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Published on: March 22, 2012
A Seminested PCR Method for the Diagnosis of Invasive Fungal Infections in Combat Injured
Graham C Ellis1, Faraz Shaikh2,3, M Leigh Carson2,3
1Walter Reed National Military Medical Center, Bethesda, Maryland, USA.
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. Here, we evaluated 3 less resource-intensive seminested assays targeting clinically relevant fungi in the order Mucorales and genera Aspergillus and Fusarium.
Methods:
Formalin-fixed paraffin-embedded tissue specimens from a multicenter trauma IFI cohort (2009-2014) were used. Cases were US 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). Seminested 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 with a panfungal assay amplifying the internal transcribed spacer 2 region of rDNA and to histopathology.
Results:
Specimens from 92 injury sites (62 subjects) were compared with control specimens from 117 injuries (101 subjects). We observed substantial agreement between the seminested 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 seminested assays were 67.4% and 96.6%, respectively (sensitivity increased to 91.7% when restricted to sites with angioinvasion).
Conclusions:
Prior studies of seminested molecular diagnostics have focused on culture-negative samples from immunocompromised patients. Our findings underscore the utility of the seminested approach in diagnosing soft-tissue IFIs using formalin-fixed paraffin-embedded tissue samples, especially with angioinvasion.
Insights
New seminested assays show promise for diagnosing invasive fungal infections (IFIs) in combat-injured patients. These molecular methods offer a valuable alternative to traditional diagnostics, particularly for soft-tissue IFIs with angioinvasion.
Area of Science:
- Medical Mycology
- Molecular Diagnostics
- Infectious Diseases
Background:
- Invasive fungal infections (IFIs) cause significant morbidity in combat-injured individuals.
- Traditional diagnostic methods like cultures and histopathology for IFIs have limitations.
- Previous research established the efficacy of a panfungal polymerase chain reaction (PCR) assay for angioinvasive IFIs.
Purpose of the Study:
- To evaluate three less resource-intensive seminested PCR assays.
- To target clinically relevant fungi: order Mucorales, and genera *Aspergillus* and *Fusarium*.
- To assess the utility of these assays in formalin-fixed paraffin-embedded tissue samples from combat-injured patients.
Main Methods:
- Utilized formalin-fixed paraffin-embedded tissue specimens from a multicenter trauma IFI cohort (2009-2014).
- Compared seminested assays (targeting Mucorales, *Aspergillus*, *Fusarium*) with a panfungal assay and histopathology.
- Included cases of US military personnel with histopathologic IFI evidence and controls with no laboratory IFI evidence.
Main Results:
- Observed substantial agreement between seminested and panfungal assays, particularly for Mucorales.
- Found moderate agreement at the genus level for *Aspergillus* and *Fusarium*.
- Seminested assays demonstrated 67.4% sensitivity and 96.6% specificity compared to histopathology, with sensitivity reaching 91.7% for angioinvasive sites.
Conclusions:
- The seminested approach is useful for diagnosing soft-tissue IFIs in formalin-fixed paraffin-embedded tissue.
- Findings support the utility of seminested molecular diagnostics in a combat-injured population.
- This approach is particularly effective for IFIs with angioinvasion.
