Evaluating a semi‐nested PCR to support histopathology reports of fungal rhinosinusitis in formalin‐fixed paraffin‐embedded tissue samples

Abstract Background Fungal rhinosinusitis (FRS) encompasses a various spectrum of diseases. Histopathology is the “reference method” for diagnosing FRS, but it cannot determine the genus and species. Moreover, in more than 50% of the histopathologically proven cases, the culture elicited no reliable results. This study was an attempt to evaluate the diagnostic efficiency of semi‐nested polymerase chain reaction (PCR) from formalin‐fixed paraffin‐embedded (FFPE) functional endoscopic sinus surgery (FESS) in FRS patients. Methods One hundred ten specimens were subjected to DNA extraction and histopathology examination. The amplification of the β‐globin gene by conventional PCR was used to confirm the quality of extracted DNA. The semi‐nested PCR was performed using ITS1, ITS2, and ITS4 primers during two steps. Sequencing the internal transcribed spacer region (ITS1‐5.8S‐ITS2) to identify causative agents was performed on PCR products. Results Sixty‐four out of 110 samples were positive by histopathology evidence, of which 56 samples (87.5%) were positive by PCR. Out of 46 negative samples by histopathological methods, five samples (10.9%) yielded positive results by PCR. Sensitivity, specificity, positive predictive value, and negative predictive value of the semi‐nested PCR method were reported 87.5%, 89.2%, 92.7%, and 85.2%, respectively. The kappa factor between PCR and histopathological methods was 0.76, indicating substantial agreements between these two tests. Conclusion Due to the acceptable sensitivity and specificity of the present method, it might be used to diagnose fungal sinusitis infections along with microscopic techniques. This method is recommended to confirm the diagnose of suspected fungal sinusitis with negative histopathology results.

On the contrary, preparing and staining tissue specimens is a time-consuming procedure that requires a trained individual with extensive knowledge. Mucorales are responsible for about 45%-75% of FRS [19][20][21][22] and have a fast invasion with significant morbidity and mortality in the event of a compromised immune system. Thus, early detection of this infection is critical. As a result, a supplementary reliable diagnostic technique on the same sample is required to support and corroborate the histopathological findings. Because the specimen obtained through endoscopy-guided biopsy is mainly used to make histopathological diagnoses, employing a reliable molecular technique for direct detection of FRS in the residual FFPE samples may enhance diagnosis. Hence in this study, a semi-nested PCR in FFPE samples was used to detect FRS compared with the histopathology examination.

| Samples and patients
The functional endoscopic sinus surgery (FESS) samples were col- Demographic data of the patients, including age, sex, type of operation, site of infection, background diseases, and final pathology report, were documented ( Table 1). The paraffin blocks were prepared for the cutting process by microtome. During this process, ten slices were randomly cut with a thickness of 5 μm from each of the PEBs. The samples were put into microtubes to further molecular investigations.

| Deparaffinization process
To reduce the contamination of samples during this process, it recommended the sterilization of microtome and other instruments using benzene and 2 M HCl rinsed with sterile water (www. specificity, positive predictive value, and negative predictive value of the semi-nested PCR method were reported 87.5%, 89.2%, 92.7%, and 85.2%, respectively. The kappa factor between PCR and histopathological methods was 0.76, indicating substantial agreements between these two tests. Due to the acceptable sensitivity and specificity of the present method,   it might be used to diagnose fungal sinusitis infections along with microscopic tech-niques. This method is recommended to confirm the diagnose of suspected fungal sinusitis with negative histopathology results.

K E Y W O R D S
formalin-fixed paraffin-embedded tissue, fungal rhinosinusitis, histopathology, semi-nested PCR, sequencing leicabiosystems.com). The 1000 µL of xylene was added to microtube containing 5 µm of a sample, which was then incubated in 56°C on a heating block for 15 min at room temperature and section subsequently centrifuged at 10,000 × g for 2 min. The supernatant was removed, and 1000 µl of absolute ethanol was added and followed by centrifugation at 10,000 × g for 3 min. The previous stage was repeated three times, and then, the tubes were incubated at 37°C on a heating block until the total evaporation of the ethanol. 23

| Histopathological assay
The FFPE-FESS samples were stained by hematoxylin and eosin (H&E), periodic acid-Schiff (PAS) stains. The staining processes were performed according to the protocols for FFPE sample staining. 24

| DNA extraction
DNA was extracted as previously described. Briley, 100 μl lysis buffer, 180 µl of ATL buffer, and 20 µl of proteinase K were added to the tube samples. After overnight incubation at 56°C, the tubes were washed via normal saline. To complete the lysis process, tubes were heated in boiling water for 5 min. The tubes were incubated in boiling water and liquid nitrogen for 1 and 2 min, respectively. This step was repeated several times. Finally, they reached room temperature.
As previously described, DNA extraction was completed by QIAamp DNA extraction from tissue mini kit (Qiagen, Hilden, Germany). 25 This process is based on the binding of the DNA to silica columns.

| PCR assay
To evaluate the quality of the extracted DNA, human β-globin gene fragments amplificated by the PCR method 26 Amplicon quality and concentrations were estimated on the agarose gel and analyzed by the Gel Doc XR system (Bio-Rad, USA).
The smart Ladder (Eurogentec, Seraing-Belgium) was used as the size and concentration marker.

| Semi-nested PCR assay
The universal fungal ITS region (ITS1-5.8S-ITS2) was targeted for evaluation by semi-nested PCR. The first PCR was performed using ITS1 (forward) and ITS4 (reverse) primers ( Table 2). The total vol-

| Sequencing
To further confirm the results, PCR products were sent to sequencing using referenced primers was performed. The obtained sequences were searched using the NBLAST algorithm (https://blast. ncbi.nlm.nih.gov/Blast.cgi), and the identity of each strain was assigned accordingly.

| Data statistics
Data were analyzed using SPSS software version 24. Briefly, descriptive data were presented as mean, standard deviation, percentages, and charts. The chi-squared and Fisher's exact tests were used to compare qualitative variables between the two groups. The Student t test was used to compare quantitative variables between the two groups. Also, the agreement between the two diagnostic methods was calculated by the Kappa test; interpretation of Kappa was based on Viera et al. 29 The p-value less than 0.05 (p < 0.05) was considered statistically significant.

| Patients and samples
One hundred ten FFPE samples were obtained from 2018 to 2020.
The patient's ages were ranged from 2 to 82 years old (mean age: 40.2 years old), with 50 being male (45.5%).

| Histopathological examinations
Fungal rhinosinusitis was found in 64 of the samples tested. Ribbonlike non-septate or slightly septate hyaline mycelium was found in 64% (41 of 64) of them, suggesting mucormycosis (Figures 1-3

| Molecular assay
As shown in Table 3, 56 of the 64 (87.5%) histologically proven specimens were positive for PCR (as shown in Figure 4)

| DISCUSS ION
Several affect the outcome of the PCR test in FFPE tissue samples.
The DNA extraction method, the inclusion of a suitable housekeeping gene, the PCR method (i.e., panfungal, specific, nested, seminested, multiplex, and real time), the target gene(s) primers, the amplicon length, the thickness of the FFPE cut, the specimen storage time, and contamination during sample preparation are all factors to consider. 30 This study found that a semi-nested PCR test targeting the ITS1-   More specifically, it is difficult to amplify the target gene when DNA is highly fragmented or cross-linked and has a large amplicon size.
Fourth, the presence of amplifiable fungal DNA in tissue does not always imply the presence of a housekeeping gene (human-globin).

F I G U R E 4
Gel electrophoresis primary PCR via β-globin (left) and semi-nested PCR for ITS region (right)