Molecular Detection of Filamentous Fungi in Formalin-Fixed Paraffin-Embedded Specimens in Invasive Fungal Wound Infections Is Feasible with High Specificity

Abstract

Trauma-related invasive fungal wound infections (IFIs) are associated with significant morbidity and mortality. Early identification and treatment are critical. Traditional identification methods (e.g., fungal cultures and histopathology) can be delayed and insensitive. We assessed a PCR-based sequencing assay for rapid identification of filamentous fungi in formalin-fixed paraffin-embedded (FFPE) specimens obtained from combat casualties injured in Afghanistan. Blinded FFPE specimens from cases (specimens positive on histopathology) and controls (specimens negative on histopathology) were submitted for evaluation with a panfungal PCR. The internal transcribed spacer 2 (ITS2) region of the fungal ribosomal repeat was amplified and sequenced. The PCR results were compared with findings from histopathology and/or culture. If injury sites contributed multiple specimens, findings for the site were collapsed to the site level. We included 64 case subjects (contributing 95 sites) and 102 controls (contributing 118 sites). Compared to histopathology, panfungal PCR was specific (99%), but not as sensitive (63%); however, sensitivity improved to 83% in specimens from sites with angioinvasion. Panfungal PCR identified fungi of the order Mucorales in 33 of 44 sites with angioinvasion (75%), whereas fungal culture was positive in 20 of 44 sites (45%). Saksenaea spp. were the dominant fungi identified by PCR in specimens from angioinvasion sites (57%). Panfungal PCR is specific, albeit with lower sensitivity, and performs better at identifying fungi of the order Mucorales than culture. DNA sequencing offers significant promise for the rapid identification of fungal infection in trauma-related injuries, leading to more timely and accurate diagnoses.

Updates on the Taxonomy of Mucorales with an Emphasis on Clinically Important Taxa

“…Only a few cases of Actinomucor infections have been reported [83,85,86]. The genus Actinomucor consists only of a single species, Actinomucor elegans, for which three varieties are described: var.…”

Diagnostic Accuracy of a Direct Panfungal Polymerase Chain Reaction Assay Performed on Stained Cytology Slides

“…5 The latter is reasonable in regard to Malassezia species, which have been reported as the most common contaminants in studies assessing panfungal PCR on FFPE tissues and as common contaminants in PCR reagents. 1,2,5 Malassezia are a normal inhabitant of the skin microbiota in both humans and animals and could easily be ruled out as a cause of infection in all cases included in this study based on clinical and cytologic findings. For this reason, when only Malassezia spp.…”

Diagnostic Utility of Polymerase Chain Reaction for Paraffin-embedded Sinus Specimens for Rhinocerebral Mucormycosis Complicated by Internal Carotid Artery Thrombosis and Cerebral Infarction

“…The specificity and sensitivity of PCR for Intern Med Advance Publication DOI: 10.2169/internalmedicine.6809-20 identifying Mucormycetes spp. are 99% and 63%, respectively (6). However, the diagnosis of mucormycosis in previous cases of cerebral ischemia was performed mainly based on histological findings.…”