A Seminested PCR Method for the Diagnosis of Invasive Fungal Infections in Combat Injured

Abstract

Background Among combat injured, invasive fungal infections (IFIs) result in significant morbidity. Cultures and histopathology are the primary diagnostic methods for IFIs, but they have limitations. We previously evaluated a panfungal polymerase chain reaction assay, which was 83% sensitive and 99% specific for angioinvasive IFIs. Herein, we evaluated three less resource-intensive semi-nested assays targeting clinically-relevant fungi in the order Mucorales and genera Aspergillus and Fusarium. Methods Formalin-fixed paraffin-embedded tissue (FFPE) specimens from a multicenter trauma IFI cohort (2009-2014) were used. Cases were U.S. military personnel injured in Afghanistan with histopathologic IFI evidence. Controls were patients with similar injury patterns and no laboratory IFI evidence (negative culture and histopathology). Semi-nested assays specific to Mucorales (V4/V5 regions of 18S rDNA), Aspergillus (mitochondrial tRNA), and Fusarium (internal transcribed spacer [ITS]/28A regions of DNA) were compared to a panfungal assay amplifying the ITS2 region of rDNA and to histopathology. Results Specimens from 92 injury sites (62 subjects) were compared to control specimens from 117 injuries (101 subjects). We observed substantial agreement between the semi-nested and panfungal assays overall, especially for the order Mucorales. Moderate agreement was observed at the genus-level for Aspergillus and Fusarium. When compared with histopathology, sensitivity and specificity of semi-nested assays were 67.4% and 96.6% respectively (sensitivity increased to 91.7% when restricted to sites with angioinvasion). Conclusion Prior studies of semi-nested molecular diagnostics have focused on culture-negative samples from immunocompromised patients. Our findings underscore the utility of the semi-nested approach in diagnosing soft-tissue IFIs using FFPE samples, especially with angioinvasion.