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.

Screening Candida auris through a multiplex stepwise PCR algorithm directly from clinical samples of patients suspected of otomycosis in south of Iran; Detection of five cases

“…Although the rate of otomycosis documented by direct examination, the standard method, was lower in the current study using molecular methods (93.6% vs. 85.5%); nevertheless, a higher percentage of molecularly associated prevalence for otomycosis was achieved in the current study than the result of a previous study conducted in the central region of Iran using the same method (85.5% vs. 81%) underlines the geographical and climatic differences that influence the incidence of the disease, as the current study was conducted in a region with high humidity and temperature 21 . Of note, in our study, 18 ear specimens remained negative by pan fungal PCR while showing fungal elements by direct examination; thus, a discordance of 8.6% between these two methods leading to an underestimation of the otomycosis in this region were noted, which is inevitably expected to happen in any PCR assay because of DNA degradation or presence of PCR inhibitor remaining from DNA extraction of clinical specimens 29 …”

Accompanying a semi‐nested PCR assay to support histopathology findings of fungal keratitis in formalin‐fixed paraffin‐embedded corneal samples

“…Moreover, we reached a Kappa factor of 0.77 between semi‐nested PCR and histopathology assays, indicating substantial agreement between these two tests in the previous study. 37 …”

Molecular Diagnostics for Invasive Fungal Diseases: Current and Future Approaches

“…When performed on FFPE tissue with features of fungal rhinosinusitis, panfungal PCR has a sensitivity of up to 87.5% with a corresponding specificity, positive predictive value (PPV) and negative predictive value (NPV) of 89.2%, 92.7% and 85.2% respectively [31]. A cost analysis study in Australia reviewed 20 months of FFPE panfungal requests.…”