Clinical Presentation of Patient 2 (K.N.)
The brother of patient E.N., born in 2003, was diagnosed with XHIM at the first month of life due to a positive family history. The lack of expression of CD40L on lymphocytes and the same point mutation as in his brother allowed confirmation of the diagnosis (Table 1). Intravenous Ig substitution therapy and P carinii prophylaxis were started. Next, a search for an MUD for HSCT was commenced. Neither signs of liver or bile duct dysfunction, nor diarrhea or malnutrition, were observed before the time of transplantation (Tables 2 and 4). He was screened for Cryptosporidium infection at the age of 11 months with negative microscopic examination (Table 4). At the age of 13 months the boy received HSCT from an MUD with full donor chimerism and good hematological reconstitution. Four weeks after the procedure, acute grade III graft-versus-host disease (GVHD) with skin and intestine involvement occurred. It was complicated by intestine perforation and development of a generalized fatal infection. After the patient's death, we retrospectively obtained positive PCR results for Cryptosporidium species from stool samples collected before transplantation (Table 4).
Clinical Presentation of Patient 3 (H.C.)
The boy, born in 1995, presented with recurrent diarrhea which started at the age of 5 months. Since the first year of life, a constantly elevated eosinophil number was observed, followed 2 years later by chronic hepatitis of unknown etiology. Other clinical manifestations were severe chronic otitis, recurrent labial herpes infection, and skin abscesses. The diagnosis of primary CD4 lymphopenia was established on the basis of the immunological status (Table 1). Genetic analysis did not reveal any mutation in the SAP gene (Table 1), CD40L expression was normal, and the molecular background of his PID remains unknown. Intravenous Ig substitution therapy and antiviral prophylaxis were started. Stunted growth, followed by chronic diarrhea and malnutrition with intestinal mucosal atrophy of grade III, were indicators for partial parenteral nutrition since the age of 9 years. At that time the boy was qualified for HSCT, but his parents refused to give consent. At the age of 10 years, he presented with recurrent bile duct inflammation followed by sclerosing cholangitis symptoms confirmed by ERCP (Table 2). At this time diagnostic tests for Cryptosporidium infection were performed and the oocysts were found in bile and fecal samples (Table 4). Azithromycin therapy was applied, with no improvement in clinical and laboratory status of the patient. Severe gallbladder inflammation was the reason for surgery. Histopathological investigation of the gallbladder showed B cell lymphoma infiltration. The first course of chemotherapy was complicated by intestine perforation, and during the surgery, intervention further dissemination of malignancy was noted. The patient underwent palliative care and died 5 months later.
Clinical Presentation of Patient 4 (A.K.)
The boy, born in 1998, was the brother of patient D.K. and presented with relevant family history (there were boys' deaths in early childhood in the maternal pedigree). The first manifestation of PID was observed in the 12th month of life, when he started to experience recurrent bronchitis. There was no suspicion of PID until the third year of life, when the boy was screened as a result of XHIM diagnosis in his brother. A deletion in the CD40L gene confirmed the diagnosis (Table 1). Despite intravenous Ig substitution therapy, chronic bronchitis is still observed.
No Cryptosporidium infection and no signs of liver or bile duct damage have been observed during follow-up (Tables 2 and 4). There is a lack of a family donor, and HSCT from an MUD is being considered.
Clinical Presentation of Patient 5 (D.K.)
The boy, born in 1994, was doing well until his 12th month of life when he started to experience recurrent upper and lower respiratory tract infections. At the age of 6 years he was hospitalized as a result of sepsis and candidiasis, and then hypogammaglobulinemia was recognized. The XHIM diagnosis was established 6 months later, with typical immunological features, and was confirmed by the detection of deletion in the CD40L gene (Table 1). Clinical improvement with a low incidence of infection was observed after intravenous Ig substitution therapy was started. No Cryptosporidium infection and no signs of liver or bile duct damage have been observed (Tables 2 and 4). There is a lack of a family donor, and HSCT from an MUD is being considered.
Clinical Presentation of Patient 6 (R.M.)
The boy, born in 2000, had recurrent bronchitis and pneumonias since his ninth month of life. Hypogammaglobulinemia was recognized at 20 months of age; 4 months later, an XHIM diagnosis was established based on detection of deletion in the CD40L gene (Table 1). Liver abnormalities were noted for the first time at the age of 3 years with periodically elevated liver enzymes and γ-glutamyltransferase. Further investigation revealed enlargement of the gallbladder, dilation of the intra- and extrahepatic bile ducts as shown by ERCP, and mild cholangitis described on liver biopsy, but no typical sclerosing cholangitis features were found (Table 4). A high eosinophil number was observed. Repeated analysis of stool samples performed at the age of 5 years revealed Cryptosporidium infection. Despite intravenous Ig therapy, there are still recurrences of respiratory infections. There is no chronic diarrhea or malnutrition present (Table 4). Long-term azithromycin treatment did not eradicate Cryptosporidium infection. In this case there is no matched family or unrelated donor for HSCT. Nevertheless, because of the severe course of XHIM, a mismatched unrelated HSCT will be performed in the near future.
Cryptosporidium infection was recognized in 4 of 6 investigated patients (Tables 3 and 4). Three sets of samples from 3 boys were positive by both methods: microscopic examination of fecal specimens and nested PCR amplification of a COWP gene fragment; however, the microscopic methods were less sensitive than the PCR assay. In patient 1, all samples (n = 11) were positive by nested PCR, whereas only 6 were positive by microscopy (Table 4). In patient 2, infection was detected only by PCR, with negative microscopic examination in 3 sets of samples.
Among these 4 infected cases, three different parasite species were identified by COWP PCR/RFLP assay (Fig. 1; Tables 3 and 4). The prolonged infection in patient 1 was caused by C meleagridis, whereas C hominis was identified in stool and bile samples from patient 3, and C parvum was associated with infections in patients 2 and 6.
The expected RFLP patterns from these species were as follows: for C meleagridis fragments of 372, 147, and 34 bp; for C hominis fragments of 284, 129, 106, and 34 bp; and for C parvum fragments of 413, 106, and 34 bp (Fig. 1).
In selected immunocompromised individuals, Cryptosporidium infection cannot be eliminated by the host (7,18). Experimental studies in severe combined immunocompromised or nude mice have shown that resolution of Cryptosporidium infection requires the activation of B and/or T lymphocytes (19). The 2 crucial factors of immune response necessary for prevention and/or resolution of cryptosporidiosis, are CD4+ lymphocytes and interferon-γ (19–22). It seems that a defective activation route for macrophages, especially the CD40–CD40L route of activation, may be the cause for the increased susceptibility to opportunistic infections (23). The lack of CD40–CD40L interaction caused by a mutation in the CD40L or in the CD40 protein in individuals with XHIM or HIGM3 syndromes is probably the main reason for their increased susceptibility to cryptosporidiosis. XHIM is a rare inherited immunodeficiency disorder characterized by recurrent sinopulmonary, bacterial, and opportunistic infections, and associated with low IgG, IgA, and IgE serum levels and normal to increased IgM serum levels (24).
In our group of patients with XHIM, no history of acute, prolonged diarrhea or malnutrition of a suspected or proven Cryptosporidium origin was recorded before the study. Chronic Cryptosporidium infection probably played an important role in chronic diarrhea, mucosal atrophy, and malabsorption requiring parenteral nutrition in a patient with CD4 lymphopenia (patient 3). In the further course of infection, the bile ducts were involved, with fatal complications. In this patient infection was caused by C. hominis, a strictly anthroponotic parasite.
Five different Cryptosporidium species have been described in immunocompromised patients all over the world (9,15,25,26). The zoonotic species C parvum (identified in 23%–88% of cases), and the human-specific C hominis (in 12%–57% of cases) are the most commonly identified ones. The other 3 species, C felis, C canis, and C meleagridis, are less common (0.2%–11% cases), but they are frequently found in their specific animal hosts, namely cat, dog, and turkey, respectively (27–29). For the first time in Polish patients with PIDs, Cryptosporidium species, including C meleagridis, were identified. Interestingly, patients 1 and 2, who are brothers, were infected with different species of Cryptosporidium (C meleagridis and C parvum; Table 3), suggesting different sources of infection.
In a recent analysis of 126 patients with XHIM syndrome reported to the European Society for Immunodeficiency registry, approximately one sixth developed liver disease, and in more than 50% cases this was associated with Cryptosporidium infection (30). In 2 boys (patients 1 and 6), the infection was correlated with the clinical course of sclerosing cholangitis and bile duct inflammation as the only manifestation.
In patient 2, infection with C parvum, which was confirmed only retrospectively, probably influenced the fatal complication of intestinal problems in the course of GVHD after hematopoietic stem cell transplantation. Fatal exacerbation of Cryptosporidium infection early after transplantation was reported in the literature (31).
Chronic infection or inflammation of bile ducts caused by Cryptosporidium species probably plays an important role in the development of malignancy. The tumors in most cases are preceded by chronic cholangiopathy and/or liver cirrhosis (32). Other mechanisms that are not well recognized are probably involved in cancer pathogenesis. It is likely that Cryptosporidium infection, which lasted many years in patient 3, was the main cause of intestinal and liver abnormalities, and was also a trigger for the development of malignancy. Several antibiotics (azithromycin, paromomycin, nitazoxanide) have shown some efficacy against the parasite. Unfortunately, clinical trials on the efficacy of treatment are discouraging: in a percentage of patients there is an improvement in the clinical condition, but it is difficult to eradicate infection. Better treatment results are achieved in HIV-infected patients presenting with intestinal involvement, but there is no relevant improvement in patients with sclerosing cholangitis (33). Symptoms may also resolve spontaneously within the restoration of immune status (eg, with antiviral therapy in HIV-infected patients). It seems that the best way to treat cryptosporidiosis in patients with PID is to correct the underlying PID syndrome. The only curative therapy for XHIM seems to be correction of the immune defect by hematopoietic stem cell transplantation (34). In long-term observation there is an extremely high risk of liver disease, leading to liver insufficiency and malignancy development requiring additional liver transplantation procedure (5,35). The 2 last factors are the main causes of deaths and are important indicators for HSCT in this group of PIDs.
It was observed in some patients that correction of immune defects yields a chance to eliminate infection and also resolve liver and bile duct inflammation (36). Immunosuppressive conditioning therapy in such children before HSCT may result in acute cryptosporidiosis and cholangiopathy associated with oocyst excretion, and can occur in patients without previous episodes of diarrhea (6,25). In the absence of a simultaneous treatment against Cryptosporidium species, this may lead to a massive amplification of the parasite, which results in rapid progress in liver disease and even death (25), as mentioned earlier. Early diagnosis of Cryptosporidium species infection, particularly during the asymptomatic phase and possibly with the use of molecular techniques, is extremely important for patients with PID who may undergo transplantation and receive proper treatment to avoid serious complications. In our group of patients, after diagnosis of cryptosporidiosis, 2 started azithromycin therapy (patients 3 and 6) and patient 1 was given combined treatment with azithromycin and paromomycin. During several months of observation, neither resolution of bile duct abnormalities nor elimination of the parasite was observed.
Conversely, children with symptoms of bile duct inflammation or hepatitis of unknown origin should be monitored for PID disorders. If possible, every case of Cryptosporidium-associated prolonged diarrhea in children should be screened for XHIM (in boys) or HIGM3 (in both sexes) (36).
In summary, this study underlines the need for a regular screening of selected patients with PID for infection by Cryptosporidium species. Unrecognized cases of cryptosporidiosis in children with PIDs may lead to serious consequences with development of sclerosing cholangitis, liver cirrhosis, and cholangiocarcinoma (32). Cryptosporidium species that are rarely found in humans, such as C meleagridis, can play an important role in the pathogenesis of cholangitis and diarrhea in PID. In agreement with a previous study (25), our data confirmed that microscopy is less sensitive than nested PCR for the detection of the parasite in asymptomatic cases (Tables 3 and 4). Therefore, molecular diagnostic tools should be considered the method of choice in screening of PIDs. Stool samples from such patients should be checked regularly, at least 3 times per year, even in the absence of diarrhea or other symptoms. Finally, because the eradication of cryptosporidial infection is difficult to obtain, even under long-lasting therapeutic regimes, new efficacious drugs are urgently needed.
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Keywords:© 2007 Lippincott Williams & Wilkins, Inc.
Cryptosporidium species; Hyperimmunoglobulin M syndrome; Primary immunodeficiencies; Sclerosing cholangitis