Assessment of immunological and hematological parameters in Blastocystis species-infected chronic leukemic patients

Blastocystis species (spp.) are anaerobic unicellular intestinal parasites with a prevalence of up to 10% in developed countries, rising to 50% in developing countries [1, 2]. This prevalence may increase remarkably to 80% in immunocompromised individuals, including those with malignancies, diabetes, and renal diseases [3]. They are highly polymorphic organisms with unclear boundaries between forms; the vacuolar form is the most common [4, 5].

Blastocystis spp. reside in the intestinal lumen of the ileum and caecum, adhering to the outer layer of the mucus membrane, with mucosal invasion rarely reported. It triggers both cellular and humoral immune responses, thereby increasing the inflammatory response of the intestinal mucosa [6]. Persistent and chronic inflammatory responses can be detrimental to the host, contributing to tissue damage through the release of various pro-inflammatory and anti-inflammatory cytokines, which may play a role in mutagenesis, carcinogenesis, and the development of inflammatory bowel disease [7, 8].

The pathogenicity of this protozoan remains controversial and inconclusive [9]. The infection may be asymptomatic or present with symptoms such as flatulence, diarrhea, vomiting, and abdominal pain [10]. Notably, symptomatic Blastocystis infections are reported more frequently in immunocompromised individuals. Immunosuppression can increase the susceptibility to infection and worsen the outcome [11, 12].

Blastocystis detection is commonly performed through microscopic examination of stool samples, either directly or following prior culture. Immunologic diagnostic methods based on coproantigen detection have been developed, offering a reliable alternative to microscopy and allowing for high-throughput screening with the potential for automation. Following infection, antibodies specific to Blastocystis antigens have been identified in both fecal and serum samples [13, 14].

Chronic lymphocytic leukemia (CLL) is the most prevalent form of leukemia in adults in developed countries, with an age-adjusted incidence of 4 to 5 cases per 100,000 of the population [15]. Infections represent a leading cause of both morbidity and mortality in leukemic patients and are often considered a leading cause of death in these patients [16]. Newly diagnosed patients with CLL often exhibit immune defects, including hypogammaglobulinemia and functional impairments in T-cells, suppressor natural killer cells, dendritic cells, neutrophils, and the complement system [17, 18]. Hypogammaglobulinemia is the main immune defect, linked to an increased risk of severe infection, with a fivefold higher risk when serum IgG levels fall below 600 mg/dL [19]. However, a clear-cut correlation between specific immunoglobulin levels and the risk of infection is not well established, and patients with normal immunoglobulin serum levels may have recurrent infections [20]. Notable alterations in the levels of interleukin-8 (IL-8), a pro-inflammatory cytokine involved in neutrophil recruitment and inflammation, and in CD4 T cell counts represent key immune defects that contribute to CLL progression [21, 22]. These abnormalities are known to increase susceptibility to a range of infections, including parasitic ones [23].

Blastocystis spp. are ubiquitous parasites with a worldwide distribution. The prevalence of Blastocystis spp. infection varies from country to country and among different communities within the same country [1, 26]. However, the question of whether it is a commensal or pathogenic parasite under certain conditions remains unresolved [6]. Many studies about the impact of Blastocystis spp. in immunocompromised individuals are available [10, 27]. Microscopic examination revealed that 30% of patients included in the present study had Blastocystis spp. infection. A similar finding was reported in Iran by Salehi Kahyesh, Alghasi [28]. Labania, Zoughbor [29], through a case-control study, reported that cancer patients have a higher risk of Blastocystis infection compared to cancer-free individuals. Sulżyc-Bielicka, Kołodziejczyk [30] reported that the prevalence of Blastocystis spp. was five times higher in colorectal cancer patients (12.5%) than in the control group (2.42%), with a significant difference. On the other hand, Essa et al. reported contradictory results and attributed their findings to the reduced exposure of leukemic children to pathogens due to lower physical activity and increased attention to food and hygiene [31].

In our study, the coproantigen detection assay identified more positive cases compared to the routinely used microscopic methods. However, a few cases were missed by the antigen assay, and there was fair agreement between these two techniques. The sensitivity of microscopic methods may be low if parasites are present in low numbers. Formalin ethyl acetate was reported to destroy some organisms during processing, potentially reducing diagnostic efficiency [32]. Additionally, irregular parasite shedding may result in a false-negative diagnosis if only one stool sample is examined. Previous studies indicated a low sensitivity of the wet mount method, ranging from 18 to 35%. Fecal antigen detection assays have shown higher sensitivities of 82–88% with no cross-reactivity with other intestinal pathogens [33, 34]. Nevertheless, false negative results may occur if antigen levels drop below the assay’s detection limit [2].

Overall, Blastocystis spp. was found to be highly prevalent among CLL patients, which could be attributed to the profound immune dysfunction associated with the disease [35]. A growing body of evidence indicates that Blastocystis colonization involves a complex interplay with the intestinal epithelium and the underlying immune system [6, 36].

Exposure to the parasite antigens elicits an antibody response [2]. IgA is the most abundant mucosal antibody that has a fundamental function in conserving homeostasis with the microbiome by binding and neutralizing invading pathogens near the mucus layer [37]. IgA secretion in the intestinal lumen is caused by parasitic infections to limit the parasite burden and enhance immune protection [38]. In the present study, only three out of 60 patients with confirmed Blastocystis infection tested positive for fecal IgA. Low secretory IgA despite active infection in the study population can be attributed to several factors. Decreased immunoglobulin production occurs in CLL, and it is often related to disease stage and duration (Ravandi and O’Brien 2006). This deficiency is likely due to direct interactions between malignant B cells and other immune cells, as well as the release of cytokines with inhibitory effects on immunoglobulin synthesis [39]. Additionally, Blastocystis spp. are known to secrete serine proteases capable of degrading secretory IgA [40], potentially contributing to lower fecal IgA levels. A previous study reported that individuals colonized with Blastocystis exhibited lower levels of fecal IgA compared to those who were not colonized [41]. Degradation of secretory IgA may lead to gut dysbiosis, which in turn increases susceptibility to intestinal protozoa, including Blastocystis spp [42, 43]. The antibody response is also influenced by the duration of exposure and antigenic variation among different parasite subtypes [2]. Notably, the presence of a positive IgG antibody response in a substantial number of patients included in the present study, despite negative stool tests, suggests resolved previous exposure.

IL-8 plays a key role in inflammatory processes by attracting polymorphonuclear leukocytes to sites of inflammation and activating monocytes [44, 45]. The present study revealed a significant elevation of IL-8 serum levels in Blastocystis spp.-infected patients. Supporting this, a previous study reported that culturing of in vitro cell lines in the presence of Blastocystis promotes the production of IL-8 along with granulocyte-macrophage colony-stimulating factor [46]. Another study demonstrated that cysteine proteases secreted by the central vacuoles of Blastocystis stimulate NF-κB-mediated IL-8 gene expression in epithelial cells of the colon [47]. Elevated IL-8 levels in CLL patients indicate that Blastocystis spp. can trigger a proinflammatory response, contributing to GIT irritation and related symptoms. These elevated levels are consistent with findings in other pathogenic protozoan infections such as amoebiasis and cryptosporidiosis, supporting the potential pathogenic role for Blastocystis spp [48‐50].

Although a low CD4 count is known to predispose individuals to many enteric infections, no statistically significant association with Blastocystis infection was observed in the present study. Similarly, Yulfi et al. (2021) reported no significant relationship between Blastocystis spp. infection and CD4 cell count in HIV patients presenting with diarrhea [42]. In another study, the average CD4 count in Blastocystis-infected HIV patients was 453 cells/µL, which is slightly below the lower limit of the reference range for healthy individuals [51].

The pathogenic potential of Blastocystis is related to several factors, such as the host’s immune status, the interaction of Blastocystis with the intestinal microbiota, and the infecting subtype. Studies carried out in different population groups suggest that Blastocystis can colonize the human intestinal tract and persist for prolonged periods without causing disease [11, 52]. In the present study, there was no statistically significant association between the presence of GIT symptoms and Blastocystis infection. Among symptomatic patients, symptoms were not related to the studied immunologic parameters. Many authors have suggested that symptomatic individuals show no correlation with Blastocystis spp. positivity [53, 54]. In contrast, some studies have confirmed a significant link between Blastocystis spp. infection and the occurrence of GIT symptoms [55, 56]. Mahmoud and Saleh reported that specific secretory IgA was not detected in asymptomatically infected individuals, whereas it was present in all cases of symptomatic Blastocystis spp. infection [57]. This discrepancy remains controversial, but it may be attributed to the wide variety of isolated subtypes and host defense factors such as age and immune status.

One of the limitations of this study is its cross-sectional design, which restricts the ability to establish a causal relationship between Blastocystis spp. infection and the observed immunological or hematological alterations. While associations were identified, it remains unclear whether Blastocystis infection contributes to these immune changes or whether the altered immune status in chronic leukemic patients predisposes them to infection. Longitudinal studies are needed to clarify the direction and nature of this relationship.

Conclusions: Our findings demonstrate that Blastocystis spp. infection is frequent among leukemic patients, but secretory IgA was detected in only a few infected cases. Serum IL-8 levels and WBC counts were significantly elevated in infected patients. Nevertheless, infection was not significantly associated with GIT symptoms or CD4 count. These results highlight the potential clinical relevance of infection by Blastocystis spp. in immunosuppressed patients and underscore the need for further research into its pathogenicity and diagnostic approaches.