Challenges and predictive value of morphologic features in panfungal sequencing of formalin-fixed, paraffin-embedded tissues: A 5-year retrospective study

Abstract

Objective: Panfungal sequencing (PFS) using formalin-fixed, paraffin-embedded (FFPE) tissue aids genus-level or species-level identification in suspected invasive fungal infections. Given the limited availability of PFS and potential risk of environmental contamination, defining histopathologic features predictive of clinically interpretable results is important.

Methods: We evaluated FFPE tissue samples submitted for PFS over a 5-year period. Histopathologic data were extracted from pathology reports; in-house cases were re-reviewed, and the burden of fungal elements was assessed using Grocott methenamine silver stain. Any available fungal culture data were also obtained for in-house cases.

Results: Of 153 cases with fungal elements reported by histopathology, 54% were positive by PFS. Of 97 cases without histologic evidence of fungal elements, only 9% were positive by PFS, and all were considered potential environmental contaminants. Culture results were available for only 9 of 461 (2%) cases, and all cultures were concordant with the PFS results. When the pathologist proposed 1 or more specific organisms based on histologic appearance alone, PFS was discrepant in 37% of cases. Of those discrepant diagnoses, and if we designate the PFS result as the true diagnosis, then 53% of misclassifications had the potential for administration of suboptimal antifungal therapy. There was no correlation between the relative abundance of fungal elements in tissue sections and detection of fungal organisms by PFS.

Conclusions: Panfungal sequencing can provide genus-level and species-level identification in the setting of visible fungal elements in FFPE tissue. It is a valuable diagnostic tool, particularly when fungal infections are clinically suspected but fungal cultures were not performed.

Keywords: fungal infection; histopathology; panfungal sequencing.

Figure 1

PFS results and availability of pathology reports, by specimen type. PFS indicates panfungal sequencing.

Figure 2

The relative proportions of fungal organisms detected by panfungal sequencing in cases where fungal elements were identified in tissue (n = 82).

Figure 3

Correlation between fungal special stains and PFS results examining (A) GMS and (B) PAS. Positivity rate of PFS based on (A) GMS and (B) PAS stain result. An indeterminate special stain result indicates that the stain interpretation was not available or was reported as indeterminate for fungus. GMS indicates Grocott methenamine silver; PAS, periodic acid–Schiff; PFS, panfungal sequencing.

Figure 4

Fungal hyphal elements with overlapping morphologic features that can have the potential to lead to incorrect histopathologic diagnoses. (A) Fusarium species (GMS, ×40); (B) Aspergillus species (GMS, ×100); (C) Scedosporium species (GMS, ×60); (D) Candida albicans (GMS, ×60). (E-G) Fungal yeasts and endospores with overlapping morphologic features. (E) Candida tropicalis (GMS, ×40); (F) Coccidioides immitus/posadasii (GMS, ×40) (note the closely approximated endospores that resemble broad-based budding yeast in the top left corner); (G) Blastomyces species (GMS, ×40; inset ×100). GMS indicates Grocott methenamine silver; PAS, periodic acid–Schiff; PFS, panfungal sequencing.

Figure 5

Sections of a lung wedge resection with broad zones of necrotizing granulomas demonstrating multiple foci of possible spherules (A, H&E, ×100;B, GMS, ×100) containing variably sized yeast-like structures (2.5-26 µm) as well hyphal forms (C, H&E, ×40; D, GMS, ×40), suggestive of Coccidioides. The patient lived in Mexico and California, and Coccidioides serologies were mildly elevated (equivocal). Coccidioides immitus/posadasii can produce hyphae and arthroconidia in pulmonary tissue sections. GMS indicates Grocott methenamine silver; H&E, hemoxylin-eosin; PAS, periodic acid–Schiff.