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Molecular Detection of Filamentous Fungi in Formalin-Fixed Paraffin-Embedded Specimens in Invasive Fungal Wound
Anuradha Ganesan1,2,3, Justin Wells3, Faraz Shaikh4,2
1Infectious Disease Clinical Research Program, Preventive Medicine and Biostatistics Department, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA anuradha.ganesan.ctr@mail.mil.
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.
Insights
Rapid PCR sequencing accurately identifies fungal infections in trauma wounds, outperforming traditional methods for certain fungi like Mucorales. This DNA sequencing approach promises faster, more precise diagnoses for invasive fungal infections in injured patients.
Area of Science:
- Medical Mycology
- Molecular Diagnostics
- Trauma Surgery
Background:
- Trauma-related invasive fungal wound infections (IFIs) cause significant morbidity and mortality.
- Early diagnosis and treatment are crucial for managing IFIs.
- Traditional diagnostic methods like fungal cultures and histopathology can be slow and lack sensitivity.
Purpose of the Study:
- To evaluate a PCR-based sequencing assay for the rapid identification of filamentous fungi in formalin-fixed paraffin-embedded (FFPE) specimens from combat casualties.
- To compare the performance of panfungal PCR with histopathology and fungal cultures for diagnosing IFIs.
Main Methods:
- A panfungal PCR assay targeting the internal transcribed spacer 2 (ITS2) region was used to analyze FFPE specimens.
- Specimens were obtained from combat casualties injured in Afghanistan.
- PCR results were compared against histopathology and/or culture findings.
Main Results:
- Panfungal PCR demonstrated high specificity (99%) but moderate sensitivity (63%) compared to histopathology.
- PCR sensitivity improved to 83% in specimens with evidence of angioinvasion.
- PCR identified Mucorales in 75% of angioinvasive sites, significantly higher than culture (45%).
- Saksenaea spp. were the most common fungi identified by PCR in angioinvasive samples.
Conclusions:
- Panfungal PCR is a specific method for identifying filamentous fungi in trauma-related IFIs.
- This molecular technique offers improved detection of Mucorales compared to traditional cultures.
- DNA sequencing holds promise for rapid and accurate diagnosis of fungal infections in complex trauma wounds.
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