Histopathological spectrum of fungal rhinosinusitis in a tertiary care center of Western India

INTRODUCTION

Fungal infections have recently emerged as an important cause of acute and chronic rhinosinusitis. Nearly 6–12% of cases of chronic rhinosinusitis have culture or histologically confirmed fungal etiology.^[1] Based on the morphological pattern of tissue injury, fungal rhinosinusitis (FRS) is broadly categorized into invasive and non-invasive forms, which are mostly dependent on the immunologic status of the patient. It is important to identify and accurately characterize FRS because management of FRS varies with the categories of FRS. Invasive FRS, both acute and chronic forms, requires aggressive surgery and antifungal treatment. In contrast, non-invasive FRS, such as fungal ball and allergic FRS (AFRS), is treated by surgery and steroids, respectively.^[2]

Variable frequency of FRS is reported from different geographical regions across the globe. High incidence of FRS has also been described in India, a tropical country with a mostly hot and humid climate. Initial reports were described from the Northern part of the country.^[3] Subsequently, studies reported FRS across different parts of the country.^[4-6] The Western part of Rajasthan has a hot arid climate, and no large studies are available in this part of the country reporting the implication of fungal infection in the causation of chronic rhinosinusitis. Our Institute is a tertiary care center in the western part of Rajasthan that routinely deals with sinonasal biopsies. Hence, we aimed to define the histopathological spectrum of FRS in this area.

MATERIALS AND METHODS

All cases of acute and chronic rhinosinusitis from January 2015 to December 2020 were reviewed. Cases of histologically proven FRS were analyzed. These specimens were received either as biopsies or debrided specimens from paranasal sinuses. Clinical details were collected from the histopathology requisition form. Data collected included age, gender, predisposing risk factors, and other relevant clinical details. Microbiological culture reports were collected whenever available.

The histological slides of all these cases were reviewed with routine hematoxylin and eosin stain, periodic acid–Schiff (PAS), and Gomori’s methenamine silver (GMS) stains. These cases were subcategorized based on the presence of mycelial elements, allergic mucin, and the type of tissue response as per the criteria given by Das et al.^[3] The morphology of the fungus was assessed on histological sections. Thin, slender fungal hyphae with acute-angled branching and septation were designated as Aspergillus spp., while broad fungal hyphae with aseptation and irregular branching were diagnosed as Mucor spp. Candida species were identified by budding yeast and pseudohyphae. The study was approved by the Institute’s Ethics Committee.

RESULTS

Of 873 cases of acute and chronic rhinosinusitis, 10.3% (90/873) were of fungal etiology. The mean age of patients was 39 years (range 1–80 years) with a male: female ratio of 1.5:1. Clinical diagnosis of fungal sinusitis or fungal granuloma was suspected in 56.6% (51/90) cases. In 25.5% cases, the clinical impression was bilateral nasal polyposis, inflammatory polyps, and mass lesions, whereas 17.7% of cases had no clinical possibilities mentioned. The histopathological examination broadly categorized the FRS into two categories: non-invasive fungal RS (52.2%, 47 cases) and invasive fungal RS (47.8%, 43 cases). Non-invasive FRS included 43.3% (39/90) cases of AFRS and 8.9% (8/90) cases of fungal ball. While invasive FRS included 33.3% (30/90) cases of acute invasive FRS (AIFRS), 10% (9/90) cases of chronic invasive granulomatous FRS, and 2.2% (2/90) cases of chronic invasive FRS. Mixed pattern of fungal rhinosinusitis was observed in 2.2% (2/90) cases. Comorbidities were present in 33.3% (30/90) of cases. Diabetes mellitus (DM) was the most common and observed in 25.5% (23/90) of cases. Details of different forms of FRS are summarized in Table 1.

AFRS was the most common FRS in the current study. The most common presentation was bilateral sinonasal polyposis noted in 18 cases, while right side involvement was noted in 3 and left side in one, and unspecified in the remaining. Grossly, the excised polypoidal tissue showed mucoid, rubber-like consistency with brown–black color. Microscopic examination showed the presence of allergic mucin with variable staining and contained enmeshed eosinophils, sloughed degenerated epithelial cells, cellular debris, and Charcot–Leyden crystals [Figure 1a and b]. Calcification and cholesterol clefts were noted in two and five cases, respectively. A total of 30 cases showed the presence of occasional septate fungal hyphae within the mucin. No invasion of the nasal mucosa is noted. The nasal mucosa was edematous and showed a mild mixed inflammatory infiltrate. Eosinophils were predominant inflammatory cells in 13 cases. Of the 15 cases with available culture reports, five cases showed growth of Aspergillus flavus, and one had Scedosporium spp. Seven cases had no growth, while the remaining two cases showed contamination with candidal overgrowth.

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Figure 1:

(a-b) Allergic fungal rhinosinusitis. Allergic mucin (yellow arrow) with enmeshed inflammatory cells (green arrow). Periodic acid-Schiff (PAS) stain highlights the occasional septate hyphae (red arrow) in allergic mucin. (c) Fungal ball. Entangled fungal hyphae, which at periphery show septate hyphae with acute angled branching (blue arrow). (a and c)- Hematoxylin and eosin stain, (b)- PAS stain, (a) x40, (b) x200, (c) x200.

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A fungal ball was identified in 8 cases. The maxillary sinus was involved in three cases, while in five cases, the site was not specifically specified. Grossly, the excised tissue has a gritty to friable texture and is brownish yellow. Histopathology revealed very tightly packed, entangled fungal hyphae; the morphology of individual hyphae was more appreciable in the periphery [Figure 1c]. In all cases, the hyphae were thin, septate with acute angled branching, suggesting possibly Aspergillus spp. Calcification was observed in four cases. Potassium hydroxide (KOH) mount was performed in 4 cases, three of which showed septate fungal hyphae, whereas only one case had culture growth of Aspergillus glaucus.

AIFRS was the most common invasive FRS. The most common clinical features observed were facial pain, swelling, and foul smell. At the time of biopsy, rhino-orbital-cerebral involvement was present in four cases and rhino-orbital in two cases, while two cases had nasal-oral fistula. Grossly, surgically debrided tissues were mostly necrotic. Histopathological examination showed extensive coagulative necrosis of the nasal mucosa and surrounding soft tissue with relatively scant inflammation. In these infarcted areas, scattered broad aseptate fungal hyphae with irregular folding were noted, morphologically resembling mucor [Figure 2a and b]. Bone destruction was observed in 20 cases and angioinvasion in 16 cases. KOH mount revealed the presence of aseptate hyphae in 15 cases, while culture growth of Rhizopus spp. was noted in three cases out of 18 cases submitted for culture.

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Figure 2:

(a). Acute invasive fungal rhinosinusitis (FRS)- low power view depicting an infarcted area with ghost like outline of mucosa (orange arrow). The right side of the image shows viable, edematous, and inflamed mucosa (green arrow). The inset shows aseptate broad fungal hyphae. (b) Gomori’s methenamine silver (GMS) stain shows aseptate broad hyphae resembling those of mucor (red arrows). (c). Chronic granulomatous FRS Low power view showing granulomatous inflammation, fibrosis and dense inflammation (black encircled area). (d) Periodic acid Schiff (PAS) stain shows septate fungal hyphae within the cytoplasm of giant cells (yellow arrow). (a and c) Hematoxylin and Eosin stain, (b) GMS stain, (d) PAS stain, (a and c) x20, (b) x400, (d) x200.

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Chronic invasive granulomatous FRS was observed in nine cases. Headache, proptosis, and mass were the common complaints. Cerebral and orbital involvement was noted in four cases, with cranial nerve palsy in one of them. Histopathology revealed granuloma formation in the mucosa with dense fibrosis and mixed inflammation in the surrounding tissue. Fungal hyphae of slender septate morphology were noted inside the multinucleated giant cells [Figure 2c and d]. A KOH mount was performed in 7 cases, of which five showed hyaline septate fungal hyphae, while no fungal hyphae were noted in 2 cases. Culture was performed in 4 cases, two of which showed A. flavus and two showed Aspergillus fumigatus.

Two cases of chronic invasive FRS were noted, one involving the right nasal cavity and the other involving the left maxilla. Both cases showed dense neutrophil-rich inflammation surrounding fungal hyphae, foci of necrotic tissue, and occasional giant cells. The hyphae were septate with acute-angled branching. However, in both cases, KOH mount and culture were not available.

Mixed FRS (invasive and non-invasive) was noted in two cases. One patient was a 56-year-old diabetic female and showed both invasive (acute fulminant FRS) and noninvasive (AFRS and fungal ball). The second case was a 30-year-old human immunodeficiency virus-positive male, who had both AFRS and chronic invasive granulomatous FRS. The former case showed the presence of both septate and aseptate hyaline fungal hyphae, whereas the latter case showed the presence of septate hyphae with growth of A. flavus on culture. Further, the data from the current study were compared to that in other parts of the country [Table 2].

DISCUSSION

FRS consists of a spectrum of sinonasal disease processes that vary in terms of clinical presentation, histopathologic appearances, and treatment modalities. The clinical course of FRS ranges from an acute, aggressive form to a subacute to chronic indolent course. On morphologic grounds, FRS is categorized as invasive or non-invasive depending on whether tissue invasion is present or not. Although FRS has been described in the literature for decades, the terminology has been better defined recently.^[2] FRS is described from various parts of the country with variable frequency.^[3-13] In one of the largest series from North India, FRS represented 42.7% of all cases of rhinosinusitis.^[3] Another study described 47 FRS cases, which consisted of 12% of all chronic rhinosinusitis.^[7] A South Indian study reported a frequency of 30% of FRS among chronic rhinosinusitis.^[6] Our study had a 10.3% prevalence of FRS.

In the present study, AFRS is the most common form of FRS and constitutes 43.3% of all FRS cases. AFRS is reported to be the most common form of FRS in several studies across the globe. It is seen more frequently in hot, humid areas such as in India, the Middle East, and the southern and southeastern United States.^[3-7,14,15] Apart from the environmental factors, a high awareness of the entity among clinicians, pathologists, and microbiologists may also have contributed. Patients with AFRS often show atopic features with chronic recalcitrant sinusitis and bilateral sinonasal polyposis. Elevated immunoglobulin E levels, peripheral blood eosinophilia, and allergic rhinitis are common features, which suggest a possible allergic hypersensitivity reaction to the noninvasive fungi present within the nasal cavity, similar to allergic bronchopulmonary aspergillosis.^[15] Sometimes, it is challenging to identify fungal hyphae in allergic mucin, which is also described in other studies.^[3-5] In our study, we were able to identify fungal hyphae in (76.9) % of cases. Hematoxylin and eosin-stained sections are more convenient to identify the hyphae, as these hyphae are often overlooked on PAS stain due to the strong staining of allergic mucin on PAS. In contrast to the Western literature, where melanized fungi such as Exserohilum roibatum, Bipolaris spicifera, and Curvularis lunata are implicated in the causation of AFRS, studies from India, including our study, found Aspergillus species as the most common etiologic agent.^[3,4,16-18] This points toward geographical variance in fungal species. In our study, the culture growth of the fungus was noted only in 26.6% cases. Low culture positivity may be attributed to impacted fungi in mucin, which causes these hyphae to form poor contact with culture media. A fungal ball is another commonly encountered non-invasive FRS. In studies from India, the frequency varies from as low as 1.6% to as high as 53%.^[3-6,8-13] One recent study from South India reported a high frequency (29.7%) of fungal ball; however, the author reported positive culture findings in only 12% cases of these cases.^[13] Making a diagnosis of a fungal ball is relatively easy on histopathology because of the numerous fungal hyphae. However, sometimes, at low power examination, these may be mistaken for necrotic debris or mucin. High-power examination and special stains such as PAS and GMS can easily identify these fungal hyphae.

In the current study, nearly half of the patients (47.7%) were diagnosed with invasive fungal sinusitis. The reported frequency in our study is almost similar to the recent study by Soundarya et al. The authors reported AIFRS as the most common spectrum of FRS and accounted for 46.4% cases in their study cohort of 85 cases.^[13] Nearly 70% of all invasive FRS cases were caused by AIFRS, which is the most severe form of FRS and results in significant morbidity and mortality.^[19] Most cases of AIFRS are noted in immunosuppressed individuals, and to some extent, this high frequency of AIFRS may be contributed to by the rising trend of diabetes.^[20] In our study, DM was the most common predisposing factor of AIFRS, and all these cases had poorly controlled diabetes at the time of diagnosis. Several Indian studies have highlighted DM as the most common predisposing factor for AIFRS.^[5] The most common fungal organism responsible for AIFRS belongs to the Zygomycetes group, among which Rhizopus arrhizus is mostly implicated.^[5,20] Apart from Zygomycetes spp., Aspergillus spp. have also been described as the causative agent for AIFRS.^[2] In our study, culture revealed Rhizopus spp. in three cases and Aspergillus fumigatus in two cases. Histopathology revealed extensive destruction of sinonasal mucosa with infarct-type necrosis, often causing underlying bone destruction, similar to the finding described by other authors.^[3-6] Whenever such a tissue reaction pattern is encountered on histology, a careful search for fungi should be performed, and appropriate fungal stains should be used.

Chronic invasive forms of FRS, including both granulomatous and non-granulomatous, follow a chronic course.^[2] In the current study, only two cases of non-granulomatous chronic FRS were identified; both cases showed tissue infiltration by septate fungal hyphae surrounded by mixed inflammatory infiltrate and occasional giant cells. No risk factor was identified in either of these cases. Unlike AIFRS, predisposing factors are not very evident in such cases. It was pointed out by Michael et al., who suggested that malnutrition may be an important factor in such patients.^[4] The granulomatous form of chronic FRS is described in both immunocompetent and immunocompromised individuals, and on histopathology shows a granulomatous response with dense fibrosis and scattered fungal hyphae mostly contained by multinucleated giant cells.^[2] In the current study, 10% cases of FRS have this morphology, and four cases had a predisposing factor. In several Indian studies, the incidence of chronic granulomatous FRS varies widely from no cases to as high as 30%.^[3-6,8-13] Most cases of chronic granulomatous FRS are described in patients from Sudan, Pakistan, India, and Africa.^[21,22] Aspergillus spp. is the most commonly cultured causative organism. In the current study, all four cases submitted for culture showed growth of Aspergillus spp. Chakrabarti et al. pointed out that A. flavus is the sole and possibly unique etiologic agent for causing granulomatous response.^[23] Both forms of chronic FRS require surgical management and treatment with antifungal medications. However, the non-granulomatous form of chronic FRS should be managed more aggressively, almost like AIFRS, with radical surgery given a worse outcome. In the present study, two cases of mixed (invasive and non-invasive) forms of FRS were identified. Das et al. reported 12 cases of mixed FRS in their cohort of 284 cases of all FRS cases. The authors suggested that FRS may be a progressive disease where the invasive form may arise from non-invasive FRS.^[3]

The main limitation of the current study is that most cases do not have culture confirmation. The primary basis for the diagnosis was morphology, which has its limits; in particular, septate molds cannot be distinguished from dematiaceous fungi solely based on morphology. However, even if fungal culture is not available, tissue reaction pattern and morphology of the fungus can offer crucial diagnostic clues.

CONCLUSIONS

FRS is a common cause of acute and chronic rhinosinusitis and should always be considered when interpreting any paranasal sinus biopsies or resections. Histopathology plays a critical role in accurately classifying the different forms of FRS. Due to the high prevalence of diabetes in India, patients may arrive with invasive sinus disease and require intensive treatment. Understanding histological characteristics is essential for accurate sub-classification and suitable treatment.