Accuracy of plasma cell-free DNA PCR versus mNGS for non-invasive diagnosis of invasive mold disease

Cell-free DNA (cfDNA) detection in plasma is a novel testing modality for non-invasive diagnosis of invasive mold disease (IMD) in immunocompromised patients. The accuracy of PCR compared to metagenomic next-generation sequencing (mNGS) for diagnosis of IMD remains unknown. Plasma samples from patients with proven and probable IMD and no IMD were tested with mold PCR and shotgun mNGS. Mold PCR detected Aspergillus species, Mucorales agents, Fusarium species, Scedosporium species, and Lomentospora prolificans . The sensitivity and specificity of mold PCR and mNGS were measured. Ninety-four patients with proven (48) and probable (48) IMD due to Aspergillus species (64), Mucorales agents (13), Scedosporium species and Lomentospora prolificans (13), and Fusarium species (6), and 62 patients with no IMD were included in this study. 99.4% (155) of the patients were immunosuppressed. The overall sensitivity and specificity were 62.5% (60/96; 95% CI 52.0–72.2) and 99.8% (535/536; 95% CI 99.0–100), respectively, for mold PCR and 43.8% (42/96; 95% CI 33.6–54.3; P < 0.001) and 99.6% (534/536; 95% CI 98.7–100; P = 1.0), respectively, for mNGS. The proportion of mold PCR-positive mNGS-negative results was 16.0% (4/25) in focal cases compared with 25.0% (11/44; P = 0.384) and 18.5% (5/27; P = 0.810) in multifocal and disseminated cases, respectively. The median PCR cycle threshold was 40.6 (IQR, 37.5–41.5) in PCR-positive mNGS-negative cases compared with 37.3 (IQR, 34.5–39.3; P = 0.004) in PCR-positive mNGS-positive cases. Plasma cfDNA PCR is more sensitive than mNGS for the diagnosis of IMD in immunocompromised patients. Lower sensitivity of mNGS was associated with lower mold cfDNA burden. IMPORTANCE Invasive mold disease (IMD) is a major cause of morbidity and mortality in immunocompromised patients. Early diagnosis and initiation of tailored antifungal therapy are critical for optimizing outcomes in patients with IMD. Detection of circulating fungal cell-free DNA (cfDNA) in the acellular fraction of blood is a novel testing modality that is increasingly used for early and non-invasive diagnosis of IMD. This study represents the first head-to-head accuracy study to compare the sensitivity and specificity of PCR with mNGS for the diagnosis of IMD. The study showed that the overall mold PCR was significantly more sensitive compared with mNGS, while both assays demonstrated very high specificity. Similar trends were observed in a smaller subgroup of patients with invasive aspergillosis, mucormycosis, and scedosporiosis. The findings have implications for assay selection when seeking to non-invasively diagnose IMD in immunocompromised patients.