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Case Report

Visceral Leishmaniasis and Other Severe Infections in an Adult Patient with p47-phox-Deficient Chronic Granulomatous Disease V. Asensi, L. Tricas, A. Meana, D. Roos, J.A. Carton, J.A. Maradona, M.F. Fresno, E. Valle, J. Fierer, J.M. Arribas Summary We report a rare case of a male patient without known immunodeficiency consecutively diagnosed with visceral leishmaniasis, brain abscess and cavitating pneumonia in the 3rd decade of life. Chronic granulomatous disease (CGD) was diagnosed by a nitroblue tetrazolium test. A p47-phox mutation of the NADPH oxidase of the leukocytes was suspected by immunoblotting and confirmed by DNA analysis. The patient was homozygous for this mutation while his mother and sister were heterozygous asymptomatic carriers. After the CGD diagnosis the patient started a chronic prophylactic regimen with subcutaneous interferon- (0.05 mg/m2 of body surface/three times a week), and oral trimethoprim-sulfamethoxazole and itraconazole (both at 5 mg/kg/day) with no subsequent infections after 12 months of follow-up.

Key Words Adult chronic granulomatous disease · p47-phox mutation · Leishmania · Brain abscess · Pneumonia

since childhood especially of the urinary tract, aphthous mouth ulcers and perianal abscesses. At 30 years of age he was diagnosed with visceral leishmaniasis or kala-azar after spending some weeks in the Valencian region, an area on the eastern Mediterranean coast of Spain that is endemic for Leishmania (Figure 1). The patient had fever, anemia and hepatosplenomegaly at the time. The diagnosis of visceral leishmaniasis was done after demonstrating Leishmania amastigotes in the bone marrow biopsy (Figure 2).The diagnosis was confirmed by serum detection of antibodies against Leishmania donovani (indirect immunofluorescent antibody assay titers of 1/640). No cultures for Leishmania were done. He was treated with iv pentavalent antimonials (meglumine antimonate 20 mg antimone/kg body weight/day for 4 weeks) and cured.At 33 years of age he underwent surgery for drainage of a large penial and scrotal abscess.At 34 years of age he was diagnosed with brain abscess, treated with iv ceftriaxone and metronidazole for 6 weeks and surgical drainage and was cured. Eight months before the present admission he was diagnosed with ecthyma gangraenosum of the right thigh. Recently he was admitted to our hospital for high fever and persistent dry cough. Thoracic radiographies and CT studies showed a pulmonary left lower lobe cavitating pneumonia. Intravenous ceftriaxone and erythromycin were administered for 12 days without clinical or radiographic improvement and then erythromycin was switched to iv ciprofloxacin.A bronchoscopy was per-

Infection 2000;28:171–174

Introduction Chronic granulomatous disease (CGD) is a rare and severe inherited immunodeficiency caused by defects in the NADPH oxidase of the leukocytes resulting in recurrent infections. Two-thirds of the patients are diagnosed in the 1st year of life, while in a small subset of patients the diagnosis of CGD is not made until the teenage or adult years due to the mildness of the symptoms [1, 2]. We report an unusual case of a patient diagnosed with CGD after severe consecutive infections in his adulthood and with no family history of CGD making the diagnosis of the immunodeficiency even more complicated.

Case Report A 37-year-old male was admitted to the hospital because of fever and cough. His family medical history was unremarkable and his parents were not related.The patient had had recurrent infections

Infection 28 · 2000 · No. 3 © URBAN & VOGEL

V. Asensi (corresponding author), J.A. Carton, J.A. Maradona, J.M. Arribas Infectious Diseases Unit, Hospital Central de Asturias, Oviedo University Medical School, c/ Celestino Villamil s/n, E-33006 Oviedo, Spain; e-mail: [email protected] L. Tricas Immunodeficiencies Unit, Hospital Central de Asturias, Oviedo University Medical School, Oviedo, Spain A. Meana Principado de Asturias Transfusion Center, Hospital Central de Asturias, Oviedo University Medical School, Oviedo, Spain D. Roos Netherlands Red Cross Transfusion Service, Amsterdam, The Netherlands M.F. Fresno Pathology Service, Hospital Central de Asturias, Oviedo University Medical School, Oviedo, Spain E. Valle Department of Biochemistry, Oviedo University Medical School, Oviedo, Spain J. Fierer Departments of Medicine and Pathology, Veterans Administration Medical Center, University of California, San Diego, USA Received: September 25, 1999 • Revision accepted: February 12, 2000

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V. Asensi et al.: Leishmaniasis and CGD

Figure 1 Visceral leishmaniasis in Spain. Shadowed areas indicate where this infection is endemic. The CGD patient spent a few weeks in the summer in rural Valencia (Leishmania-endemic eastern Mediterranean coast).

formed showing a normal bronchial tree. Blood and bronchoalveolar lavage (BAL), bacterial, fungal and viral cultures and blood serologies, including Legionella, Mycoplasma, Chlamydia and Q fever were negative. Gram and Ziehl-Neelsen stains of the BAL were negative. HIV-1 and -2 serum antibodies and HIV viral load were also negative. A PPD test was negative. No other skin tests were done. Blood levels of IgG, IgM, IgA, complement proteins and granulocytes were normal. Fluorescent-activated cell sorter (FACS) analysis of lymphocytic subpopulations in peripheral blood was normal: CD3+ 860/µl (80.2%), CD4+ 379/µl (35.4%), CD8+ 306/µl (28.6%), ratio CD4+/CD8+ 1.2, CD19+ B cells 106/µl (9.9%), CD 16+ NK cells 86/µl (8%). Lymphocytic response to the mitogens phytohemagglutinin, pokeweed, to phorbol myristate acetate+ionomycin and to anti-CD3 monoclonal antibody was normal. Mixed lymphocyte culture stimulated with alloantigens was also normal. Table 1 summarizes the different infections and pathogens isolated. A nitroblue tetrazolium test (NBT) done with the patient’s peripheral WBC was suggestive of CGD (Figure 3) and confirmed by flow cytometry using dihydrorhodamine-123 (DHR) as indicator [3, 4]. DNA from our case, his mother and sister was sent to the Laboratory of Experimental Immunohematology of the Netherlands Red Cross Blood Transfusion Service, Amsterdam. A GT mutation at the beginning of the exon 2 of the p47 gene (NCF1), suspected by previous immunoblot analysis [5], was confirmed by restriction enzyme analysis and sequencing of the patient’s DNA. He was then diagnosed with the p47-phox mutation, an autosomal recessive form of CGD [6]. After the diagnosis, subcutaneous recombinant human interferon- administration, 0.05 mg/m2 of body surface, three times a week, was begun along with 3 weeks of iv ceftriaxone and ciprofloxacin and then 5 additional weeks with oral amoxicillinclavulanic acid with complete resolution of the pneumonia. Trimethoprim-sulfamethoxazole (5 mg/kg/day), twice a day and itraconazole (5 mg/kg/day) once a day, both orally, were administered as prophylaxis against bacterial and fungal infections. No new infections were detected with this chronic prophylactic therapy after 12 months of follow-up.

Discussion Figure 2 Bone marrow biopsy smear of the CGD patient, then 30 years old, admitted for anemia and splenomegaly. Leishmanial amastigotes are seen within the macrophages (arrows). (Stain, hematoxylin and eosin; original magnification * 1,000 with immersion). He was diagnosed with visceral leishmaniasis or kala-azar.

Table 1 Summary of the different infections and pathogens isolated in the reported case.

Year

Infection

Pathogen

1991

Visceral leishmaniasis

Leishmania donovani

1994

Genital abscess

Escherichia coli

1995

Brain abscess

Unknown

1997

Ecthyma gangraenosum

Pseudomonas aeruginosa

1998

Cavitating pneumonia

Unknown

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CGD is a very infrequent disorder (incidence one in 250,000 persons) characterized clinically by severe recurrent bacterial and fungal infections due to a defect in the NADPH oxidase of the neutrophils, monocytes, eosinophils and certain fixed tissue macrophages. The normal array of microbicidal oxidants (superoxide, hydrogen peroxide, hypochloric acid) is not produced intracellularly and the phagocytic cells ingest but do not kill the ingested microorganisms [1, 2]. CGD is transmitted by X-linked inheritance in 66% and by autosomal recessive inheritance in 33% of the cases. Neutrophils from most X-linked and autosomal CGD patients lack one of the subunits (gp91-phox, p22-phox) of the membrane-associated cytochrome b558, a flavo-hemeprotein which represents the enzymatic unit of the NADPH oxidase, or some cytosolic components (p47-phox, p67-phox) [2, 6]. Most commonly the identified infectious agents in CGD patients are Staphylococcus aureus and less frequently Aspergillus, Nocardia, Pseudomonas, Serratia species and other gram-negative rods. However, in more than 55% of infections no microorganisms are re-



Figure 3 Peripheral white blood cells collected on endotoxin-coated coverslips and incubated with a solution of nitroblue tetrazolium (NBT) and serum. In a normal control (left) NBT was reduced to blue formazan and seen as blue dots within the cytoplasm of the white cells (arrows). More than 90% of the cells were NBT-positive in the control, while no NBT-positive cells were seen in the CGD patient (nuclei were stained in red with safranin; original magnification * 400).

covered as in our case where the etiological agents for the cavitating pneumonia and for the previous brain abscess could not be demonstrated. The good response to antibiotics and the negative BAL cultures ruled out a fungal pneumonia in our case. Although infection by Leishmania, an intracellular parasite, has been associated with AIDS and other states of Tlymphocyte dysfunction (hematologic malignancies, renal transplantation, autoimmune diseases, or treatments with steroids or immunosuppressors) [7, 8], it has not been described associated with CGD, where T- and B-lymphocyte functions are normal. Furthermore, it has been shown that the killing of L. donovani by human mononuclear phagocytes is oxygen-independent and therefore not affected in the CGD [9], making our case even more unusual and probably the first of visceral leishmaniasis in a CGD patient reported in the world literature. A possible explanation for the infection by L. donovani in our case is that patients with CGD are often malnourished as a result of recurrent severe infections as in this case, and also often have granulomatous disease of the bowel that mimics Crohn’s disease, with malabsortion, especially likely if they had perianal abscesses as our patient had. It is therefore likely that these facts might have increased the predisposition to visceral infection with Leishmania. However, the chance association of this infection in a patient who lived temporarily in an endemic area for leishmaniasis (the eastern coast of Spain) cannot be dismissed. He helped farming outdoors during summer time with a high risk of exposition to the sandfly Phlebotomus papataci, the vector of L. donovani infection in Spain, and the link between humans and the animal reservoirs (canines and rodents). CGD can be diagnosed easily by the nitroblue tetrazolium (NBT) dye reduction slide test [3]. The NBT test performed for the patient was negative but those of his mother and sister were normal. Therefore an X-linked


CGD form was unlikely, although it could not be ruled out completely because about one third of the CGD cases develop as a spontaneous mutation. The patient was homozygous for the p47-phox mutation, found in 90% of the autosomal recessive CGD patients, while his mother and sister were heterozygous asymptomatic carriers. After the patient was diagnosed with CGD, subcutaneous recombinant human interferon- and chronic prophylaxis with trimethoprim-sulfamethoxazole and itraconazole were started. Interferon- treatment for at least 1 year reduced in 70% of patients with CGD the incidence of serious infections in a large multicenter randomized placebo-controlled assay [10, 11].The significant clinical improvement of the study patients was not due to improvement of superoxide production of the neutrophils, suggesting a different mechanism of action. Increased tumor necrosis factor- (TNF-) production which activates the intracellular killing of microorganisms in the macrophages, as it does in patients with visceral leishmaniasis treated with interferon- and pentavalent antimonials [12, 13], and the TNF- priming of CGD neutrophils with increased antimicrobial activity by an oxygen-independent mechanism [14] are possible explanations. Prophylactic trimethoprim-sulfamethoxazole therapy diminishes the frequency of life-threatening bacterial infections in patients with CGD [15, 16] and prophylactic itraconazole diminishes the infections by Aspergillus and other fungi as well [17]. Although many CGD patients, especially those with the p47-phox mutation as our case, survive well into adulthood [18], bone marrow transplantation and gene therapy [2, 19] can be considered for more lethal mutations of the NADPH oxidase. In conclusion, it seems reasonable to rule out CGD in those patients with multiple and severe recurrent infections when other immunodeficiencies are discarded, in spite of their adult age and in the absence of a family history of CGD as in our case. An early CGD diagnosis can save the patient’s life and certainly will make his chronic disease much more manageable.

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