Rapid respiratory cryptococcosis detection using targeted next-generation sequencing

Cryptococcus neoformans, a WHO critical-priority and neglected fungal pathogen, causes cryptococcosis primarily via inhalation. Clinical manifestations range from asymptomatic infection to fatal meningitis; suboptimal diagnostics cause delayed detection and high meningitis mortality. This retrospective study evaluated the clinical utility of targeted next-generation sequencing (tNGS) for diagnosing pulmonary cryptococcosis using respiratory specimens, compared to serum cryptococcal antigen (CrAg) testing and fungal culture. Over 38 months, 39 patients with confirmed cryptococcosis were enrolled. tNGS detected Cryptococcus neoformans in 92.3% (36/39) of respiratory samples, significantly outperforming fungal culture (23.1%, 9/39; P < 0.05). Serum CrAg testing was positive in 64.7% (22/34) of tested patients. tNGS identified C. neoformans in 12 CrAg negative cases and 75% (9/12) of culture-negative cases, enhancing diagnostic yield. The median turnaround time for tNGS (23 [IQR: 21-43] hours) was substantially shorter than for fungal culture (112 [IQR: 77.5-120.5] hours; Mann-Whitney U test, P < 0.05). Higher tNGS read counts correlated with culture positivity (P < 0.05) but not with CrAg status. Polymicrobial infections were detected in 76.9% (30/39) of tNGS tests, underscoring its utility in comprehensive pathogen identification. tNGS of respiratory specimens demonstrates superior sensitivity and markedly shorter turnaround time than fungal culture for cryptococcosis diagnosis. Crucially, its ability to detect C. neoformans in serum CrAg negative patients enables earlier diagnosis in cases missed by standard serology.