Case Report: Neonatal-onset NCF1-related chronic granulomatous disease with pulmonary Aspergillus infection: an apparent discordance between DHR response and molecular findings

Background Chronic granulomatous disease (CGD) is caused by impaired phagocyte NADPH oxidase activity. NCF1 -related CGD results from p47 phox deficiency and shows substantial phenotypic variability. Diagnosis may be challenging when neutrophil oxidative burst testing appears partially preserved and when molecular analysis is complicated by the highly homologous NCF1B and NCF1C pseudogenes. Case description A 27-day-old term boy presented with cough and fever and rapidly developed acute hypoxemic respiratory failure. Chest computed tomography showed multiple bilateral pulmonary nodules and mass-like consolidations. Aspergillus fumigatus was detected by targeted sequencing of bronchoalveolar lavage fluid and recovered from sputum culture, supporting pulmonary Aspergillus infection. Dihydrorhodamine 123 (DHR) testing showed an unusual pattern: 72.26% of neutrophils were classified as activated after phorbol 12-myristate 13-acetate (PMA) stimulation (reference, >90%), whereas the stimulation index was only 33.60 (reference, >100), with an insufficient rightward fluorescence shift. Whole-exome sequencing (WES)-based quantitative analysis of the NCF1/NCF1B/NCF1C GTGT/ Δ GT differential site yielded a markedly reduced GTGT/ Δ GT ratio of 0.013. Targeted multiplex ligation-dependent probe amplification subsequently demonstrated 0 GTGT and 6 Δ GT copies in the patient and 1 GTGT and 5 ΔGT copies in each parent, supporting biallelic ΔGT-associated NCF1 deficiency. Voriconazole therapy was associated with resolution of fever and respiratory distress, and the patient completed a 6-month antifungal course. During follow-up, the patient developed methicillin-susceptible Staphylococcus aureus suppurative cervical lymphadenitis. Conclusion This case shows an apparent discrepancy between the proportion of neutrophils classified as activated on DHR testing and molecular evidence of NCF1 deficiency. The activation percentage should not be interpreted as a direct estimate of residual NADPH oxidase activity. When CGD remains clinically suspected despite a partially preserved DHR response, the stimulation index and fluorescence distribution should also be considered. Pseudogene-aware molecular testing may be required when short-read sequencing does not adequately resolve abnormalities at the NCF1 locus.