Iranian Journal of Medical Microbiology

The present study was conducted at the Department of Microbiology (Mycology section) at Mamata General and Super Specialty Hospital affiliated with Mamata Medical College and Hospital, Khammam, Telangana. The study was conducted for a period of 1 year from June 2021 to June 2022 year after due to ethical committee clearance (IEC/IRB:19/22). This was a retrospective study on samples obtained from patients suspected of fungal rhinosinusitis.
Inclusion and Exclusion Criteria for Study Participants
Deep nasal swabs or tissue received from patients suspected of fungal rhinosinusitis were included in the study. All other clinical samples like bronchoalveolar lavage, sputum, tracheal aspirates, Endotracheal secretions, and blood that were received for infections other than FRS were excluded from the study. COVID-19 infection from the patient records were cross-checked with RTPCR reports and data available from the Molecular laboratory attached to Mamata Medical College.
Clinical records of patients from Outpatient (OPD) / in-patient (IPD) departments and Intensive care unit (ICU) were noted. Patient data and relevant clinical history were recorded. Associated comorbidities like Diabetes mellitus, COVID-19 status, use of steroids, immunosuppression, iron-chelation therapy, neutropenia, solid organ transplant, chronic kidney disease, chronic alcoholism, chronic respiratory disease, stem cell transplantation, and HIV status were noted.
Protocol at the Tertiary Care Center for Diagnosing FRS
Patients were suspected of fungal rhinosinusitis when one of the following symptoms and signs were present: facial pain, postnasal drip, nasal pain/stuffiness, eye pain, worsening headache, nasal discharge, eyelid edema/ facial edema, eyelid/ periorbital/ facial discoloration, proptosis/ ptosis causing changes in visual acuity, sudden loss of vision, restricted eye movement. Relevant investigations like Computerised tomography (CT) scan of the paranasal sinuses (PNS) and Magnetic resonance imaging (MRI) scan were obtained. CT findings suggestive of fungal rhinosinusitis included intralesional hyperdensity or calcification within an opacified sinus/ Bone erosion or expansion/ mucosal thickening in an asymmetric fashion, and invasion of the fat surrounding the maxillary antrum for sinus involvement. MRI scan was done for invasive cases when extension into tissues/ brain/ orbit was suspected.
They were then subjected to diagnostic nasal endoscopy. Indications for collection of deep nasal swabs included inflamed/ crusted nasal mucosa, purulent discharge/ oedematous mucosa, and blackish crusts/ eschar not extending into sinuses during endoscopy itself. Tissue was collected when invasion was noted, which included blackish crusts/ eschar/ septal perforation/ mass (polyp) in the nasal cavity or paranasal sinuses after patient admission.
Mycological Evaluation
To confirm the clinical/ radiological findings of FRS, deep nasal swabs and tissue samples were obtained. Both the samples were then subjected to gram stain, 10% KOH mount, and culture on Sabouraud dextrose agar (SDA). Mucormycosis was identified by the presence of broad, ribbon-like aseptate/pauci-septate hyphae with right-angled branching on KOH mount/Gram stain. Non-Mucorales were identified by the presence of narrow, septate hyphae with acute angled branching. Preliminary reports of KOH mount and gram stain findings were issued and communicated to consultants telephonically. The fungus was further identified by growth on SDA slants kept at 37^⁰C and 25^⁰C by noting colony characteristics on the obverse and reverse for pigmentation. Slide culture was performed for further species identification.
Thus, for this retrospective study, a diagnosed case of FRS was when clinical/ radiological and microbiological (microscopy and culture) findings were met in the case of nasal swab samples, for tissue samples, similar findings as for swabs along with histopathological evidence. The treatment and outcome of the patients were checked by medical records or by direct telephonic conversation.
Data was entered in Microsoft Excel sheet 2019. The data was transformed into IBM SPSS software version 26 and analyzed. The data were represented by means, frequency, and percentages. Fisher's exact test with continuity correction was used to analyze categorical data for smaller values. The association was considered statistically significant if the P-value was <0.05.

 

Of the 41 cases studied, 19.5 % had culture-proven rhino-orbito-cerebral infection by Mucorales (ROCM) and 19.5% cases had non-invasive fungal rhinosinusitis caused by other fungal genera. Youssif et al. (25) reported 8.25% CAM (COVID associated mucormycosis) and Rawson et al. (26) found 8% of patients with secondary fungal infection with COVID-19. Both these studies had less incidence compared to our study (19.5%). This could be due to less sample size in the present study along with the inclusion of COVID negative cases.
Male gender, old age, COVID-19, diabetes, and steroid use had a high risk of developing not only mucormycosis but also other fungal rhinosinusitis which was seen in the present study. A study done by Aranjani JM et al. (9) in 2021 emphasized the significance of the above risk factors like our study. A study done by Shetty S et al. (27) in 2022 on invasive aspergillosis of the nose and paranasal sinuses in COVID-19 convalescents had 8 patients with associated comorbidities like diabetes and male preponderance which was similar to the present study. Facial pain (78.04%), facial edema (58.5%) followed by headache (29.2%) was the most common symptom in the present study which is similar to the study done by Abdollahi A et al. (28) in 2016 in the pre-COVID era. The present study contrasts a study done by Shetty S et al. in 2022 (27) where nasal obstruction and headache was the commonest symptom among patients with Aspergillus infections.
Two types of samples were received in the present study which included deep nasal swabs and biopsy-tissue. Of the total 41 samples received maximum were deep nasal swabs (70.7%) followed by tissue samples (29.3%). In a study by Ahirwar S et al. (20) in 2021 different samples like swabs, sputum, and BAL were received for diagnosing mucormycosis in post-COVID-19 patients in which 22.8% (8/35) were positive for fungal smear which is similar to the present study where CAM was seen in 25.9 % (7 /27) cases. In total, 58.5% of cases in our study were reported as having no fungal growth, this could be because non-invasive fungal rhinosinusitis tends to have low fungal growth as reported by few studies (12, 29).
Rhizopus spp, Aspergillus spp, Rhizopus spp with Aspergillus spp., and Penicillium spp were isolated in the present study from nasal samples (tissue & and nasal swabs) received from both COVID-19 positive and COVID-19 negative patients which can be compared to study done by Nazari T et al. (30) in 2022 which was a systemic review of 169 patients on COVID-19 associated fungal infections (CAFI) where Mucormycosis, Aspergillosis and Fusariosis were diagnosed. As both COVID-19-positive recovered patients and COVID-19-negative patients were included in our study, we analyzed that these fungal infections in the pandemic era in COVID-19-negative cases were due to another most common risk factor like Diabetes mellitus. In a study done by Chaganti PD et al. (31) in 2020, Aspergillus spp was the most common cause of fungal rhinosinusitis in non-COVID-19 patients which was seen in the present study also. Penicillium spp is also one of the known causes of rhinosinusitis which was seen in our study (32).
In the present study, the utility of deep nasal swabs in isolating Mucorales or other fungi concerning gender, COVID-19 status, diabetes mellitus, and steroid use did not contribute to any significant finding but was significantly associated with outcome (P=0.041). Most of the patients who were diagnosed with Mucormycosis by microscopy and culture from deep nasal swabs had bad outcomes (prognosis) compared to patients diagnosed with other fungal infections.
The tissue sample obtained for biopsy is the sample of choice for microbiological diagnosis in mucormycosis as it can be subjected to histopathological examination simultaneously to look for tissue invasion. But this requires patient admission. Although 29.3% of tissue samples were received in our study, 50% (6/12) of them showed growth of Mucorales or other fungi which was better compared to 37.9% (11/29) growth reported from deep nasal swabs. Deep nasal swabs although not recommended as per many literatures and guidelines are still samples of (33)choice in many institutes due to their ease of collection during DNE and adaptability involved (23, 34, 35). In a study done by Gade N et al. (22) in 2022, deep nasal swabs constituted 57% of samples which is less compared to the present study (70.7%).
Mucor /Aspergillus spores are present widely in the environment. They may exist in healthy individuals as commensals by inhabiting nasal mucosa/ paranasal sinuses, due to their low virulence. When such a patient’s immune system is suppressed, they may germinate and spread (6). Fungal growth in culture without microscopic detection of the fungal agent usually indicates false positives as these fungi are known saprophytes (8). In addition to this, swabs collected from non-sterile sites like nasal mucosa can further accentuate diagnostic dilemmas in the absence of microscopic findings due to the non-availability of tissue for histopathology (33). In the present study, 10.3 % of false positives were observed from deep nasal swabs from mycological viewpoint and retrospective analysis of cases. The false negative rate from all nasal samples was 51.2%. A study by Watkinson, J.C et al. (36) in 2018 showed that the sensitivity of culture in fungal ball/AFRS/Acute invasive fungal rhinosinusitis was low indicating false negatives and they concluded that if fungal cultures were negative, the presence of fungi cannot be ruled out which correlates with the present study.
For invasive FRS, a delay in commencing antifungal therapy can significantly increase the mortality of the patients. As radiological investigations may yield non‑specific results, surgical intervention aids in mitigating disease burden, and improves drug bioavailability, limiting it from spreading further. It also has an important role for confirmation of diagnosis as samples can be sent for histopathological and microbiological examination intraoperatively (37). Thorough saline irrigation of sinuses with subsequent follow-up on an OPD basis was done in our study in most cases without the need for antifungal drugs. Extensive debridement with Liposomal Amphotericin B/ Posaconazole was the drug of choice for mucormycosis and Posaconazole/ Isuvaconazole/ Liposomal Amphotericin B along with surgical debridement was the drug of choice for aspergillosis when indicated at our setup (38, 39). The outcome in our patients was poor who were diagnosed with mucormycosis compared to other fungal rhinosinusitis. Management of mucormycosis is often ineffective even after early diagnosis, aggressive medical and surgical management leading to progression of infection and high fatality (6, 40).
This study analyzed nasal samples from both COVID-positive and negative patients, so all suspected cases of fungal rhinosinusitis were taken into consideration. Deep nasal swabs aided in early diagnosis due to rapid microscopy findings along with positive culture results. False positives and false negatives from deep nasal swabs could be identified from this study. As this was a retrospective study with small sample size, the utility of nasal swabs in comparison with tissue could not be done. Relation of fungal isolates with risk factors or outcomes from biopsy-tissue samples could not be attempted again due to the smaller sample size. More studies are thus needed to assess the incidence and prevalence of FRS in COVID or non-COVID patients in this pandemic era along with the utility of deep nasal swabs for diagnosing them.

 

This was one of the studies in which both COVID and non-COVID patients were included to diagnose invasive and non-invasive fungal rhinosinusitis from nasal samples. Risk factor like diabetes mellitus was a major contributor to infections in non-COVID-19 patients. Poor outcome was significantly associated with isolation of mucormycosis obtained from deep nasal swabs. False positives and false negatives could be seen with nasal samples for FRS as the sensitivity of culture is low. In the present study, we observed that deep nasal swabs can be useful samples for expeditious diagnosis of fungal rhinosinusitis on an OPD basis during diagnostic endoscopy itself helping for better patient management.

 

We would like to express our heartfelt thanks to Professor Dr. Siva Subba Rao Pakanati (Head, ENT department) for his guidance.
 

The present study was conducted at the Department of Microbiology (Mycology section) at Mamata General and Super Specialty Hospital affiliated with Mamata Medical College and Hospital, Khammam, Telangana. The study was conducted for a period of 1 year from June 2021 to June 2022 year after due ethical committee clearance (IEC/IRB:19/22).

 

None.

Conflicts of Interest

The authors declared no conflict of interest.