Host-specific fungal target detection by triplex PCR with contemporaneous EBV and HCMV testing

The fungal triplex real-time PCR assay detects three opportunistic fungal targets, but their distribution across host groups and relationships with Epstein-Barr virus (EBV) and human cytomegalovirus (HCMV) DNA detection in clinical practice remain unclear. We conducted a single-center retrospective study of fungal triplex real-time PCR testing from January 2025 through June 2026. Hospital-wide testing was described, and Firth logistic regression was used to examine host-specific and viral associations in 332 adults with contemporaneous fungal, EBV and HCMV testing. Among 5,724 fungal triplex PCR test events from 3,558 patients, at least one fungal target was detected in 1,321 (23.1%); Pneumocystis jirovecii, Aspergillus spp. and Cryptococcus neoformans were detected in 16.5%, 7.9% and 0.7%, respectively. In the patient-level cohort, 126/332 adults (38.0%) had at least one fungal target detected. Compared with patients without a major immunocompromising condition, AIDS and transplantation were associated with higher odds of any fungal target detection (adjusted odds ratio [aOR], 7.64; 95% confidence interval [CI], 2.88-23.72; and aOR, 6.50; 95% CI, 2.51-19.85, respectively), as well as P. jirovecii detection. Other immunocompromising conditions were associated with Aspergillus spp. detection (aOR, 7.20; 95% CI, 1.84-40.63). HCMV associations with any fungal target and Aspergillus spp. were not significant after multiple-testing correction (both adjusted P = 0.063), and EBV showed no clear association. These findings support organism- and host-specific interpretation of fungal triplex PCR results; molecular detection alone does not establish active fungal disease.