A 11-year-old boy with Blastocystis hominis infection, presents as immune thrombocytopenia

Immune thrombocytopenia (ITP) is an acquired autoimmune disease characterized by low platelet counts (< 100 × 10⁹/L) and an increased risk of bleeding [1]. First-line therapy for ITP is are corticosteroids and intravenous immunoglobulins [2]. However, sometimes first-line treatment failure is observed in the patients. ITP can occur in isolation (primary ITP) or in association with a predisposing condition (secondary ITP) [1]. Some predisposing conditions are closely related to infections (such as viruses, bacteria, and Mycoplasma pneumoniae) [3, 4]. Parasitic infections are often rarely mentioned and therefore easily overlooked. Our case firstly describes a boy with ITP associated with Blastocystis hominis (BH) infection.

The patient was an 11-year-old male who visited a local hospital in August 2021 after discovering skin bleeding spots and ecchymosis that had been present for 3 days. The boy had paroxysmal epigastric pain and constipation for two weeks before his presentation. He denied any recent history of respiratory infection or vaccination. The physical examination revealed hemorrhagic spots and ecchymosis on the skin of the calf. The superficial lymph nodes, liver, and spleen were not palpable or enlarged. Routine blood tests showed that the platelet count (PLT) was 13 × 10⁹/L; the white blood cell count, red blood cell count, and hemoglobin were normal (Table 1). ALT, AST, creatinine, urea, PT, APTT, and electrolytes were normal. B-scan ultrasound of the liver, gall bladder, pancreas, spleen, intestine, and appendix failed to identify any abnormalities. At the same time, the platelet IgG was tested, and the result was positive. The boy was considered to have ITP, and a series of treatments were instituted (Fig. 1). First, gamma globulin was administered at 1 g/kg. The next day, routine blood tests showed that the PLT count had increased to 23 × 10⁹/L. The gamma globulin infusion was administered again at the same dose. On the following day, the PLT count dropped to 17 × 10⁹/L. Considering this treatment failure, the local physician conducted screening for possible underlying infectious factors and immune diseases. On the following day, the PLT count was 19 × 10⁹/L, and a plain chest X-ray showed no abnormalities. Mycoplasma pneumoniae IgM, Chlamydia pneumoniae IgM, respiratory syncytial virus IgM, adenovirus IgM, and Coxsackie virus IgM antibodies were negative; HIV, HBV, HCV, HAV, and Helicobacter pylori antibodies were negative (Table 1). In addition, the boy's C13 urea breath test was negative and the urine test was normal. Autoantibody and thyroid function showed no abnormalities. In order to rule out acute leukemia and aplastic anemia, they tested the bone marrow examination. The bone marrow examination showed plate-producing megakaryocytopenia, which was consistent with thrombocytopenic bone marrow. According to these results, the patient was commenced on oral prednisolone at 1 mg/kg/d. Over the next twenty days, the PLT count was fluctuated around 21 × 10⁹/L. During the above treatment, the patient was also given an infusion of 10 mg/kg of sulfoethylamine (Because of a lot of skin bleeding spots) and 200 mg of cimetidine (Fig. 1). However, the boy continued reporting episodic abdominal pain and constipation, and the thrombocytopenia could not be relieved.

At this point, the boy presented to our hospital for treatment. We found that the PLT count was 17 × 10⁹/L. Considering the failure of first-line treatment and the patient's accompanying gastrointestinal symptoms, we conducted a stool pathogen examination. BH was found in the stool (Fig. 2). Subsequently, we performed a DNA pathogenic microbial macro analysis of the boy's stool and blood. The results showed that BH infection was found in the stool and negative in the blood. In addition, we found that in the boy's blood the level of cytokines (IL-6, IL-17, IFN-γ) was increased (Table 1); TNF-α, IL-4, IL-8, IL-10, IL-5 and IL-2 were not significantly altered. Then, metronidazole was administered against BH. The following day, the PLT count increased to 42 × 10⁹/L and to 74 × 10⁹/L and 105 × 10⁹/L over the next two days. After 1 week of continuous oral metronidazole, the PLT count increased to 417 × 10⁹/L. At the same time, the abdominal pain and constipation were relieved. Re-examination of stool showed negative. The metronidazole was ceased and the patient’s PLT count was maintained at 350–420 × 10⁹/L during follow-up over the following six months.

BH, an intestinal parasitic protozoan, exhibits primarily vacuolar, granular, and amoebic features [5]. Clinical presentations of BH infection include abdominal pain and diarrhea. A minority of patients experience symptoms such as anorexia, abdominal distension, constipation, and stomach pain [6]. BH was historically considered to be clinically inconsequential, requiring little attention. However, has emerged as an opportunistic pathogen with relevance to intestinal disease. For instance, a systematic review suggested that intestinal colonization by BH may exacerbate colorectal cancer by altering the host immune response and increasing oxidative damage [7]. Additionally, BH is implicated in the development of irritable bowel syndrome through its influence on serum IgA levels [8]. Moreover, BH infection has been linked with several extraintestinal diseases [9, 10]. In a case involving a boy with Henoch-Schönlein Purpura, who experienced recurrent abdominal pain and rash after steroid treatment, BH was detected in the stool; symptoms improved following BH eradication with a compound triazole [9]. Additionally, BH contributes to the occurrence of Hashimoto's thyroiditis by engaging in immune responses, and BH eradication has shown promise in ameliorating Hashimoto's thyroiditis [10, 11]. Notably, in our case, we detected BH in the patient's stool.

In our case, the boy's platelet count did not improve following first-line treatment. Intriguingly, only after metronidazole was administered to eradicate BH did we observe a recovery in his platelet count. These findings suggest an unidentified relationship between BH infection and the pathogenesis of ITP. Although the mechanism of BH infection in some immune diseases has been elucidated, this is the first instance that BH infection was identified in the stool of patients with ITP. Consequently, the precise manner in which BH contributes to the onset of ITP remains unclear.

The disruption of intestinal microflora is a critical mechanism in understanding ITP. A study involving 16 children with ITP showed an increased abundance of Bacteroides and actinomyces in their stool compared to healthy children. This shift in population potentially contributes to the development of ITP by regulating IgG levels [12]. Remarkably, a study comparing gut flora in BH positive and BH-negative individuals reported alterations in the abundance of Bacteroides and Firmicutes in the former, resembling patterns seen in dysbiotic flora [13]. In addition, another study demonstrated that the presence of BH was associated with increased gut bacterial diversity, with a negative correlation observed with bacteroides levels [14]. This leads us to speculate that BH may play a role in the onset of ITP by influencing the equilibrium of intestinal microflora and the abundance of specific intestinal bacteria. However, since our patient did not undergo a complete stool microbiota examination, further research is necessary to test this relationship.

ITP patients exhibit an imbalance of CD4 + T cell subsets and abnormal secretion of associated cytokines [15]. CD4 + T cells differentiate into TH1 cells (IFN-γ, IL-2, TNF-β) and TH2 cells (IL-4, IL-5, IL-6, IL-10, IL-13), while TNF-α is secreted by macrophages. Collectively they play an important role in the pathogenesis of ITP. Additionally, Th17 cells (IL-17A, IL-17F, IL-21, and IL-22) are known to play an inflammatory role in mediating autoimmune diseases.[15].

One study showed elevated levels of IL-6, IL-17, and IFN-γ in the serum of ITP patients, along with decreased levels of IL-4 and TGF-β [16]. In our patient, we observed an increase in IL-6, IL-17, and IFN-γ, while TNF-α, IL-4, IL-8, IL-10, IL-5 and IL-2 were not significantly altered. It has been shown that BH can modulate immune responses, leading to the activation of immune cells and an increase in various interleukins [17]. The diverse BH subtypes may exert varying effects on intestinal flora and cytokines, resulting in distinct outcomes. One study suggested that the BH3 subtype shows greater therapeutic potential than others, promoting an increase in IFN-γ levels and inducing inflammatory responses [18]. IL-17, a key cytokine involved in regulating autoimmune disease, is secreted by Th17 cells and CD8-positive T cells, both of which are increased in ITP [19]. In cases of steroid-refractory ITP, there is an elevated IL-17 expression [20]. Studies have demonstrated high IL-17 expression in the intestinal mucosa of BH-infected mice [21]. The BH7 subtype has been shown to trigger ERK and JNK pathways in vitro, thereby influencing the expression of pro-inflammatory cytokines in macrophages, including IL-1β, IL-6, and TNF-α [22]. Based on these findings, we speculate that the observed cytokine abnormalities may be a potential mechanism by which BH infection could contribute to the development of ITP. Unfortunately, we did not go further to conduct a genetic classification. In addition, because the father did not agree to draw the patient's blood again, no results were obtained on cytokine levels after ITP remission.

In short, the exact mechanism of thrombocytopenia due to BH infection in this patient is unclear. But this interesting case reminds us of the importance of screening for potential predisposing factors in all ITP patients especially in those who did not respond well to the standard first-line treatment. The optimal treatment strategy for secondary ITP should include the management of the underlying disease.