Molecular identification of pathogenic fungi in formalin-fixed and paraffin-embedded tissues

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.

Reports are increasing on the emergence of COVID‐19–associated mucormycosis (CAM) globally, driven particularly by low‐ and middle‐income countries. The recent unprecedented surge of CAM in India has drawn worldwide attention. More than 28,252 mucormycosis cases are counted and India is the first country where mucormycosis has been declared a notifiable disease. However, misconception of management, diagnosing and treating this infection continue to occur. Thus, European Confederation of Medical Mycology (ECMM) and the International Society for Human and Animal Mycology (ISHAM) felt the need to address clinical management of CAM in low‐ and middle‐income countries. This article provides a comprehensive document to help clinicians in managing this infection. Uncontrolled diabetes mellitus and inappropriate (high dose or not indicated) corticosteroid use are the major predisposing factors for this surge. High counts of Mucorales spores in both the indoor and outdoor environments, and the immunosuppressive impact of COVID‐19 patients as well as immunotherapy are possible additional factors. Furthermore, a hyperglycaemic state leads to an increased expression of glucose regulated protein (GRP‐ 78) in endothelial cells that may help the entry of Mucorales into tissues. Rhino‐orbital mucormycosis is the most common presentation followed by pulmonary mucormycosis. Recommendations are focused on the early suspicion of the disease and confirmation of diagnosis. Regarding management, glycaemic control, elimination of corticosteroid therapy, extensive surgical debridement and antifungal therapy are the standards for proper care. Due to limited availability of amphotericin B formulations during the present epidemic, alternative antifungal therapies are also discussed.

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