Inherited CARD9 Deficiency in a Patient with Both Exophiala spinifera and Aspergillus nomius Severe Infections

Abstract

Purpose: Caspase-associated recruitment domain-9 (CARD9) deficiency is an inborn error of immunity that typically predisposes otherwise healthy patients to single fungal infections and the occurrence of multiple invasive fungal infections is rare. It has been described as the first known condition that predisposes to extrapulmonary Aspergillus infection with preserved lungs. We present a patient that expands the clinical variability of CARD9 deficiency.

Materials and methods: Genetic analysis was performed by Sanger sequencing. Neutrophils and mononuclear phagocyte response to fungal stimulation were evaluated through luminol-enhanced chemiluminescence and whole blood production of the proinflammatory mediator interleukin (IL)-6, respectively.

Results: We report a 56-year-old Argentinean woman, whose invasive Exophiala spinifera infection at the age of 32 years was unexplained and reported in year 2004. At the age of 49 years, she presented with chronic pulmonary disease due to Aspergillus nomius. After partial improvement following treatment with caspofungin and posaconazole, right pulmonary bilobectomy was performed. Despite administration of multiple courses of antifungals, sustained clinical remission could not be achieved. We recently found that the patient's blood showed an impaired production of IL-6 when stimulated with zymosan. We also found that she is homozygous for a previously reported CARD9 loss-of-function mutation (Q289*).

Conclusions: This is the first report of a patient with inherited CARD9 deficiency and chronic invasive pulmonary aspergillosis (IPA) due to A. nomius. Inherited CARD9 deficiency should be considered in otherwise healthy children and adults with one or more invasive fungal diseases.

Keywords: Aspergillus nomius; Chronic invasive pulmonary aspergillosis; Exophiala spinifera; Inherited CARD9 deficiency; Phaeohyphomycosis.

Figure 1.. Identification and characterization of CARD9 deficiency.

A. Pedigree of the family. I-IV (left margin) indicate generations; arrow indicates proband; black symbols indicate patients with phaeohyphomycosis. M, mutant; WT. wild-type; “E?” indicates those whose genetic status could not be evaluated. (Collection of pedigree information may be incomplete). B. Clinical and radiological features of the patient. Vegetating, verrucous skin lesions due to E. spinifera (a-d): on the back (a), on the knee (b), on the cheek and outer ear (c), and on the forehead (d). Phaeohyphomycosis involving the bone: osteolytic lesion at tibia’s proximal region (e). Imaging findings in pulmonary aspergillosis (f,g): chest radiography showing dense pulmonary infiltrate in the middle lobe of right lung (f). Chest computed tomography scan showing pseudotumoral lesion in the right lung (g). C. Microscopic and histological features of E. spinifera (a,b,c) and A. nomius (d,e,f) infection. Histopathological study of a lymph node biopsy specimen showing numerous giant cells with round brown bodies inside (H&E stain; original magnification, X400) (a). Microculture of Exophiala spinifera (Lactophenol cotton blue stain; original magnification, X1000) (b). Giemsa stained smear taken from the draining fistula arising from the osteolytic lesion in the tibia to the skin, showing neutrophilic accumulation around Exophiala hyphae (c). Histopathological study of lung surgical piece showing an epithelioid granuloma with a giant cell surrounding fungal hyphae within an arteriole (H&E stain; original magnification, X400) (d). Histopathological preparation of tissue sections of lung. Fungal hyphae within an arteriole are observed (Grocottś methenamine-silver stain, original magnification, X400) (e). Histopathological study of lung surgical piece showing an epithelioid granuloma with giant cells. No neutrophils are seen (H&E stain, original magnification, X200) (f). D. Macro- and micromorphological aspects of Aspergillus nomius. Rough colonies of yellowish-green color and wooly texture (a). Radiate heads with uniseriate and biseriate conidiogenous cells, hyaline rough-walled conidiophores and echinulate, spherical conidia (b).

Figure 2.. ROS and IL-6 production in blood cells of CARD9 deficient patient.

A. Normal NADPH oxidase activity in the patient’s neutrophils after activation with unopsonized zymosan (Z) (5μg/ml), as assessed by the monitoring of luminol-dependent chemiluminescence for 60 minutes at room temperature. B. Interleukin 6 (IL-6) production in the supernatants of cultured whole blood cells stimulated for 48 hours with unopsonized zymosan (Z) (5 μg/ml); LPS from Salmonella Minnesota (100ng/ml) or phorbol 12-myristate 13-acetate (PMA) – Ionomycin (I), 10⁻⁷ M and 10⁻⁵ M), as measured by enzyme-linked immunosorbent assay (ELISA). The results are representative from one experiment performed using duplicate samples involving two healthy age matched controls.