these metabolites can impact the risk of developing CRC.   Another carcinogenic mechanism of the microbiota is the production of DNA-damaging toxins [40,45,46]. In a driver-passenger model of CRC, the mucosa of the colon is colonized by pathogenic driver bacteria, which produce genotoxins that induce inflammation and, consequently, the adenoma-carcinoma sequence [47]. On the other hand, opportunistic passenger bacteria might proliferate in CRC tumours and stimulate the infiltration of immune cells [40,47]. Disruptions in the gut microbiota and changes in its relative abundance can alter the balance, leading to many diseases, including inflammatory bowel disease (IBD) and Clostridium difficile infection [48,49]. Impairment of the symbiotic relationship between the microbiota and the host leads to immune dysregulation and can induce chronic inflammation, resulting in IBD [48]. The microbiota composition varies between certain subtypes of IBD (Crohn's disease, colitis ulcerosa) and the presence of an active phase [50][51][52]. The role of gut microbiota alterations in IBD has been widely examined [53,54]. However, in our study, we focused on patients with CRC, and none of our patients suffered from IBD.
To the best of our knowledge, this is the first report to estimate the prevalence of Blastocystis sp. infection in CRC patients before oncological treatment in Europe. Few studies have examined Blastocystis sp. infection in CRC patients (those that have were from Uzbekistan, Saudi Arabia, Turkey, and Malaysia) [64][65][66][67]. Most studies indicate opportunistic characteristics of Blastocystis sp. infection, but there are also reports indicating that Blastocystis sp. is a component of the healthy gut microbiome [14].
Our results were obtained from a homogeneous group of patients with CRC before oncological treatment, and individuals in the control group were matched by age to the CRC patients. The results showed that the odds of Blastocystis sp. infection patients with CRC were 5 times higher than those in the control group; these results were obtained not only by LM but also by PCR, and the proportion of individuals infected with Blastocystis sp. was significantly higher in the CRC group (12.63%) than in the control group (2.63%). Chandramathi et al. [15] demonstrated the opportunistic characteristics of Blastocystis sp. infection among patients with CRC (n = 15). Our results suggest that CRC is related to an increased risk of opportunistic infection with Blastocystis sp. even before oncological treatment, which may have additional effects on the immunological system.
At the time of planning of our study, we could not predict what the results would be, because Blastocystis sp. can be considered a marker of a healthy gut microbiota [57]; however, it is not possible to exclude opportunistic Blastocystis sp. infection in patients with CRC.
A significantly higher prevalence of Blastocystis sp. (80%) was found using LM in 200 CRC patients compared to the control population in Tashkent, Uzbekistan [64]. The prevalence of Blastocystis sp. in CRC patients was fourfold higher than that in the control population. However, the authors did not conduct PCR analysis, which could have enabled the determination of the ST of Blastocystis sp. [64]. Yersal et al. [66] detected Blastocystis sp. in 6.5% of 232 stool samples from cancer patients suffering from different types of cancer (lung, breast, CRC) by LM, but in the CRC patients, Blastocystis sp. was detected using PCR in 7.5% of the 66 examined cases [66], among which ST1 was the predominant ST (3 cases, 60%), followed by ST3 (2 cases, 40%). In our study, ST3 was the predominant ST in CRC patients (9 patients, 75%), followed by ST1 (2 patients, 16.7%) and ST2 (1 patient, 8.3%).
On the other hand, Beghini et al. [14] showed a lower frequency of Blastocystis sp. infection among patients with CRC (5.7%) compared to the healthy control group in their analysis of 12 studies of patient populations from different continents (n = 1689) with different diseases of the GI tract, including CRC. It is worth noting that only 53 of those patients suffered from CRC [3,14].
Differences in the results of studies on the prevalence of various Blastocystis sp. STs may be related to the genetic diversity of Blastocystis sp., non-homogeneity of the analysed patient groups, and ethnic diversity among the examined populations inhabiting different parts of the world [56].
A possible role for Blastocystis sp. in CRC pathogenesis has been suggested [15,64,68,69]. Postulated potential carcinogenic effects of Blastocystis sp. infection in humans, especially CRC patients, was examined by Chan et al. [70], and the ability of Blastocystis sp. to induce the growth of CRC cell lines by inhibiting the apoptotic effects of CRC cells has been documented [70]. Furthermore, isolated antigens of Blastocystis sp. isolates were shown to promote the proliferation of cancer cells via downregulation of host immune cellular responses [22,68,70]. Chan et al. [70] showed that antigens isolated from symptomatic human hosts caused a more extensive inflammatory reaction and a higher proliferation rate of CRC cells than isolates from asymptomatic human hosts. Chandramathi et al. [68] also showed that solubilized antigen of Blastocystis sp. facilitates growth in the human HCT116 CRC cell line. The antigen isolated from ST3 had the most prominent effect on the proliferation of CRC cells [71], which confirmed the case of severe ST3 Blastocystis infection in a 35-year-old man at the time of CRC diagnosis, as described by Padukone et al. [72].
The possible impact of Blastocystis sp. infections on CRC carcinogenesis remains unclear. The pathogenicity of Blastocystis sp. is suspected to be caused by the release of cysteine proteases by this protozoan. These proteases stimulate mucosal cells to release interleukin 8, which has been associated with gut inflammation [56].
Chronic inflammation is an established risk factor for CRC [73]. Among the molecular mechanisms of CRC pathogenesis, oxidative stress plays an important role and has been shown to be associated with Blastocystis sp. infection [69].
In our study, Blastocystis sp. infection was confirmed by both LM and PCR in all nine samples positive for ST3 from patients with CRC, but no such confirmation was found for the ST1 or ST2 genotypes. In three CRC patients, Blastocystis sp. infection detected by LM was not confirmed by PCR, possibly due to the presence of other organisms in the stool samples and non-specific amplification, which was revealed by sequencing of the obtained PCR products. A possible explanation for this discrepancy could also be a misdiagnosis of the parasite by light microscopy.
However, there was a strong positive correlation between the LM and PCR results in the CRC group (phi coefficient = +0.71). The presented results show that LM is an inexpensive, sensitive, and accessible method in daily practice. On the other hand, the PCR method allows for the identification of STs that may have different pathogenic potentials.

Conclusions
We demonstrated that the prevalence of Blastocystis sp. was higher in CRC patients than in the control group, independent of the diagnostic method used. In addition, ST3 was the predominant Blastocystis sp. ST among Polish CRC patients. Furthermore, Blastocystis sp. infection occurred five times more often in the CRC group than in the control group, independent of age, sex, and diagnostic method. No association was found between Blastocystis sp. infection and age, sex, staging, grading, or CRC location. Together, these results show that CRC is associated with an increased risk of opportunistic Blastocystis sp. infection even before oncological treatment. The potential relationship of Blastocystis sp. with CRC carcinogenesis needs further study (Additional file 1).