MYCOBACTERIUM FORTUITUM-CHELONAE COMPLEX INFECTION IN A CHILD WITH COMPLETE INTERLEUKIN-12 RECEPTOR BETA 1 DEFICIENCY

A 10-year-old boy had chronic diarrhea, abdominal pain, severe weight loss and hepatomegaly; multiple enlarged para-aortic and mesenteric lymph nodes. Mycobacterium fortuitum-chelonae complex was identified in the culture of the lymph nodes. Interleukin-12 receptor beta 1 expression could not be observed in phytohemagglutinin-driven T cell blasts. A homozygous missense interleukin-12 receptor beta 1 mutation was found (R173P). The major effector mechanism of cell-mediated immunity against intracellular pathogens, including mycobacteria, is thought to be the activation of infected macrophages by interferon-gamma (IFN-gamma). IFN-gamma is produced by natural killer cells and T helper 1 lymphocytes, and its production is up-regulated by interleukin-12 (IL-12) which is produced and released by macrophages and dendritic cells. 1–3 It has been reported that IL-12-dependent IFN-gamma secretion is essential and specific for protective immunity to mycobacterial infections. 4–6 In recent reports idiopathic disseminated infections caused by nontuberculous mycobacteria have been described in a few children without well-defined immunodeficiencies such as severe combined immunodeficiency and chronic granulomatous disease. 1, 2 The underlying immune disorder was genetically heterogeneous and related to cell-mediated immunity to intracellular pathogens. 1, 2 Altare et al. 4 diagnosed a homozygous deletion within the IL-12 p40 subunit gene in a child with disseminated bacille Calmette-Guérin (BCG) and Salmonella enteritidis infection, which was the first discovered human disease resulting from a cytokine gene defect. Besides mutations in the IL-12 p40 gene, mutations causing complete IFN-gamma receptor (R) 1 and R2 deficiencies and partial IFN-gamma-R1 deficiency were also found. 1 IL-12 binds to high affinity beta 1/beta 2 heterodimeric IL-12 receptor (IL-12R) complexes on natural killer cells and T helper 1 lymphocytes. 3, 7 Recently recessive mutations in IL-12 beta 1 chain genes have been identified in a number of patients with disseminated mycobacterial infections such as Mycobacterium avium and Mycobacterium bovis. 3–6, 8 Rapidly growing mycobacterial species have been cultured in patients with complete IFN-gamma-R1 and IFN-gamma-R2 deficiency. 3–6 A 10-year-old boy with severe Mycobacteriumfortuitum-chelonae complex infection lacked IL-12 beta 1 chain expression, and his cells were deficient in IL-12R signaling and IFN-gamma production. Because of his remarkable clinical course and history and excellent success as a result of treatment, this case is presented and discussed. Case report. A 10-year-old boy was referred to the Pediatric Immunology Unit, Ege University Faculty of Medicine, İzmir, Turkey, for chronic diarrhea, abdominal pain and weight loss of 6 kg in the previous 6 weeks. Before admission to our clinic he had been hospitalized for 2 weeks in another hospital with the diagnosis of giardiasis and iron deficiency anemia and had been given metronidazole and iron treatment without improvement. He had had chickenpox and mumps in early childhood. His immunization status was up to date, and he had been given BCG vaccines twice (at birth and at 7 years of age in the primary school). He had been healthy until 7 years of age at which time he was diagnosed with pulmonary tuberculosis and received isoniazid, rifampin and streptomycin. We were unable to obtain his medical records pertaining to that illness. The parents were first degree relatives (cousins), and they had three healthy daughters. Another female sibling died when she was 1 year old because of severe persistent diarrhea. The patient weighed 24 kg (3 to 10%), and his height was 137 cm (50 to 75%). He had two BCG scars on the left deltoid region. The physical examination was otherwise normal except for pallor and hepatomegaly 5 cm below the costal margin. Laboratory studies revealed normal leukocyte and platelet counts. The hemoglobin value was 8.3 g/dl. Erythrocyte sedimentation rate (56 mm/h) and C-reactive protein (5.1 mg/dl) concentrations were elevated. Total serum protein, albumin and globulin concentrations were slightly decreased, and the alanine aminotransferase (39 IU/l) and aspartate aminotransferase (73 IU/l) values were mildly elevated. Lactate dehydrogenase was 470 IU/l, prothrombin time was 1 min, activated partial thromboplastin time was 30 min and blood urea was 31 mg/dl. Anti-nuclear antibody, anti-gliadin and anti-endomysium antibodies were normal. No pathogens were detected in stool and blood cultures. Antibodies against HIV-1 and HIV-2 were negative. Antibody examinations against viral agents were as follows: cytomegalovirus, IgM−, IgG+; Epstein-Barr virus (viral capsid antigen), IgM−, IgG+; rubella, IgM−, IgG+; parvovirus B19, IgM−, IgG+. Salmonella and Brucella serologies were negative. The chest radiograph was normal, and the tuberculin skin test was negative at 72 h. Cultures for three consecutive early morning gastric aspirates for Mycobacterium tuberculosis were negative. A computed tomography of the abdomen demonstrated hepatomegaly, multiple enlarged para-aortic and mesenteric lymph nodes and mucosal thickening of the ileum, cecum and ascending colon. Excisional biopsy samples were obtained from lymph nodes and a needle biopsy sample from the liver at the time of explorative laparotomy. The biopsy samples from the lymph nodes revealed large, pale histiocyte layers with small, hyperchromatic nucleoli and foamy large cytoplasms. These cells were also observed in the portal regions of the liver. Acid-fast bacteria were demonstrated in the cytoplasm of the histiocytes (Ziehl-Neelson). The patient’s family history revealed no known cases of tuberculosis, and both parents and siblings had negative tuberculin tests although they were known to have been vaccinated with BCG at least once. Because the initial microbiologic findings and pathology results suggested an atypical mycobacterial infection, the patient was treated with rifampin (15 mg/kg/day), ethambutol (15 mg/kg/day), clarithromycin (25 mg/kg/day) and ciprofloxacin (40 mg/kg/day). The results of lymphocyte phenotyping (CD3+, 69%; CD4+, 16%; CD8+, 43%; CD19+, 12%; CD16+56+,14%; alpha beta T cell receptor (TCR), 66%; gamma delta TCR, 14%) and oxidative burst test by flow cytometry and serum immunoglobulins, IgG subgroups were normal, ruling out severe combined immunodeficiency and chronic granulomatous disease. Low CD4 and high gamma delta TCR values were thought to be in accordance with the natural progression of the mycobacterial infection. To determine the suspected underlying immunodeficiency, peripheral blood samples were analyzed for the flow cytometric expression of IL-12R and IFN-gamma-R in Hôpital Necker-Enfants Malades, Paris. Phytohemagglutinin-driven T cell blasts did not show expression for IL-12R beta 1, although IFN-gamma-R expression was normal. IL-12R beta 1 sequence analysis was performed with the previously described technique of Altare et al. 5 It revealed a genetic mutation that resulted in premature stop codons in the extracellular domain. The IL-12R beta homozygous mutation was found to be R173P, in which an arginine becomes a proline at amino acid position 173. Two weeks after the onset of antimycobacterial therapy, the diarrhea and abdominal pain disappeared, but liver function tests (transaminases) were highly elevated. Rifampin and ciprofloxacin were stopped, and streptomycin was administered. In addition to antibiotic therapy, recombinant IFN-gamma treatment (Imukin, 50 μg/m 2 subcutaneously three times a week; Boehringer Ingelheim) was started. However, continuous elevation of transaminases suggested that granulomatous hepatitis could not be treated with these drugs. In addition multiple cervical, axillary and inguinal lymphadenopathies appeared 2 months after admission. Cervical lymph node biopsy demonstrated granuloma formation, histiocytic infiltration and acid-fast bacilli within the macrophages. The spleen was also enlarged. The leukocyte (2000/mm 3) and platelet (68 000/mm 3) counts decreased. Hypersplenism associated with mycobacterial infection was diagnosed, and a total splenectomy was performed. The histopathologic examination of the spleen showed findings similar to those of the liver and lymph node examinations. The causative agent was cultured from specimens of cervical and mesenteric lymph nodes and identified as M. fortuitum-chelonae complex (a rapidly growing mycobacterium). To identify this microorganism Accu-Probes (Gen-Probe, USA) were used to exclude M. tuberculosis complex, M. avium, Mycobacterium intracellulare and Mycobacterium kansasii. Then by using biochemical tests and methods defined by Kent and Kubica, 9 the causative agent was identified in the Microbiology and Clinical Microbiology Department, Mycobacteriology Laboratory, Ege University. The agent was differentiated from Mycobacterium smegmatis because it grew on MacConkey agar at 28°C. After identification treatment was changed in accordance with results of susceptibility studies (amikacin, doxycycline and clofazimine). For susceptibility studies we used Mueller-Hinton agar supplemented with oleic acid-albumin-dextrose-catalase (10%) to match the MIC ranges on the respective Etest strips as it has been reported before. 10 In addition IFN-gamma treatment was continued. One month later the patient began to gain weight, and the hepatomegaly and enlarged lymph nodes disappeared. Liver function tests, C-reactive protein and erythrocyte sedimentation rate returned to normal. Antimicrobial and recombinant IFN-gamma therapy was given for 6 months at which time the patient was totally recovered and had gained 12 kg. Discussion. Seven cases of IL-12R beta 1 deficiency with mycobacterial infections have been reported through 1998. 1 Insufficient IFN-gamma production appears to be the main pathogenic mechanism in IL-12R beta 1-deficient patients. Complete IFN-gamma-R deficiencies can also lead to more severe and fatal infections with mycobacteria of low grade virulence, usually before 3 years of age. 11–13 In contrast to these disorders patients with IL-12R beta 1 deficiency often develop milder infections, which manifest at a later age and can usually be cured by extensive chemotherapy. 1 These patients resemble children with partial IFN-gamma-R deficiency, and they display a milder histopathologic phenotype, at least in the case of infection with BCG. 1 Unlike children with complete IFN-gamma-R deficiency, both children with partial IFN-gamma-R and complete IL-12R beta 1 deficiency retain some residual IFN-gamma-mediated immunity, which probably accounts for both the tuberculoid granulomas and milder course of infections. 2 In view of these results exogenous IFN-gamma treatment appears to be the best therapeutic option for IL-12R beta 1-deficient patients. Our patient also responded well. Splenectomy might have played an essential role in the success of therapy in our case and should be considered in complete IL-12R beta 1 deficiencies showing findings of hypersplenism. 14 The previous patients reported to have been infected with M. fortuitum-chelonae complex are known to have complete IFN-gamma-R1 and IFN-gamma-R2 and partial IFN-gamma-R2 deficiency. 15 To our knowledge our case is the first patient reported to have complete IL-12R beta 1 deficiency who was infected with a rapidly growing mycobacterium. Exogenous IFN-gamma therapy, besides effective antibiotics and splenectomy if indicated, is critical in the control of such mycobacterial infections.