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 disease, which vary in clinical presentation, histologic features, and biological significance. FRS is commonly classified in two categories, i.e., invasive and non-invasive infection. Histopathology is the “gold standard” diagnostic method, but it is not able to determine the genus and species. Almost 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) to confirmation of pathology reports obtained 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 β-globin gene by conventional PCR was used for confirming the quality of extracted DNA. The semi-nested PCR was performed using ITS 1 and ITS4 primers during two steps. In order to identification of causative agents, sequencing of the internal transcribed spacer region (ITS1-5.8S-ITS2) 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 and 8 (12.5%) samples were negative. Out of 46 negative samples by histopathological methods, 41 samples (89.1%) were negative in PCR while 5 (10.9%) were positive. 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. Aspergillus flavus, Rhizopus oryzae, Lichtheimia corymbifera and Candida albicans have identified as common fungal species. Conclusion Based on staining and direct examination that are time-consuming, applying molecular methods might be an appropriate choice for rapid diagnosis and support the consequences of histopathology examinations. We suggest that fresh biopsy specimens elicit more reliable results in comparison with FFPE samples. It is recommended that semi-nested PCR assays be performed in a single tube, which showed less prone to contamination when compared with assays that were carried out in two stages and in separate tubes. Trial registration: Not applicable..
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Preprint| Erscheinungsjahr: |
2022 |
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| Erschienen: |
2022 |
| Enthalten in: |
ResearchSquare.com - (2022) vom: 29. Juli Zur Gesamtaufnahme - year:2022 |
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| Sprache: |
Englisch |
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| Beteiligte Personen: |
Ashraf, Mohammad Javad [VerfasserIn] |
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| doi: |
10.21203/rs.3.rs-558660/v1 |
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| PPN (Katalog-ID): |
XRA033770174 |
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| 520 | |a Abstract Background Fungal rhinosinusitis (FRS) encompasses a various spectrum of disease, which vary in clinical presentation, histologic features, and biological significance. FRS is commonly classified in two categories, i.e., invasive and non-invasive infection. Histopathology is the “gold standard” diagnostic method, but it is not able to determine the genus and species. Almost 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) to confirmation of pathology reports obtained 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 β-globin gene by conventional PCR was used for confirming the quality of extracted DNA. The semi-nested PCR was performed using ITS 1 and ITS4 primers during two steps. In order to identification of causative agents, sequencing of the internal transcribed spacer region (ITS1-5.8S-ITS2) 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 and 8 (12.5%) samples were negative. Out of 46 negative samples by histopathological methods, 41 samples (89.1%) were negative in PCR while 5 (10.9%) were positive. 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. Aspergillus flavus, Rhizopus oryzae, Lichtheimia corymbifera and Candida albicans have identified as common fungal species. Conclusion Based on staining and direct examination that are time-consuming, applying molecular methods might be an appropriate choice for rapid diagnosis and support the consequences of histopathology examinations. We suggest that fresh biopsy specimens elicit more reliable results in comparison with FFPE samples. It is recommended that semi-nested PCR assays be performed in a single tube, which showed less prone to contamination when compared with assays that were carried out in two stages and in separate tubes. Trial registration: Not applicable. | ||
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