Complete Recovery from Cryptosporidium Parvum Infection

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Case Report Complete recovery from Cryptosporidium parvum infection with gastroenteritis and sclerosing cholangitis after successful bone marrow transplantation in two brothers with X-linked hyper-IgM syndrome

S Dimicoli1, D Bensoussan1, V Latger-Cannard2, J Straczek3, L Antunes4, L Mainard5, A Dao6, F Barbe3, C Araujo7, L Cle´ ment7, P Feugier7, T Lecompte2, JF Stoltz1 and P Bordigoni7

1Unite´ de The´rapie Cellulaire et Tissus, CHU de Nancy, Alleé du Morvan, 54511 Vandoeuvre les Nancy, France; 2Laboratoire d’He´matologie Biologique, CHU de Nancy, Alleé du Morvan, 54511 Vandoeuvre les Nancy, France; 3Laboratoire de Biochimie, CHU de Nancy, Alleé du Morvan, 54511 Vandoeuvre les Nancy, France; 4Laboratoire d’Anatomo-Pathologie, CHU de Nancy, Alleédu Morvan, 54511 Vandoeuvre les Nancy, France; 5Service de Radiologie Pe´diatrique, CHU de Nancy, Alleé du Morvan, 54511 Vandoeuvre les Nancy, France; 6Laboratoire de Parasitologie et Mycologie CHU de Nancy, Alleé du Morvan, 54511 Vandoeuvre les Nancy, France; 7Service de Me´decine infantile 2, CHU de Nancy, Alleé du Morvan, 54511 Vandoeuvre les Nancy, France

Summary: parvum, Histoplasmosa capsulata and Leishmania. Dysimmunity and malignancy are classical complica- We describe two brothers who suffered from hyper-IgM tions.1,2 syndrome (HIGM1) with similar clinical features: recur- CD40L, a membrane glycoprotein, is transiently ex- rent infections, especially cryptosporidium gastroenteritis pressed on activated CD4 þ and CD8 þ T cells, mast cells, with cholangitis. Their activated T cells did not express basophils, eosinophils and platelets. CD40, the CD40L CD40L. Nucleotide sequencing revealed a mutation in receptor, is a member of the TNF receptor family found on both boys with respect to intron 4 and exon 5 boundaries B cells, monocytes, dendritic cells, epithelial cells, fibro- of the CD40L gene in Xq26. They underwent successful blasts and some malignant cells. CD40 expression can also bone marrow transplantation (BMT) from HLA-geno- be induced on different cells by gamma interferon (IFNg).3,4 identical siblings. The Cryptosporidium infection and Thus, a potential mechanism contributing to C. parvum cholangitis resolved thereafter. At 6 months after BMT, immunity could be a direct triggering of apoptosis in expression of CD40L on activated T lymphocytes was CD40 þ infected cells by CD40L expressed on activated normal. After 1 year, both boys are well, and immune T cells. More recently, Hayward et al 4 have shown in a reconstitution has improved. Based on these two successful mouse model that CD40 expression on intestinal epithelial experiences, BMT with a genoidentical sibling seems a cells is not required for C. parvum clearance. Moreover, C. reasonable therapeutic approach for HIGM1, if Crypto- parvum infection can be cleared only when CD40 is present sporidium infection occurs. on dendritic and mononuclear phagocyte cells. Interleukin Bone Marrow Transplantation (2003) 32, 733–737. 12 production by these antigen-presenting cells leads doi:10.1038/sj.bmt.1704211 activated T cells (via CD40-CD40L pathway) to produce Keywords: X-linked immunodeficiency with hyper-IgM; TH1 cytokines such as IFNg. CD40 Ligand; cryptosporidum parvum gastroenteritis; C. parvum, a coccidian parasite, infects the intestine sclerosing cholangitis and sometimes the respiratory tract. In immunocompro- mised patients, it may cause severe intestinal fluid loss, with dehydration, chronic diarrhea and malnutrition. Hyper-IgM Syndrome (HIGM1) is a primary immuno- Several antimicrobial treatments have been tried but deficiency associated with mutations in the gene encoding none has proven effective. As previously reported by 5 the CD40L, located at Xq26. Patients suffer recurrent Hayward et al, patients with HIGM1 are at increased sinopulmonary and gastrointestinal infections, opportunis- risk of cholangiopathy, especially with C. parvum tic infections due to Pneumocystis carinii, Cryptosporidium infection. Curative therapy of HIGM1 relies on bone marrow transplantation (BMT), from an HLA-matched sibling, as first reported by Thomas et al,6,7 or from matched unrelated donors.8 However, the presence of C. parvum Correspondence: Dr D Bensoussan, Unite de Therapie´ Cellulaire et infection, especially when complicated by sclerosing cho- tissus, CHU de Nancy, Alle´ e du Morvan, 54511 Vandoeuvre-Les-Nancy. langitis, impairs the results of BMT. Khawaja et al 9 E-mail: [email protected] The first two authors contributed equally to this work reported three cases where patients suffering from severe Received 6 November 2002; accepted 8 April 2003 C. parvum infection died soon after BMT.9 BMT for HIGM1 and Cryptosporidium infection S Dimicoli et al 734 In this report, we describe two brothers with HIGM1 who were both suffering from severe C. parvum infection with gastroenteritis and sclerosing cholangitis. They underwent successful BMT from a genoidentical HLA sibling and recovered from the C. parvum infection.

Materials and methods

Case report Two brothers, had a family history of immune deficiency: two cousins suffered from HIGM1, one of whom died of diffuse C. parvum infection despite an HLA-sibling allogeneic bone marrow transplant together with an orthotopic liver transplant. Thanks to family screening, the two brothers were diagnosed with HIGM1, confirmed by the absence of CD40L expression on activated T lymphocytes. Sequencing of PCR-amplified CD40L genomic DNA revealed the Figure 1 Sclerosing cholangitis due to Cryptosporidium infection. A large bile duct is surrounded by a cuff of oedematous, inflamed fibrous tissue same mutation for the two brothers: a 10-nucleotide with an onion-skin appearance (arrow). Unaffected interlobular portal deletion (gTGTTACAGT) and a dinucleotide insertion tract (arrow head). Needle biopsy, H&E. (aa) at the intron 4/exon5boundary. This mutation predicts a premature termination of translation and generation of a truncated protein. The brothers presented similar clinical features. Treatment was initiated with intravenous (i.v.) serum aspartate and alanine amino transferase levels were immunoglobulin and P. carinii prophylaxis. five times above normal. Neutropenia and eosinophilia At 6 years of age, brother A had Cryptosporidium were also present. gastroenteritis with sclerosing cholangitis resulting in After considering the poor prognosis of this disease, persistent diarrhea and severe weight loss requiring especially when it is complicated by Cryptosporidium continuous enteral nutrition. The sclerosing cholangitis infection, and given the family history, the parents gave worsened and at MR-cholangiography, dilated extra and informed consent for their two sons to undergo BMT. The intrahepatic bile ducts were observed. Needle liver biopsy donors were two different HLA-identical sisters. revealed sclerosing cholangitis with prominent peri-portal The boys were conditioned with busulfan (4 mg/kg body concentric fibrosis, eosinophilic infiltration but no biliary weight per day for 4 days) followed by cyclophosphamide cirrhosis (Figure 1). Liver function tests were abnormal (50 mg/kg body weight per day for 4 days). Graft-versus- (Table 1). Alkaline phosphatase was elevated and serum host disease (GVHD) prophylaxis consisted of short-term aspartate and alanine amino transferase were six times methotrexate (15mg/m 2 on day 1 and 10 mg/m2 on days 3, above normal. There was also chronic, but not severe, 6, 11 after BMT), and cyclosporine A (3 mg/kg/day as a neutropenia and eosinophilia. continuous i.v. infusion on days À1to þ 30 and 6.25mg/kg Brother B had recurrent common sinus infections. At the twice daily from days 31 to 180 post-BMT). Infection age of 8 years, he also suffered from a Cryptosporidium prophylaxis included isolation in a laminar air-flow unit, infection with cholangiopathy, requiring enteral nutrition. oral administration of nonabsorbable antibiotics and The results of MR-cholangiography were similar to those treatment with i.v. immunoglobulins for 1 year (200 mg/ of A (Figure 2). Needle liver biopsy revealed cholangitis kg weekly for 3 months and then 400 mg/kg per month). with mild, nonconcentric, fibrotic enlargement of the portal The boys continued with enteral nutrition and anti- tracts and eosinophilic infiltration. Liver function tests were Cryptosporidium treatment (paromomycin 500 mg/day and also abnormal. Alkaline phosphatase was elevated and azithromycin 500 mg/day).

Table 1 Liver function tests (patient A)

Before C. Before BMT Day of BMT Day +15 post BMT Day +30 post BMT Day +60 post BMT Normal values parvum infection

ALP 423 962 397 893 586 279 70–260 UI/l Total bilirubin 2 2 2 18 8 2 2–11 mg/l Direct bilirubin 1 1 1 9 2 1 o5 mg/l ALT 31 17546 12 8 12 5–40 UI/l AST 16 99 72 31 31 26 5–35 UI/l 50-nucleotidase 3 40 7 44 26 6 0–9 UI/l LDH 356 595 534 739 801 695 210–400 UI/l

ALP: alkaline phosphatase, ALT: alanine amino transferase, AST: aspartate amino transferase.

Bone Marrow Transplantation BMT for HIGM1 and Cryptosporidium infection S Dimicoli et al 735 Results

Clinical course after BMT The clinical course for A was uneventful. At day 17, his absolute neutrophil count was over 500 per cubic millimeter and platelet counts were self-sustaining at levels above 50 000/ml at day 26. Neither GVHD nor veno-occlusive disease was observed. He had suffered from sinusitis (no organism identified) successfully treated by i.v. antibiother- apy. The Cryptosporidium infection disappeared soon after engraftment and was undetectable after day 60, despite immunological defects due to the allograft procedure. At 4 months after BMT, MR-cholangiography was significantly improved. After 19 months, stabilization of the cholangitis and decreased dilation of the extra and intrahepatic bile ducts was confirmed (Figure 2). Liver function tests normalized by day 60 post-BMT (Table 1). On day 200 post BMT, the quantitative and qualitative expression of CD40L on T cells was normal. DNA analysis revealed that 100% of lymphocytes and polynuclear cells were of donor origin. Immune functions have improved regularly. Today, the patient is well, requires no parenteral nutrition, has had no further infection, no GVHD, and is going to school. At day 10, B had sinusitis that resolved with i.v. antibiotics. At day 13, he had grade 2 veno-occlusive disease that resolved with appropriate treatment. At day 49, he developed acute GVHD of the gut, successfully treated by corticotherapy. On day 15, an absolute neutrophil count higher than 500 per cubic millimeter was observed, and platelet counts were above 50 000/ml at day 20. Cryptosporidium was detectable until day 16 after BMT and then, as with his brother, the infection resolved. Figure 2 (a) MR-Cholangiogram from patient B at 7 years of age, before Results of MR-cholangiogram analysis and liver function Cryptosporidium infection; intra- and extrahepatic bile ducts (’) are tests were similar to those of A. On day 200 post BMT, normal according to age. (b) At 8 years of age during Cryptosporidium infection. MR-Cholangiogram shows dilated extra- and intrahepatic bile quantitative and qualitative expression of CD40L on T cells ducts with strictures of intrahepatic bile ducts. (c) At 19 months after BMT, was normal. DNA analysis revealed that 100% lympho- MR-Cholangiogram shows stabilization of cholangitis and a decrease in cytes and polynuclear cells were of donor origin. Immune the dilatation. functions have improved regularly. Today, the patient is well, requires no parenteral nutrition, has had no infections, no recurrence of GVHD, On 27 July 2001, B received 1.6 Â 108 nucleated marrow and is going to school. cells/kg and 3.36 Â 106 CD34 þ cells/kg, and A received 0.78 Â 108 nucleated marrow cells/kg, and 5.8 Â 106 CD34 þ cells/kg on 3 August 2001. Immunological reconstitution At 1 month after cessation of i.v. immunoglobulin, normal Cryptosporidium detection production of IgM, IgG, IgG subclasses and specific antibodies to tetanus and diphteria toxoid occurred. The (1) Immunofluorescence detection same time course of lymphocyte reconstitution was Stools were prepared and processed using the observed in the two brothers. The T-cell count, normal monofluokit Cryptosporidium (Sanofi Pasteur, before BMT, improved gradually and was completely France), based on fluorescein isothiocyanate normal 19 months post BMT (1618 Â 106/l for A and (FITC)-conjugated monoclonal antibodies directed 1123 Â 106/l for B). The T-cell subset, evidence of thymic against the oocyst wall. Cryptosporidium sp oocysts function (CD4 þ CD45RA þ ) simultaneously reached (5-7 mm) were detected by fluorescence microscopy. normal levels (412 Â 106/l for A and 312 Â 106/l for B). (2) PCR detection The absolute B-cell count, low before BMT, normalized Stools were processed and DNA was extracted with within the first year after BMT. Moreover, memory B cells QI Amp stool MiniKit (Quiagen). The reaction (CD19 þ CD27 þ ), absent before BMT, were first observed was performed using primers SB012F and SBR012R 1 year post BMT. that amplified a 458 bp fragment specific for C. Before BMT, in vitro proliferative responses to T-cell parvum.10 mitogens were normal but no proliferative response to

Bone Marrow Transplantation BMT for HIGM1 and Cryptosporidium infection S Dimicoli et al 736 tetanus toxoid antigen could be observed. At 1 year post tion of the primary immune defect and secondary liver BMT, response to mitogen completely normalized. The complications. Martinez Ibanez et al 12 described three same normalization was observed after stimulation with HIGM1 patients with sclerosing cholangitis who under- tetanus toxoid antigen. went only liver transplantation and suffered relapse of sclerosing cholangitis 10, 18 and 25months later. 12 Owing to the T-cell deficiency, HIGM1 patients are predisposed to Discussion autoimmune disease triggered by opportunistic infections. Thus, in HIGM1 patients, a liver transplant alone cannot We describe two brothers with HIGM1. Both had a severe prevent sclerosing cholangitis relapse and must be accom- form of Cryptosporidium infection with sclerosing cholan- panied by a BMT to correct immune defect.12,13 gitis requiring continuous enteral nutrition. Both under- When Cryptosporidium infection occurs during the wait went successful BMT without serious complications. for a transplant, antimicrobial treatment can be initiated. Despite weak immune defences, the Cryptosporidium Many treatments have been studied in AIDS patients, infection did not worsen but disappeared soon after including Paromomycin, Clarithromycin, Azythromycin BMT. After 1 year, immune reconstitution has gradually and Sinefungine, but without any demonstrable benefit. improved and both patients are currently well with a good In our two patients, Paromomycin did not clear the quality of life. Cryptospridium infection. As previously mentioned, the The absence of serious, lesions in the liver or other CD40L-CD40 pathway is vital for C.parvum clearance.4 organs allowed us to perform a standard BMT with However, we have successfully used Nitazoxamide in a myeloablative conditioning. This was not the case for the child with Cryptosporidium infection secondary to cord cousin who also suffered from HIGM1, complicated by blood transplantation for acute myeloblastic leukemia frequent pulmonary infections which caused bronchiecta- (unpublished). This may be an interesting alternative sis. He also had a C. parvum infection with liver lesions antimicrobial treatment. Additional intravenous CD40L including sclerosing cholangitis. At the age of 12 years, he could be useful in temporary Cryptosporidium infection underwent orthotopic liver transplantation, followed by management, although Vestereng et al14 did not observe P. BMT. This was unsuccessful and he died of diffuse carinii clearance using recombinant CD40L in SCID mice. C. parvum infection with pulmonary and hepatic failure. Optimal HIGM1 management appears a challenge when This was probably due to the parasite, the advanced severe hepatic lesions occur, especially in the presence of sclerosing cholangitis, as well as to the pulmonary C. parvum.8,9,11,12,13 Myeloablative, or perhaps nonmyelo- insufficiency and patient’s age at BMT. Owing to this case, ablative stem cell transplantation, should be performed as the two brothers underwent BMT at a younger age, before soon as C. parvum infection occurs. In cases of advanced serious lesions had occurred and in the absence of any other hepatic damage, liver transplantation together with non- organ injury. myeloablative BMT should be considered. Nitazoxamide Khawaja et al 9 reported two HIGM1 patients with could decrease C. parvum infection before BMT, limiting severe liver disease and C. parvum infection, both of whom conditioning regimen toxicity, and could be maintained died soon after myeloablative BMT. One had chronic during the profound post BMT immunodeficiency. Regular Cryptosporidium infection with diarrhea, sclerosing liver function checks and early C. parvum detection would cholangitis, cirrhosis and pancreatitis, requiring total be helpful for HIGM1 patients. PCR analysis together with parenteral nutrition. After BMT, he died from fulminant immunofluorescence studies seem promising and sensitive liver failure associated with veno-occlusive disease, Cryp- tools for early parasite detection.10 PCR sensitivity could be tosporidium infestation, and GVHD. The other had a improved by using competitive, quantitative or nested similar clinical pattern, and also extensive bilateral PCR. Early diagnosis could allow clinicians to consider bronchiectasis. On the other hand, Amrolia et al 8 reported BMT–especially when a genoidentical sibling is available– nonmyeloablative stem cell transplants on eight patients before any severe hepatic lesions occur. with congenital immunodeficiency and severe organ dys- function. Two, who suffered from HIGM1 with sclerosing cholangitis, were successfully treated. One of these had a Acknowledgements Cryptosporidium infection before BMT that cleared after transplantation, suggesting significant recovery of func- We thank Mrs D Binot Saintot and S Lapuyade for their tional immunity. However, Khawaja et al 9 described an excellent technical assistance. HIGM1 patient with a severe Cryptosporidium infection who died soon after nonmyeloablative BMT. He had cryptosporidial ascending cholangitis with portal inflam- References mation, confirmed by liver histology. The Cryptosporidial enteritis worsened after BMT and this, together with a 1 Puck JM. A disease gene for autosomal hyper-IgM syndrome: Clin capillary leak syndrome, resulted in the patient’s death. more genes associated with more immunodeficiencies. Immunol 2000; 97: 191–192. When severe liver failure is associated with HIGM1, 2 Fuleihan RL. Hyper IgM syndrome: the other side of the coin. orthotopic liver transplantation, together with nonmyeloa- Curr Opin Pediatr 2001; 13: 528–532. 11 blative BMT, could be an option. Hadzic et al reported 3 Fries KM, Sempowski GD, Gaspari AA et al. CD40 the successful treatment of an 18-year-old HIGM1 patient expression by human fibroblasts. Clin Immunol Immunopathol with serious cirrhosis: they accomplished combined correc- 1995; 77: 42–51.

Bone Marrow Transplantation BMT for HIGM1 and Cryptosporidium infection S Dimicoli et al 737 4 Hayward AR, Levy J, Facchetti F et al. Cholangiopathy and 9 Khawaja K, Gennery AR, Flood TJ et al. Bone marrow tumors of the pancreas, liver, and biliary tree in boys with transplantation for CD40 ligand deficiency: a single centre X-linked immunodeficiency with hyper-IgM. J Immunol 1997; experience. Arch Dis Child 2001; 84: 508–511. 158: 977–983. 10 Wu Z, Nagano I, Matsuo A, Uga S et al . Specific PCR primers 5Hayward AR, Cosyns M, Jones M, Ponnuraj EM. Marrow- for Cryptosporidium parvum with extra high sensitivity. Mol derived CD40-positive cells are required for mice to clear Cell Probes 2000; 14: 33–39. Cryptosporidium parvum infection. Infect Immun 2000; 69: 11 Hadzic N, Pagliuca A, Rela M et al. Correction of the hyper- 1630–1634. IgM syndrome after liver and bone marrow transplantation. 6 Thomas C, De Saint Basile G, Le Deist F et al. Brief report: N Engl J Med 2000; 342: 320–324. correction of X-linked hyper-IgM syndrome by allogeneic 12 Martinez Ibanez V, Espanol T, Matamoros N et al. Relapse of bone marrow transplantation. N Engl J Med 1995; 333: sclerosing cholangitis after liver transplant in patients with 126–129. hyper-IgM syndrome. Transplant Proc 1997; 29: 432–433. 7 Bordigoni P, Auburtin B, Carret AS et al. Bone marrow 13 Levy J, Espanol-Boren T, Thomas C et al . Clinical spectrum transplantation as treatment for X-linked immunodeficiency of X-linked hyper-IgM syndrome. J Pediatr 1997; 131: 47–54. with hyper-IgM. Bone Marrow Transplant 1998; 22: 14 Vestereng VH, Kovacs JA. Recombinant CD40 ligand 1111–1114. administration does not decrease intensity of Pneumocystis 8 Amrolia P, Gaspar H, Hassan A et al. Nonmyeloablative stem carinii infection in SCID mice. J Eucaryot Microbiol 2001: cell transplantation for congenital immunodeficiencies. Blood (Suppl.): 153S–154S. 2000; 96: 1239–1246.

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