Mycobiome analysis in fungal Infected formalin-fixed and paraffin-embedded tissues for identification of pathogenic fungi: A pilot study

Background Fungal organisms are frequently observed in surgical pathological diagnosis. In order to more accurately identify fungi in formalin-fixed and paraffin-embedded (FFPE) tissues, it is necessary to use genomic information. The purpose of our pilot study is to identify the factors to be considered for the identification of pathogenic fungi using mycobiome analysis in FFPE tissues. Methods We selected 49 cases in five hospitals. In each case, FFPE tissue was cut into 50 µm and DNA was extracted. Multiplex PCR with four primers (ITS1, ITS2, ITS3 and ITS4) was performed. Multiplex sequencing was performed using MinION device according to the manufacturer’s protocol. Sequences of each case were searched using BLASTN with an ITS database from NCBI RefSeq Targeted Loci Project with default parameter. Results A total of 2,526 DNA nucleotides were sequenced. We were able to identify 342 fungal nucleotides in 24 (49.0%, 24/49) cases. The median value of the detected fungal DNA per case was 3 (1Q: 1 and 3Q: 14.25). The 215 (62.87%) fungal DNA contained the entire region of ITS1 or ITS2. The remaining 127 fungal DNAs were identified as fungi using partial sequence of ITS1, ITS2, 5.8S, LSU or SSU. Conclusion In conclusion, we have identified the possibility of finding pathogenic fungi through mycobiome analysis in fungal infected FFPE tissues using nanopore sequencing method. However, we have also found several limitations to be solved for further studies. If we develop a method to characterize pathogenic fungi in FFPE tissues in a follow-up study, we think it will help patients to use appropriate antifungal agents.


Introduction
Fungal organisms are frequently observed in surgical pathological diagnosis. Recent developments in technology have enabled the identification of fungi using a variety of methods. 1 These novel methods are based on analyzing fungal genomes or proteomes using fresh tissues. 2,3 However, these methods are difficult to apply in formalin-fixed and paraffin-embedded (FFPE) tissues used in surgical pathology. Sanger sequencing and IHC staining have been used in FFPE tissue, but most medical institutions only identify fungi based on morphological findings. [4][5][6][7][8][9] The identification of fungi only by morphological findings can lead to inadequate treatment for patients due to misdiagnosis, which can often result in fatal consequences. 10 Because of the different antifungal agents preferentially used at the initial infection stage depending on the fungus, it is necessary to identify the exact fungi present through testing methods in addition to the morphological findings. 11 In order to more accurately identify fungi in FFPE tissues, it is essential to use genomic information. DNA markers that could be used to identify fungi include the internal transcribed spacer (ITS) region, small subunit (nrSSU-18S), large subunit (nrLSU-26S or 28S), elongation factor 1-alpha (EF1α), and the largest (RPB1) and second largest (RPB2) subunits of RNA polymerase. 1,2 Among these markers, ITS could be relatively easily and effectively used for fungal identification, and the database of the ITS regions of fungi is available. [12][13][14] In general, sequencing equipment is classified into first-(Sanger sequencing), second-(massively parallel sequencing), and third-generation (real-time and single molecule sequencing) equipment according to key analytical methods. 15 Third generation sequencing technology is characterized by direct sequencing of nucleotides without PCR amplification. Oxford Nanopore Technology (ONT) introduced several sequencing equipment using the nanopore sequencing technology, which measures the change in current that occurs when a nucleotide sequence passes through a narrow channel. 16 Since there is an advantage of identifying each DNA sequence without PCR amplification, it is expected that the DNA can be effectively detected in spite of DNA degradation during FFPE tissue preparation and storage.
Extracting only fungal DNA from the fungal infection site of FFPE tissue is very difficult. Therefore, we decided to use the mycobiome analysis method. However, no studies have attempted to analyze mycobiome in FFPE tissue. Therefore, this study was conducted as a pilot study on the development of a method for finding pathogenic fungi using mycobiome analysis in the fungal infected FFPE tissues.

Sample collection
The cases were extracted from the pathological examination reports of five medical institutions. We first extracted the pathology report that mentioned the presence of fungi. We then selected typical cases for use as positive controls, cases reported to be difficult to differentiate (in briefly, fungi with branched-hyphae, such as Aspergillus species and Mucor species, are often difficult to distinguish morphologically), and cases with additional information related with fungal identification on pathology report (i.e., identification using culture or sequencing). Through this process, we finally selected 49 cases (case 3-02 and 5-10 were able to confirm the fungal identification results using sequencing). Cases included 21 lungs, 8 paranasal sinuses, 7 gastrointestinal tracts, 5 orbits, 2 skin and mouths, 1 adrenal gland, bone, gum and liver. The average storage period of FFPE tissue was 4.4 years. This study was exempted from obtaining informed consent by Institutional Review Board of Hallym University Dongtan Sacred Heart Hospital (NON2018-005).

DNA extraction, PCR amplification, Sequencing and Base calling
In each case, FFPE tissue was cut into 50 µm (5 µm x 10) sections and collected in a 2.0

Data analysis
We used BLAST+2.10.0 on a local PC (Ubuntu 19.10) for analysis 18 . In brief, we downloaded the ITS database file from NCBI RefSeq Targeted Loci Project (last modified Mar 3, 2020) and then converted it using "makeblastdb" of BLAST+2.10.0. This database file contained full or partial ITS sequence of 11,133 genus of fungi. The sequences of Pneumocystis jirovecii, which is not found in this database, were downloaded RU7 genomic reference sequences and compared. The sequence of Actinomyces israelii, a bacterium that looks morphologically similar to that of a filamentous fungus, was downloaded (Actinomyces israelii DSM 43320, whole genome shotgun sequencing project; NZ_JONS00000000.1) and compared. We confirmed the BLASTN search using GRCh38 to determine if the detected DNA corresponds to the human genome.
We converted FASTQ files to FASTA format using "seqtk" (https://github.com/lh3/seqtk). Sequences of each case were searched using BLASTN using ITS database file without default parameter modification. When the sequence matched in the ITS database, the result with the highest bit score value was selected. We selected five results in order of higher bit score when five or more fungal DNAs were detected in one case.
We downloaded the GenBank files of all fungi contained in the database and separated the base sequences of ITS1, 5.8S and ITS2 for each fungus. When fungi were detected, they were classified into three categories ("entire", "partial", and "none" match) according to the relationship between the detected fungal DNA and ITS1/ITS2. "Entire" means that all ITS1 or ITS2 base information is used for fungal identification, "partial" means that even if only one sequence information is used, and "none" means that base information other than ITS1 or ITS2 is used. Representative microscopic images of some cases with inconsistent pathologic diagnosis and mycobiome analysis are summarized in Figure 3. Aspergillus and Candida species were detected in case 1-01 ( Figure 3A).

Results
Compared to Aspergillus species commonly found in nasal cavities, thinner hyphae were observed, which may be misleading as sulfur granule of Actinomyces. Aspergillus and Acremonium species were detected in case 1-03 ( Figure 3B). Contrary to case 1-01, it is thought to be misdiagnosed as Mucor species because of its slightly wider hyphae than Aspergillus species. Case 1-08 ( Figure 3C) was also misdiagnosed as Actinomyces, which is thought to be similar to case 1-01. Alternaria, Fusarium and Aspergillus species were commonly detected. In case 2-08 ( Figure 3D), yeast-form fungi were observed in alveolar macrophage on Gomori Methenamine-silver stain and Starmerella cellae was identified. Starmerella cellae is a relatively recently identified ovoid to ellipsoidal fungus. 19 Case 2-09 ( Figure 3E) is a fungus found in pharynx, which shows morphological findings different from those of Candidda, Aspergillus, and Mucor. In other words, yeast-form or short branching-type, fungal nuclei were found inside fungi like Pneumocystis jiroveci. Cladosporium coloradense was identified.
Case 3-04 ( Figure 3F) is a yeast-form fungus found in subcutaneous tissue and Candida glabrata were frequently identified by sequencing.

Discussion
We performed mycobiome analysis in fungal infected FFPE tissues using nanopore sequencing. The detected fungal DNA occupied approximately one-third of the ITS1 entire region, the ITS2 entire region, and other regions, respectively. The advantages of nanopore sequencing compared with Sanger sequencing are as follows.
First, unlike Sanger's method, which requires a lot of DNA, nanopore sequencing can be performed with a small amount of DNA (in theory, even with one strand). Second, nanopore sequencing method could sequenced DNA separately, even if the sample contains a variety length of DNA. 20 Third, nanopore sequencing equipment (i.e., MinION) could be operated at lower cost (about $1,000) compared to Sanger sequencing equipment. This low initial cost is a critical factor in the introduction of equipment in small pathology laboratories. Compared to the 2nd generation sequencing equipment, nanopore sequencing has the advantage of sequencing with damaged DNA because there is no PCR amplification process in the sequencing process itself. In this study, about one third of the fungal DNA was not an ITS1 entire match or an ITS2 entire match. Therefore, these fungi can be effectively detected using nanopore sequencing.
It is necessary to find pathogenic fungi in various fungi detected by mycobiome analysis. bp gap open at ITS1 in Candida albicans. Two mismatched nucleotides are located in ITS2, where the DNA base "C" in Candida albicans is "T" in Candida africana. It is generally known that nanopore sequencing has a slightly higher error rate than second-generation NGS sequencing. 23 DNA extracted from FFPE tissue is known to have a higher C:T conversion than DNA extracted from fresh frozen tissue. 24 Because of these, our method seems to be difficult to identify precise fungi, especially at the species level.
In summary, we have identified the possibility of finding pathogenic fungi through mycobiome analysis in fungal infected FFPE tissues. However, we have found problems to be solved for further studies, such as increasing sequencing output, increasing fungal DNA concentration, excluding normal flora, and expanding fungal DB. If we develop a method to characterize pathogenic fungi in FFPE tissues in a follow-up study, we think it will help patients to use appropriate antifungal agents.

Declaration of interest
The authors declare no competing interests.