Case Report: Manual review of a gray-zone multiplex PCR targeted sequencing signal in clinically diagnosed pulmonary cryptococcosis

Background Pulmonary cryptococcosis may be difficult to diagnose when clinical manifestations are nonspecific and conventional microbiological tests are negative. Multiplex PCR-based targeted next-generation sequencing (mp-tNGS) can provide rapid pathogen evidence, but weak fungal signals require careful interpretation. Case presentation A 47-year-old apparently immunocompetent woman was admitted with persistent bilateral pulmonary lesions after empirical antibacterial treatment. Bacterial and fungal cultures, fungal smears, acid-fast staining, and Mycobacterium tuberculosis nucleic acid testing were all negative for both sputum and bronchoalveolar lavage fluid (BALF) specimens. BALF mp-tNGS yielded low-confidence Cryptococcus spp. signals. The putative C. neoformans assignment had 1,414 normalized reads and one positive primer pair among six; the putative C. gattii assignment had 78 normalized reads and two positive primer pairs among three. Neither established species identity. Manual review considered run quality and negative controls alongside positive serum cryptococcal antigen and supportive India ink microscopy. Management and outcome The patient was managed as having clinically diagnosed pulmonary cryptococcosis. HIV testing was negative, and the CD4T-cell count was 600 cells/µL. Blood culture was negative. She had no headache, vomiting, or focal neurological signs; however, no CSF CrAg, culture, India ink microscopy, or mp-tNGS was performed. She received intravenous fluconazole 400 mg once daily after an 800-mg loading dose from 18 to 23 December 2025, followed by oral fluconazole 600 mg once daily. Symptoms improved; imaging showed early progression followed by partial improvement, with persistent cavitary changes. Conclusion Low-level fungal mp-tNGS signals warrant multidisciplinary review integrating technical quality, specimen type, antigen testing, microscopy, culture, and clinical findings. Species-specific confirmation should be pursued when feasible. This proposed BALF review framework requires prospective validation and should not be transferred directly to CSF or other specimens.