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Introduction
Cryptosporidiosis is a disease caused by Cryptosporidium spp., the intracellular obligate protozoan parasites with worldwide distribution[1, 2]. These parasites invade the gastrointestinal and respiratory epithelium of many vertebrates, including humans[3]. Cryptosporidiosis has public health implications globally but has shown higher prevalence in the developing world (2.98%–25.9%) than the developed countries (0.1%–9.1%)[1, 4].
Infection occurs when an oocyst, which is the infective stage of the parasite, is ingested. The excystation of Cryptosporidium oocysts in the intestine is triggered by the host’s gastrointestinal conditions, such as pH and temperature[5]. Sporozoites released from the oocyst are attached to the epithelial cells of the hosts. The invasion of the sporozoites results in recruitment of epithelial cells into the site of invasion and the formation of a parasitophorous vacuole[6]. A feeder organelle develops in sporozoites, and the morphology of the parasite gradually becomes spherical. Trophozoite is formed and later develops into a Type I meront which can in turn develop into Type II meront. In some instances, the Type I meront may produce 6–8 merozoites. These merozoites may either re-infect the host cells and replicate asexually or develop into Type II meront[7]. The sexual phase is initiated by Type II meronts, which release a set of four merozoites. The merozoites are differentiated into macrogamonts which produce macrogametes. The invading merozoites also form microgamonts which produce microgametes. Fertilisation occurs when a microgamete penetrates a macrogamete to form a zygote. The zygote divides meiotically to produce sporozoites, which in turn develop into oocysts. The oocysts are differentiated into two types; the thin and the thick-walled oocysts. The latter produce sporozoites, which reinvade the host cells, whilst the former are released into the environment through feces[7].
Cryptosporidiosis is significantly associated with diarrhoea and infections, and the causal parasites could be opportunistic and could cause life-threatening enteric disease, especially in immunocompromised individuals[8].
Several reports have linked cryptosporidiosis to malignancies[9, 10] with severe Cryptosporidium infection more frequently observed in cancer patients[11]. A study also showed a 2.8 times greater rate of predisposition to Cryptosporidium infection in children with malignancy compared with healthy patients[12]. Importantly, some studies have related cryptosporidiosis with colorectal adenocarcinoma[13, 14]. However, the correlation between Cryptosporidium infection and human gastrointestinal malignancies is currently unclear. So this review aims to evaluate the possible association between cryptosporidiosis and colorectal cancer using available epidemiological, pathological, and immunological evidence. This may chart a new research direction in this relatively neglected field.
Epidemiology of Cryptosporidium infection
Cryptosporidiosis is currently endemic in more than 70 countries in the world[15]. There are more than 30 species of Cryptosporidium; however, only two species, C. parvum and C. hominis, are known to frequently infect humans. Whilst the earlier species is associated with zoonotic transmission, the latter exclusively causes human infection[16, 17]. The prevalence of Cryptosporidium infection is significantly higher in developing countries compared with the developed world since many areas in the former still lack potable water and the level of sanitation is generally poor[8, 18]. Cryptosporidium infection rarely occurs in immunocompetent persons but the infection results in approximately 10%–15% of cases of severe diarrhoea in malnourished children < 5 years old[8, 18]. A very high prevalence of cryptosporidiosis (77%) was reported among slum-dwelling children in Bangladesh[19]. In Zambia, Cryptosporidium infection was diagnosed in 30.7% of all pre-schoolers in a peri-urban area[20]. Some other epidemiological studies spanning two decades (2000–2020) documented the prevalence of Cryptosporidium in the human population, as presented in Table 1.
Distribution of Cryptosporidium infection in different countries (2000 – 2020).
Pediatric units of Teaching Hospitals, Kandy and Peradeniya, Sirimawo Bandaranayake Childrens’ Hospital, Peradeniya and District General Hospital, Matale
In addition to children and those living in areas with poor sanitation facilities, other groups at greater risk of this infection are transplant recipients, AIDS patients, and a host of others whose immune systems have been compromised. The disease could also be nosocomial. Two or more of these factors often interact to determine the severity of the disease. White[21] identified Cryptosporidium spp. as a common aetiology and a major cause of chronic diarrhoea among immunocompromised patients, and about 60,400 deaths were accrued to the infection. Ninety-three percent (93%) of the 64,818 reported deaths in 2015 in sub-Saharan Africa were mainly among children under the age of five infected with Cryptosporidium[22]. Nevertheless, the 2010 diarrhoeal mortality rate in children under five reported in the Mediterranean African countries was meaningfully lower, varying from 1% to 12% in Liberia and Sudan. Poor surveillance, under diagnosis, or underreporting and poor bookkeeping could be responsible for the lower rate of infection detected in the Mediterranean African countries. Death rate in children below the age of five attributable to cryptosporidiosis in India is about 7.0% based on a multicenter study carried out by Ajjampur et al.[23].
Colorectal cancer epidemiology and risk factors
Cancer that affects either the colon or rectum is called colorectal cancer. Colorectal cancer is a major public health concern with cases increasing worldwide[38]. It is the third highest leading cause of cancer death globally[38]. In Africa, it used to be a rare disease but over the years with changing lifestyle and the adoption of westernized diet, the incidence has increased over time[39]. According to the data recorded in the last five years, annual age-standardised colorectal cancer incidence was 38.7 per 100,000 persons (2012–2016), and the mortality rate was 13.9 per 100,000 persons (2013–2017)[40]. Several risk factors can predispose individuals to colorectal cancer including age, gender, unhealthful diet, sedentary lifestyle, obesity, environmental factors, and other underlying diseases such as bowel inflammation[41,42,43,44]. According to Sulżyc-Bielicka et al.[45], the risk of developing colorectal cancer increases with age and has been observed to reach its peak in the eighth decade of life. Though there have been no data showing significant differences in the age range of patients suffering from cryptosporidiosis and colorectal cancer, studies have shown that the median age was between 57.7[13] and 64.7 years[45].
Other than the aforementioned factors, there are other genetic predispositions to colorectal cancer: MHL1, MSH2, MSH6, PMS2, EPCAM, APC, MUTYH, PTEN, POLE, POLD1, SMAD4, GREM1, BMPRIA, and STK11 are genes associated with hereditary colorectal cancer[46, 47]. Table 2 shows the prevalence or incidence of colorectal cancer in different regions of the world.
Prevalence or incidence of colorectal cancer in different parts of the world.
Is there a correlation of risk factors for cryptosporidiosis and colorectal cancer?
It appears the risk factors for cryptosporidiosis and colorectal cancer do not overlap. For example, while Cryptosporidium infection is likely to be higher among children, colorectal cancer is more common in the older population. Studies have also revealed that cryptosporidiosis is poverty-related, occurring in many low-middle income countries (Table 1). On the other hand, colorectal cancer is more endemic in the developed countries (Table 2). Each disease, hence, has different drivers, and Cryptosporidium appears to be one of the promoters of colorectal cancer.
Cryptosporidiosis as a risk factor for colorectal cancer
The contribution of Cryptosporidium to benign tumours has been established in vertebrates such as reptiles and birds[62,63,64]. The first report of the link between cryptosporidiosis and malignant tumours was established in mice[65]. Since then, several other mouse models have supported this association[66, 67]. A recently developed three-dimensional (3D) in vivo-like culture model from an adult murine colon also allowed for the maintenance of Cryptosporidium. The system allowed for in vitro development of low-grade intraepithelial neoplasia after 27 days post-infection[68]. Other evidence that suggested the possible association between Cryptosporidium and colorectal cancer was extensively discussed in a recent work by Başak et al.[6]. The up-regulation of certain oncomiRNAs and the down-regulation of tumour suppressor miRNAs in the host epithelial cells due to Cryptosporidium infection may lead to cancer initiation in humans[6]. Since Cryptosporidium spp. are localised in the colon-rectal region, the most likely cancer-type it may induce is the colorectal cancer.
Although there are limited studies showing a higher prevalence of Cryptosporidium spp. in colorectal cancer patients, growing evidence from epidemiological data has shown that Cryptosporidium spp. is highly associated with the development of colorectal cancer, thus, indicating that the protozoan parasite could be a potential risk factor for colorectal cancer. For example, a study by Sulżyc-Bielicka et al.[46] has shown that Cryptosporidium spp. infections occur more often in patients with colorectal cancer than those without a neoplastic disease. The study corroborated with report findings from a previous study published in 2012 on the ‘Prevalence of Cryptosporidium sp. in patients with colorectal cancer’ by Sulżyc-Bielicka et al.[45]. According to Sulżyc-Bielicka et al.[45], there’s a close relationship between cryptosporidiosis and many cases of human colorectal cancer. Similarly, Osman et al.[13] have shown that DNA and oocysts of C. parvum and C. hominis are more prevalent in digestive biopsies of patients diagnosed with colon neoplasia/adenocarcinoma compared with patients without digestive neoplasia. Berahmat et al.[10] also reported that the rate of cryptosporidiosis in children with cancer undergoing chemotherapy was higher (3.8%) than those in the control group (0%). Additionally, Shebl et al.[42] reported that cryptosporidiosis was associated with a rare squamous cell carcinoma of the colon in AIDS cases. The findings of Sulżyc-Bielicka et al.[46] have led to the conclusions that (1) Cryptosporidium spp. infections appear significantly more frequently in patients with colorectal cancer, before oncological treatment, than in controls, regardless of age and sex; (2) amongst patients with colorectal cancer, the Cryptosporidium spp. infection is not associated with the colorectal cancer stage, grade, or location, or with patient age; and (3) males were significantly likely to have an increased frequency of Cryptosporidium spp. infections, with the presence of colorectal cancer old or young. It has been observed by Shebl et al.[69] that the risk for developing colorectal cancer is higher in patients who have HIV infection and cryptosporidiosis than in patients who have HIV infections without cryptosporidiosis. In a study conducted by Sulżyc-Bielicka et al.[45], it was observed that Cryptosporidium sp. infection was more common in colorectal patients who had tumours on the left side of their colon compared with those who had tumours on the right side. Amongst the patients who had tumours on the left side, it was observed that Cryptosporidium sp. infection affected mostly those with tumours located in the sigmoid colon and descending colon. The prevalence of Cryptosporidium among colorectal cancer patients has been documented in different populations (Table 3).
Prevalence of Cryptosporidium spp. among colorectal human cancer patients.
Pathophysiology of colorectal cancer and a possible contribution from Cryptosporidium
Colorectal cancer is a very common malignancy with a high mortality rate, making it one of the leading causes of cancer-related death[73]. It often arises sporadically but a significant number of cases are correlated with a specific genetic background[73]. The development of colorectal cancer is a multi-stage event that involves the stages of initiation, promotion, and progression. The initiation stage involves the development of a stable alteration in the DNA sequence, which is subsequently followed by the uncontrolled expansion of mutated clones which characterises the growth of the tumour[74]. These events occur over a period of time involving the progression from benign neoplasm to colorectal cancer[75].
Can Cryptosporidium contribute to the pathogenesis of colorectal cancer?
The frequent occurrence of Cryptosporidium spp. infections in patients with colorectal cancer[45, 46] suggests its involvement in the pathogenesis of colorectal cancer. Cryptosporidium infection mostly affects those with tumours located in the sigmoid colon and descending colon[45]. Moreover, Cryptosporidium infection was observed mostly in patients with grade 2 colorectal cancer[45]. Cryptosporidium infection induces intestinal dysplasia, which is significantly correlated with parasite load, particularly in immunosuppressed mice[76]. Overexpression of Cyclin I D was observed in Cryptosporidium-induced intestinal dysplasia[76], which implied modulation of gene expression in infected patients. The ability of some protozoans to interfere with signaling pathways in host cells has been reported[77]. The mechanisms of Cryptosporidium infection in the pathogenesis of colorectal cancer, however, still remain largely unclear.
Immunomodulation in Cryptosporidium infection and risk of colorectal cancer
Immunomodulation in cryptosporidiosis
The process of immune response begins with the recognition of Cryptosporidium by toll-like receptors (TLRs) on epithelial cell surfaces. As reviewed by Ludington and colleagues, several toll-like receptors, including TLRs 2, 4, 5, and 9, have been shown to be involved, and they subsequently aid in parasite clearance[78]. This brings in other associated proteins that trigger the activation of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways[79], resulting in the release of inflammatory chemokines or cytokines as well as the antimicrobial peptides[6]. The inflammatory cytokines, such as IL-8 secreted by the intestinal epithelial cells (IECs) and phagocytes, are very crucial to the innate immune response to the parasite[80]. Likewise, the chemokines CXCL10, CCL2, and CX3CL1 are implicated in the recruitment of inflammatory cells and activation of adaptive immune cells in Cryptosporidium infection[80, 81]. The antimicrobial peptides, such as β-defensins, hinder the growth activity of the parasite and also aid in destruction of the sporozoites in vitro[82, 83].
The adaptive immune responses are triggered by the reactions of innate immunity. Cells involved in the adaptive immune response include CD4+ and CD8+ lymphocytes, natural killer (NK) cells, macrophages, dendritic cells, and innate lymphoid cells[6].
Despite the activation of both the adaptive and innate immune responses of the host, the parasite is able to evade them with its own defense mechanism. For example, after Cryptosporidium infection, there is a depletion of the transcription factor, signaling transductor and activator of transcription1α (STAT1α) in infected cells, thus, affecting the production of IFN-γ, which is important in the activation of both innate and adaptive immunity[6]. Likewise, in C. parvum infection of neonatal mice, there is a down-regulation of the chemokine CCL20, which is responsible for parasite clearance[84].
Alterations in the immune profile and development of cancer
Cryptosporidium spp. has largely been associated with diarrhoea and particularly the life-threatening form in immune-compromised individuals. The pathophysiological mechanisms of Cryptosporidium infection are multifactorial and not completely understood. One of the challenges is the difficulty in translating what obtains in mice models to the human immune system. The invasive process involved in obtaining tissue samples from the intestines of humans is a major limitation in carrying out such studies[80]. This is due to the variation in clinical symptoms between mice and humans.
The repeated inflammation generated due to the immune responses to Cryptosporidium can result in cell proliferation and a destabilised genetic process causing oncogenic mutations[85]. Quite a number of experimental and epidemiological studies have suggested a link between colorectal cancer and Cryptosporidium[13, 65, 71].
Immunological evidence associating this pathogen with colorectal cancer is based on the established activation of the NF-κB pathway during Cryptosporidium infection[86]. Increased activation of this pathway potently affects the initiation, development, and metastasis in human cancer. Thus, the down-regulation of the let-7 family and the modulation of IL-6/STAT3 signaling in response to these inflammatory signals may lead to an uncontrolled proliferation of cells that results in cancer initiation[87].
Other parasites as risk of colorectal cancer
A wide variety of protozoan and helminth parasites that colonise the gastrointestinal tract are known to cause severe pathology in the region, and colorectal cancer is occasionally identified as one of parasite-induced morbidities in endemic areas. Although Cryptosporidium spp. appears to be the most frequently encountered protozoa in CRC patients, evidence from Europe, Middle-East, and Central Asia[88,89,90] has implicated the opportunistic protozoan Blastocystis sp. as a risk factor for CRC. The amoeba Endolimax nana, which is well known not to be pathogenic, has also been reported to be associated with CRC[89]. These two opportunistic protozoans are known to coexist and cause bowel functional disorder and chronic diarrhoea in immunocompetent individuals[91, 92]. Although the impact of their concomitant infection on CRC is yet to be explored, the aggravated disease spectrum in the form of severe diarrhoetic condition during concurrent infection is suggestive of a higher risk of colon neoplasia/adenocarcinoma when the two protozoans coexist. Another protozoan linked to adenocarcinoma colon cancer is Entamoeba histolytica, but the body of evidence available to support this proposition suggests that the parasite is an unlikely cause of CRC[93].
It is not certain whether Blastocystis sp. is directly involved in the development of colon neoplasia and adenocarcinoma, but studies have shown that the organism alters the gut microbiota community[94, 95]. The disruption of gut microbiota could induce CRC carcinogenesis through mechanisms such as inflammation, immune regulation, metabolism of dietary components, and genotoxin production[96]. The interaction of gut microbiota with the host immune system can modulate inflammation in the gastrointestinal tract[97]. The microbiota produces metabolites that could promote the development of CRC[98]. Microbiota can also induce the production of DNA-damaging toxins[99]. The ability of Blastocystis sp. to disrupt the gut microbiota (or microbiome) to induce mechanisms that could potentiate the development of CRC may be similar for other earlier discussed protozoan organisms.
Several case-control studies, epidemiological studies, case series, and case reports have shown the helminth Schistosoma japonicum as a probable cause of CRC. Much of the pathogenesis of S. japonicum-associated CRC still remain unknown, but it shares some similarities to that seen arising in the setting of inflammatory bowel disease[96]. More importantly, a higher proportion of base-pair substitutions were found at CpG dinucleotides in S. japonicum-associated rectal cancers when compared with non-schistosomal rectal cancers[96]. S. mansoni is weakly associated with CRC and the malignant factor Bcl-2 appeared to be the most significantly expressed cancer-related biomarker in S. mansoni-associated CRC. Others such as p53 and c-myc were not significantly expressed when compared with non-schistosomal-associated rectal cancer (similar to what was observed in S. japonicum)[96, 100].
Conclusion
This review attempts to find a link between Cryptosporidium and colorectal cancer. Although the clinical data presented in this review suggest an association between the two, more evidence-based case-control studies will be needed to reach a conclusion. A direct causal link between virulent Cryptosporidium factors and the mechanisms of colorectal tumour development is necessary. Recent advances in genomic studies, 3D culture models, gene manipulation using CRISPR/Cas9 system, and clinical trials involving the application of sensitive diagnostic tools can be adopted to study the association between the two. The establishment of a link between Cryptosporidium and colorectal cancer will promote public health measures targeted against cryptosporidiosis which in turn could reduce the risk of colon cancer. In addition, this could stimulate interest in drug and vaccine discovery research, thus, reducing the number of children suffering from cryptosporidiosis-related enteric diseases and mortality. We recommend more research efforts in this field as cryptosporidiosis and colorectal cancer have become persistent public health issues globally.
