Fungal Integrated Histomolecular Diagnosis Using Targeted Next-Generation Sequencing on Formalin-Fixed Paraffin-Embedded Tissues

Abstract

Histopathology is the gold standard for fungal infection (FI) diagnosis, but it does not provide a genus and/or species identification. The objective of the present study was to develop targeted next-generation sequencing (NGS) on formalin-fixed tissue samples (FTs) to achieve a fungal integrated histomolecular diagnosis. Nucleic acid extraction was optimized on a first group of 30 FTs with Aspergillus fumigatus or Mucorales infection by macrodissecting the microscopically identified fungal-rich area and comparing Qiagen and Promega extraction methods through DNA amplification by A. fumigatus and Mucorales primers. Targeted NGS was developed on a second group of 74 FTs using three primer pairs (ITS-3/ITS-4, MITS-2A/MITS-2B, and 28S-12-F/28S-13-R) and two databases (UNITE and RefSeq). A prior fungal identification of this group was established on fresh tissues. Targeted NGS and Sanger sequencing results on FTs were compared. To be valid, the molecular identifications had to be compatible with the histopathological analysis. In the first group, the Qiagen method yielded a better extraction efficiency than the Promega method (100% and 86.7% of positive PCRs, respectively). In the second group, targeted NGS allowed fungal identification in 82.4% (61/74) of FTs using all primer pairs, in 73% (54/74) using ITS-3/ITS-4, in 68.9% (51/74) using MITS-2A/MITS-2B, and in 23% (17/74) using 28S-12-F/28S-13-R. The sensitivity varied according to the database used (81% [60/74] using UNITE compared to 50% [37/74] using RefSeq [P = 0.000002]). The sensitivity of targeted NGS (82.4%) was higher than that of Sanger sequencing (45.9%; P < 0.00001). To conclude, fungal integrated histomolecular diagnosis using targeted NGS is suitable on FTs and improves fungal detection and identification.

Keywords: formalin-fixed paraffin-embedded tissues; fungal identification; histopathology; integrated histomolecular diagnosis; massive parallel sequencing; next-generation sequencing.

FIG 1

(A to I) Histopathological patterns 1, 2, and 3: yeasts without pseudomycelium (pattern 1), pseudomycelium with or without yeast (pattern 2), and hyalohyphomycetes (pattern 3); HES, hematoxylin-eosin-saffron.

FIG 2

(A to F) Histopathological patterns 4, 5, and 6: Mucorales (pattern 4), phaeohyphomycetes (pattern 5), and spherules (patter 6); HES, Hematoxylin-eosin-saffron.

FIG 3

Example of a fungal integrated histomolecular diagnosis: an ileal coccidioidomycosis after liver transplantation. (A) Ileal resection (before formalin fixation) in a 7-year-old immunocompromised female with a history of liver transplantation. The presence of a tissue thickening, without mucosa ulceration, is evident. A slice is made along the yellow dotted line. (B) Sliced surgical specimen (along the yellow dotted line) after formalin fixation. (C) Hematoxylin-eosin-saffron (HES) tissue analysis (×10). The tissue thickening is located below the mucosa and concerns the peritoneum. Inflammation of all layers under the mucosa is observed, with a predominant thickening of the subserosa/peritoneum, which is abraded and covered with fibrin. (D) HES tissue analysis (×400). In this inflammatory infiltrate, there are spherules, suggestive of the genus Coccidioides (pattern 6): mature spherules with (yellow arrow) or without (black arrow) endospores. (E) Grocott tissue analysis (×400). Spherules are stained by Grocott. Mature forms with endospores (black arrow) and small immature forms without endospores (red arrow) are present. (F) Periodic acid-Schiff (PAS) tissue analysis (×400). Spherules are stained by PAS (yellow arrows). The liver transplant was from a patient who had a positive coccidioidomycosis serology. The patient was not from a coccidioidomycosis area of endemicity (European) but had traveled to Peru 4 months before his death. DNA extraction was performed on FTs, and NGS found Coccidioides immitis/Coccidioides posadasii, which is compatible with the morphological features (pattern 6), leading to the integrated histomolecular diagnosis of a disseminated coccidioidomycosis caused by Coccidioides immitis/Coccidioides posadasii, transmitted by the donor (liver transplant). Culture on fresh tissue identified the same fungus.

FIG 4

Detailed workflow and perspectives of the histomolecular integrated approach. The histomolecular approach combines morphological features (fungal patterns) that must be compatible with the fungal molecular identification obtained from formalin-fixed tissues. The implementation of such an approach in the clinical routine feasible and should allow for the prescription of the most appropriate antifungal therapy.

FIG 5

Algorithm for the management of formalin-fixed paraffin-embedded samples (FTs) with a histopathological diagnosis of fungal infection in Lyon, France. On FTs, the diagnosis of fungal infection is based on the evidence of fungi in necrotic and/or inflamed tissues, observed on the hematoxylin-eosin-saffron and Grocott/Periodic acid-Schiff stains. Depending on the fungal pattern identified, different additional investigations should be performed. For patterns 1 and 5, special stains must be used, Alcian Blue and Fontana-Masson, respectively. For pattern 3, A. fumigatus being one of the most prevalent fungal pathogens in human pathology, a specific PCR must be performed first, followed by next-generation sequencing (NGS) if the result is negative. Similarly, a specific Mucorales PCR should be performed first when pattern 4 is observed. If Mucorales PCR is negative, NGS using ITS-3/ITS-4 primers should be done (no detection using MITS-2A/MITS-2B in the present study). However, the distinction between patterns 3 and 4 is sometimes difficult (e.g., see Fig. S3C in the supplemental material), and both specific PCRs, completed or not by NGS, must be performed in these cases (*). Moreover, if a coinfection is suspected, NGS must be performed. In any case, histopathological observations should be confirmed and/or specified by panfungal sequencing (if no specific PCR is available) to achieve an integrated histomolecular diagnosis.