Fungal Profile in Non-Invasive Fungal Sinusitis Using PCR in Post-Covid Scenario at a Tertiary-Care Centre in India

Abstract

Objective: To detect the presence of Mucorales in the sinuses of healthy individuals and non-invasive fungal sinusitis patients. Materials and methods: Post FESS specimens with appearance suggestive of fungal ball or allergic mucin from 30 immunocompetent patients was sent for KOH smear, HPE, fungal culture and PCR. Results: Fungal culture was positive for Aspergillus flavus in one specimen. PCR demonstrated Aspergillus (21), Candida (14), and Rhizopus in one case. HPE detected mainly Aspergillus in 13 specimens. No fungal presence in four cases. Discussion: There was no significant undetected Mucor colonisation. PCR proved to be the most sensitive test in reliably detecting the organisms. No significant variation in the pattern of fungi were detected between COVID-19 infected and non-infected, but for the slightly higher detection of candida in the COVID-19 infected group. Conclusion: There was no significant presence of Mucorales in non-invasive fungal sinusitis patients in our study.

Introduction

The term “mucormycosis” was coined by R.D. Baker in 1957 [1]. The infection caused by eukaryotic fungi belonging to Mucorales Order are collectively called Mucormycosis [2]. India undoubtedly had the highest prevalence of mucormycosis in the world even before Coronavirus disease 2019 (COVID-19) pandemic [3]. Poor socioeconomic status, air contamination, nosocomial infections, high prevalence of diabetes in community, etc. were all put forward as reasons for this [4–7] The drastic increase in incidence of mucormycosis during the COVID-19 pandemic was totally unforeseen even by the experts.

Cutaneous mucormycosis in immunocompetent hosts being described following trauma, contaminated injections, etc. are common in the Indian subcontinent [8]. There were a few case reports of mucor fungal balls and aggressive mucormycosis in healthy hosts published even before the pandemic [9–11]. Interestingly, an increasing number of case reports on immunocompetent patients with COVID-19 infection suffering from mucormycosis were being published from credible sources [11–13]. COVID-19 induced immunosuppression was stated as the probable cause for this notable occurrence.

The possible source of Mucorales infection is ambiguous in immunocompetent individuals. There is the logical possibility of a significant presence of Mucorales in the sinuses of healthy individuals and also in non-invasive fungal sinusitis patients. Such a presence of undetected mucor colonisation is significant as these patients can become Immunocompromised or may be exposed to a similar pandemic in future which makes them vulnerable to mucormycosis. For this reason, we decided to undertake this study to perform a detailed evaluation of specimens from immunocompetent patients undergoing functional endoscopic sinus surgery (FESS) using potassium hydroxide (KOH) smear, fungal culture, histopathological examination (HPE) and polymerase chain reaction (PCR).

Materials and methods

The study was performed in Department of Otorhinolaryngology (ENT), Government Medical College, Kottayam, Kerala, India. Ethical clearance was obtained from Institutional Review Board (IRB) and funding secured from State Board for Medical Research (SBMR). Study was tentatively proposed to accurately ascertain the apparent prevalence of Mucorales in paranasal sinus specimens of immunocompetent patients with non-invasive fungal sinusitis voluntarily undergoing FESS in our institution.

We carefully collected specimens from 30 patients who underwent FESS for suspected non-invasive fungal sinusitis (clinical or radiological) in our hospital during the seven months extending from March to October, 2022. Only immunocompetent patients were selected. Susceptible individuals with history of diabetes, cancer, chemotherapy, radiotherapy, steroid use, HIV infection, prolonged antibiotic intake and any known immunodeficiency were excluded from the study. There were 12 patients with documented history of COVID-19 infection. All of them had only mild symptoms and there was no apparent necessity for hospital admission, corticosteroids or humidified oxygen.

Consent was obtained from the patient, with one bystander as witness, for both the procedure as well as the extensive evaluation of the sample. During the elective surgery if any material with appearance suggestive of fungal ball or allergic mucin was seen then the specimen was carefully collected and promptly sent for KOH smear, HPE, fungal culture and PCR. Chosen samples were collected in saline for KOH smear, fungal culture and PCR. For HPE, specimens were promptly sent in 10% buffered formalin. As part of infection control protocol operation theatres (operating tables, microscope lens, instruments, overhead light, air conditioning, etc.) were routinely monitored for aerobic, anaerobic and fungal organisms which helped to rule out contamination of samples.

For KOH smear, 10% KOH wet mount was carefully prepared and the slide was observed under microscope (low and high power) to detect the visible presence of fungal hyphae. For fungal culture the specimens were inoculated into two tubes of Sabouraud Dextrose Agar (SDA) with cycloheximide and two tubes of SDA without cycloheximide. One tube each from the two groups was incubated at 37^oC for a period of three weeks. The other two tubes at 22^oC for three weeks. Zygomycetes routinely requires 2–4 days to grow. Colony morphology, topography and pigment production remain the basis for identification. Tease mount and slide culture were used for species identification.

PCR was performed at Xcyton diagnostics private limited, Peenya, Bengaluru, Karnataka. Specimens for PCR were given a code number and no personal details were shared with the laboratory as per IRB guidelines. Nucleic acid extraction, amplification and detection were performed as per standard protocols. To monitor the accuracy of amplification and subsequent hybridization reactions, three controls, positive, negative and test, were used for each batch. Each sample was analysed for the presence of Candida, Aspergillus, Cryptococcus and Mucorales (Fig. 1). Using a multiplex of five sets of primers including ß-globin as an internal control. Positive Control DNA used in all the tests were procured from Microbiologics Inc, St Cloud, Minnesota, USA, Microbial Type Culture Collection (MTCC), Chandigarh, India, and National Collection of Industrial Microorganisms (NCIM), Pune, India. A total of 21 American type culture collections (ATCC) Fungal Cultures (eight Candida species, two Cryptococcus species, four Aspergillus species and seven Mucorale organisms) was employed.

Fig. 1.

Template of syndrome evaluation system from Xcyton diagnostics private limited technology: multiplex nucleic acid amplification of virulent specific signature genes followed by sequence specific hybridization

For HPE specimens were sent in 10% buffered formalin. Stages of fixation, dehydration, clearing and impregnation as per standard protocols were carried out. Tissues were cut into thin sections with microtome followed by attachment to surface of slides and then staining (haematoxylin and eosin) was carried out (Fig. 2). Grocott – Gomori’s methenamine silver (GMS) stain was used for identification of fungus as it imparts a black colour to fungus and a green shade to the background (Fig. 3).

Fig. 2.

Hpe of fungal ball showing necrotic debris and narrow septate acute angle branching fungi consistent with aspergillus. (haematoxylin and eosin stain)Labomed LX 500 microscope (400x magnification)

Fig. 3.

Hpe slide with gms stain showing fungal hyphae in non-invasive fungal sinusitis Labomed LX 500 Microscope (400x magnification) scale: 100 μM

Results

A total of 30 patients (13 males and 17 females) was selected for the study. Age of participants ranged from 5.5 to 73 years (Mean: 45.35; median:46.5). In case of male patients, it was from 23 to 73 years (Mean:45.31; median:48) and among females 5.5 to 65 years (Mean: 45.38; median: 45). Twenty-three patients were undergoing FESS for the first time. Five patients underwent first revision surgery, and two had their second revision surgery. Documented COVID-19 infection was present in 12 (six males and six females) and two of the patients suffered infection twice with the time gap between infections exceeding 12 months.

KOH smear reliably detected septate hyphae only in one case. Fungal culture was positive in one specimen which showed a growth of Aspergillus flavus. But KOH smear did not detect any fungal elements in this specimen. PCR was positive for Aspergillus in 21 specimens, Candida in 14 cases and Mucorales (Rhizopus) in one case. HPE detected fungal presence in 13 specimens, majority of which was Aspergillus. Four specimens were negative for fungal presence in all investigations. (Chart 1).

Chart 1.

Fungal presence based on pcr analysis of 30 samples asp - aspergillus asp & cnd - aspergillus and candida cnd - candida asp, cnd & mcr - aspergillus, candida and mucorales nofd - no fungi detected

When fungi were considered separately, it was recorded that HPE reported presence of Aspergillus in nine cases and PCR in 21 cases (Chart 2). In 12 cases it was the only fungus detected in PCR. There were no cases with negative PCR and certain identification of Aspergillus in HPE. Tissue invasion was described in two specimens. Both cases on follow up (clinical examination and diagnostic endoscopy) did not show any features suggestive of invasive fungal sinusitis. Actinomycosis was also detected in one case along with Aspergillus in HPE. Based on this limited number, it was estimated that HPE had only a 60% accuracy when compared to PCR (more sensitive tests) in case of Aspergillus.

Chart 2.

Comparison of number of cases of aspergillus and candida detected by pcr and hpe

There were two specific instances of Mucormycosis reported in HPE but PCR failed to detect Mucorales in both these cases. In one case mucormycosis was stated as differential diagnosis and Aspergillus with degeneration as another likely possibility. Aspergillus was positive in PCR for this particular case. Patients on follow up didn’t exhibit any clinical features suggestive of mucormycosis. PCR detected Mucorales (Rhizopus) in one specimen along with Candida and Aspergillus but HPE reported only Aspergillus. Here again patient had no abnormality on follow up.

Presence of Candida was reported only in PCR (Chart 3). KOH smear, fungal culture and HPE were negative for Candida. A total of 14 specimens was PCR positive for candida. Of these eight were positive for Aspergillus also. One selected sample was positive for all three fungi. Remaining five cases had only Candida.

Chart 3.

Comparison of fungal distribution (as percentage of total cases in each group) based on pcr between patients with and without history of COVID-19 infection

Hyphae was detected in one HPE report but fungus was unidentified and the corresponding PCR showed presence of Aspergillus and Candida. Fungal exudate with inflammation and Splendore Hoeppli material was reported in one specimen that tested positive for Candida in PCR. Inverted papilloma was reported in HPE for two cases. In these specimens PCR detected Aspergillus in one and Candida in the other. Four cases that were negative for fungus in all investigations had mucinous material, necrotic bone, chronic sinusitis with inflammatory polyp and focal squamous metaplasia, etc. being reported. All these cases are on follow-up at regular intervals.

In 10 patients out of the total 12 with documented COVID-19 infection, fungal presence was detected with PCR. Three (30%) were positive for Aspergillus and Candida, three (30%) positive only for Aspergillus and four (40%) only for Candida. Mucorales were undetected in this group. Out of the 18 cases without documented COVID-19 infection, 16 were positive for fungus. Here only one (6.25%) case was positive for Rhizopus along with Candida and Aspergillus. Five cases were positive for Aspergillus and Candida, one (6.25%) case had only candida and the rest for Aspergillus alone (Chart 3).

Discussion

Severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) is an infection caused by COVID-19 which is an enveloped positive-stranded RNA virus [14]. Initial cases were reported from Wuhan, China, in December 2019 [15]. In India, the first case was reported in Kerala on January 30, 2020 [16]. The health infrastructure in India was hard-pressed during the pandemic which undoubtedly affected both the patients and the healthcare providers alike [17]. In this background the sudden increase in incidence of mucormycosis further worsened the desperate situation.

This was popularly attributed to the high prevalence of diabetes and other Immunocompromised states in community coupled with the universal presence of the fungi in the environment [18]. Extensive use of steroids, antibiotics, were also projected as alleged causes for the remarkable rise in incidence after active COVID-19 infection [19, 20]. Immunosuppression due to COVID-19 was attributed to decrease in T-cell counts (CD4 + and CD8+) [21]. Abnormalities in iron metabolism, endothelialitis, increase in glucose related protein 78 kDa (GRP78) protein, etc. stand the other proposed reasons for mucormycosis [22–24].

Fungi in the order Mucorales are classified into 55 genera with 261 species [25]. Among these Rhizopus, Rhizomucor, Cunninghamella, Lichtheimia and Apophysomyces are typically known to cause majority of mucormycosis reported in India [26]. Mucorales, in general, are thermotolerant, eukaryotic and saprophytic. Mucormycosis is classified based on anatomical location into rhino-orbital-cerebral, pulmonary, gastrointestinal, cutaneous, renal and disseminated types [26]. The COVID-19 pandemic led to a surge in rhino-orbital and rhino-orbital-cerebral variety [20]. In addition, the fatality rate of this debilitating disease was scientifically documented at 90% in cases with intracranial involvement [27].

HPE is the most depended upon in diagnosis of fungal sinusitis as it is cost-effective and available in almost all institutions. GMS and periodic acid–Schiff (PAS) are the commonly used special stains for fungus [28]. Invasive fungal sinusitis is exclusively reported in HPE as it convincingly demonstrates angioinvasion [29]. But the significant drawback is the apparent lack of sensitivity in reliably detecting the fungi. Scanty fungi in the specimen, possible loss of fungal material during processing and inter observer variability are the few factors mentioned as apparent reasons for this drawback [30]. In our study there were 21 specimens positive for Aspergillus in PCR and only 12 in HPE. There were two specific cases with mucor in HPE that tested positive for Aspergillus in PCR. Likely explanation remains the practical difficulty in correctly identifying the acute angle septate hyphae of Aspergillus. Candida was not reported in HPE most likely due to a lack of sufficient concentration of fungal elements.

There were a few case reports of mucor fungal balls published in peer-reviewed literature before the pandemic [9]. Majority of these cases did not show growth on culture. So, the practical difficulty in identifying the septations might have given an incorrect diagnosis [30]. In our study there were reports of tissue invasion in HPE. But all of these patients on follow-up did not demonstrate any features suggestive of invasive fungal sinusitis. There is a possibility that biopsy method, staining artefacts, tissue reaction due to infection, etc. led to an erroneous interpretation of tissue [31].

The study by Ling Ma et al. in the Chinese population using Mucin 5B, Oligomeric Mucus/Gel-Forming (MUC5B) immunohistochemical staining demonstrated mucor presence in a significant number of fungal balls when compared to the routine GMS staining [32]. They came to the conclusion that majority fungal balls had a combination of Aspergillus and Mucor. Furthermore, the study by Ying-Shi Piao et al. demonstrated only a 4% positivity for Mucorales in paraffin-embedded samples with MUC5B which was way lower than that for Aspergillus and Candida which helps in differentiating these fungi clearly using MUC5B staining [33]. But in our study, we did not see such a substantial number of undetected Mucor colonisation even in post-covid patients. Only one sample out of the 26 (3.85%) with fungal presence in PCR had Mucorales.

KOH is used to clear cellular material in clinical specimens and for faster visualisation of fungal elements. But the major drawback is the artefacts in the background [34]. In our study only one KOH smear reported septate hyphae. A reduced concentration of fungal material in the sample studied could have constituted the reason for this result. Fungal culture yielded result only in one case and the fungi identified was Aspergillus flavus. Slow growth of fungus, inhibitory agents for bacteria in media affecting the fungi, scanty fungal density in the sample, sample processing methods, etc. may have contributed to this poor result [35].

PCR proved to be the most sensitive test in our study as far as reliably detecting the organisms. Though it is expensive, the substantial advantage of PCR is prompt detection of fungus especially in mucormycosis where timely intervention improves the patient outcome. But a positive PCR result without a growth in culture can involve contamination rather than actual infection [36]. In addition, PCR detects the local presence of fungal DNA only and it is impossible to comment on the viability of the organism. The need for standardisation and rigorous controls required are also considered drawbacks in the routine use of PCR [37].

Finally, we were unable to demonstrate a significant variation in the pattern of fungi detected between COVID-19 infected and non-infected individuals except for the slightly higher detection of candida in the COVID-19 positive group. In the COVID-19 positive group 70% of samples with fungus had candida presence compared to 38.5% in the COVID-19 negative group. There was no evidence to suggest that COVID-19 immunosuppression led to a significant dysbiosis in the paranasal sinuses of immunocompetent individuals. Based on this limited number of specimens studied the authors are of the opinion that COVID-19 immunosuppression by itself may not be responsible for the invasive mucormycosis in healthy individuals. Moreover, we were unable to demonstrate a reservoir of Mucorales in healthy individuals with non-invasive fungal sinusitis in our study.

Our study had a few limitations. Sample size was limited to 30 due to financial constraints. A larger sample size may report a more significant number of Mucorales in non-invasive fungal sinusitis. Lack of immunohistochemical staining methods like MUC5B in HPE which could have improved the sensitivity in detecting the fungus. In the author’s opinion expression of GRP78 in nasal epithelial cells of affected healthy individuals and the presence of virulence factor spore coat protein (CotH3) in the mucor samples received are to be studied to comprehend why only a few immunocompetent patients are affected by mucormycosis. Species identification of each fungus is also a necessity. All these limitations can be overcome with future studies with adequate funding and availability of required expertise.

Conclusion

In conclusion, there was no significant presence of Mucorales in non-invasive fungal sinusitis patients in our study. Routine use of PCR and other molecular diagnostic methods are recommended as an indispensable adjunct to HPE especially in cases or studies where an increased presence of Mucorales is detected. Use of antifungals and other interventions needed to be decided upon only after all investigative modalities. Lastly, there was no significant dysbiosis in healthy individuals due to COVID-19 infection evident in our study.

Abbreviations

COVID-19

Coronavirus disease 2019

FESS

Functional Endoscopic Sinus Surgery

KOH

Potassium hydroxide

HPE

Histo Pathological Examination

PCR

Polymerase Chain Reaction

IRB

Institutional Review Board

SBMR

State Board for Medical Research

SDA

Sabouraud Dextrose Agar

MTCC

Microbial Type Culture Collection

NCIM

National Collection of Industrial Microorganisms

ATCC

American Type Culture Collection

GMS

Grocott-Gomori’s Methenamine Silver

SARS-CoV-2

Severe acute respiratory syndrome corona virus 2

GRP78 protein

Glucose related protein 78 kDa

MUC5B

Mucin 5B, Oligomeric Mucus/Gel-Forming

CotH3

Spore coat protein

Funding

The study was funded by a grant from the State Board of Medical Research, under the Medical Education Services of the Health and Family Welfare Department, Government of Kerala, India(Order No. A/01/R1/2022–2023/GMCK-SBMR-IRC dated 28/03/2023). There are no competing financial interests regarding this study.

Declarations

The authors declare no potential conflicts of interests with respect to the research, authorship and/or publication of this article.