Introduction
Fungi are a diverse group of saprophytic (i.e. derive nourishment from dead organic matter) and parasitic eukaryotic organisms. Fungal pathogenicity is determined by the host's immune response and virulence factors specific to the fungus. Fungal features that are not present in other microorganisms are a rigid cell wall and the formation of large hyphae, which are frequently present in clinical specimens. Both features enable fungi to resist the host's defence mechanisms. In normal hosts, fungal isolation commonly reflects colonisation rather than infection. However, in patients with an abnormal immune response, a wide spectrum of fungal-related diseases can develop.Reference Shoham and Levitz1
Fungal infection of the ear is most commonly restricted to the external auditory canal.Reference Morgan, Warnock, Gleeson and Jones2 Middle-ear cleft cholesteatoma is a gradually expanding, destructive epithelial lesion. Established cholesteatoma contains keratinous debris, which is probably an ideal medium for saprophytic fungal colonisation.Reference Bottone, Hong and Zhang3
In this prospective study, we performed a fungal analysis of keratinous debris samples obtained during primary mastoid surgery from patients with cholesteatoma presenting to El-Sahel Teaching Hospital, Cairo. We determined the frequency and nature of fungal elements associated with cholesteatoma. A PubMed and Medline search from 1980 until 2013 was performed using the search terms ‘cholesteatoma’ and ‘fungi’. The literature was reviewed to determine the clinical and pathological relevance of fungal colonisation in middle-ear cleft cholesteatoma. As far as we are aware, this is the first prospective mycological study to be specifically performed on cholesteatoma keratin samples obtained during primary mastoid surgery.
Materials and methods
Approval from the local ethics committee and informed consent from the patients or guardians of children participating in the study were obtained. Patients with cholesteatoma who presented consecutively during the 7 months from 30 March 2013 onwards and were operated on at the El-Sahel Teaching Hospital in Cairo were included.
Cholesteatoma was clinically diagnosed by otomicroscopy in patients with chronic otitis media. Microsuction of keratin flakes from the tympanic membrane defect or retraction pocket confirmed the diagnosis. Patients presenting with occluding aural polyps had the polyps excised under general anaesthesia, and keratin was detected in the tympanic membrane defect. Pure tone audiometry was performed before and after mastoid surgery; however, audiometric data will not be reported in this work. All patients diagnosed with cholesteatoma underwent computed tomography imaging of the temporal bone to determine disease extent and the associated bone destruction. All patients with cholesteatoma encountered during the study period had advanced disease, requiring canal wall down mastoidectomy for extirpation of the disease. No cases of congenital cholesteatoma were encountered. Only patients that underwent primary mastoid surgery were included in the study.
During the procedure, keratinous debris was removed from the mastoid antrum using sterile micro-cup forceps, placed in a sterile container and transported promptly to the Medical Microbiology and Immunology Department in the Faculty of Medicine, Cairo University. Initially, a portion of each specimen was examined under high power microscopy after treatment with 10 per cent potassium hydroxide to degrade the proteinaceous components. This enabled the early detection of any fungal elements present in the specimen. The remainder of the specimen was cultured on Sabouraud's dextrose agar medium at 25°C for up to 21 days for fungal growth assessment. Fungal identification was based on colony morphology and pigmentation. Microscopic identification of fungi was performed after lactophenol cotton blue staining. If yeasts were encountered, the germ tube test was used to assay for Candida albicans.
Results
Clinical data for patients in the study group and the results of the mycological analysis are shown in Table I. There were 18 patients with middle-ear cleft cholesteatoma (19 ears were operated on): 13 males and 5 females. Patient age ranged from 9 to 45 years (mean 23 years). The right ear was affected in 10 patients, the left ear in 7 patients, and 1 patient had bilateral disease. Patients most frequently presented with hearing loss and offensive purulent aural discharge. One patient presented with a left-sided Bezold's neck abscess and a right-sided Luc's zygomatic abscess. Another patient presented with a left mastoid subperiosteal abscess, while one patient presented to the neurosurgical service with a cerebellar abscess. All patients presenting with abscesses required drainage of the pus one week before mastoid surgery. Four patients had occluding aural polyps at presentation; polyps were excised permeatally one week before mastoid surgery. Fungal cultures were obtained from cholesteatoma keratin from 17 ear samples (89 per cent; fungal isolates shown in Table I). Five fungal isolates belonged to the dermatophyte group (21 per cent).
Table I Mycological data
Pt no = patient number; yr = years; F = female; M = male; R = right; L = left
Discussion
Middle-ear cleft cholesteatoma is an aggressive form of chronic otitis media that requires surgery for extirpation. Clinically, an infected cholesteatoma is associated with a malodorous ear discharge which adversely affects the patient's quality of life.Reference Wang, Nadol, Merchant, Austin and Gliklich4 The cause of fetor has been attributed to anaerobic bacterial infection.Reference Chole, Sudhoff, Cummings and Harker5 However, anaerobic bacteria have rarely been cultured from mastoid granulations from ears with cholesteatoma.Reference Albert, Job, Kuruvilla, Joseph, Brahmadathan and John6 In a previous study, we postulated that the fetor associated with primary atrophic rhinitis was caused by saprophytic fungal colonisation of the nasal cavities of affected patients.Reference Effat and Madany7 We therefore hypothesised that the fetor associated with infected cholesteatomas might be caused by saprophytic fungal colonisation within the keratinous debris.
In the current study, fungal elements were detected in 89 per cent keratinous debris samples obtained during primary mastoid surgery. In a previous study, fungi were isolated from 25 per cent of discharge samples from ears affected by active chronic suppurative otitis media.Reference Ibekwe, Shareef and Benayam8 In a mycological study on otomycosis, Vennewald and colleagues reported the presence of Aspergillus spp. determined histologically in keratinous debris in four cases of cholesteatoma that required mastoidectomy.Reference Vennewald, Schönlebe and Klemm9 However, to our knowledge, no prospective mycological study has been previously undertaken in cholesteatoma. The external ear canal can harbour various fungal commensals, and fungi have been isolated from unstable post-mastoidectomy cavities.Reference Gluth, Tan, Santa Maria and Atlas10 We chose to investigate fungal colonisation in mastoid antrum keratin obtained during primary mastoid surgery. We propose that the saprophytic fungal colonisation in our patients was a result of germination of fungal spores that entered through the external ear canal. However, an alternative route might have been via the eustachian tube.Reference Murakami, Tutumi and Watanabe11 In the current study, Microsporum canis and Epidermophyton spp. comprised 21 per cent of the total fungal isolates. Both species belong to the dermatophyte group, which uses keratin as its source of nutrition.Reference Bottone, Hong and Zhang3
An established cholesteatoma presents pathologically as a keratin-containing sac surrounded by a keratinising epithelial layer (i.e. matrix) and an adjacent subepithelial connective tissue layer with a mucosal epithelial layer boundary (i.e. peri-matrix).Reference Louw12 Current cholesteatoma studies focus on the peri-matrix–matrix interactions necessary for the progression of this condition. A chronic inflammatory process occurring in the peri-matrix is a cardinal feature in established cholesteatomas that plays a role in disease onset, maintenance, growth and expansion.Reference Louw12 Bacteria, identified by microbiological cultures from diseased granulations or detected as bacterial biofilms by electron microscopy, have been reported in infected cholesteatomas.Reference Albert, Job, Kuruvilla, Joseph, Brahmadathan and John6, Reference Saunders, Murray and Alleman13 Bacterial products are responsible for maintaining an inflammatory response in the peri-matrix region.Reference Louw12
• Cholesteatoma keratin samples were obtained from 19 ears during primary mastoid surgery
• Fungi were isolated from 89 per cent of samples
• Fungal colonisation may be related to the clinical and pathological features of cholesteatoma
In chronic rhinosinusitis, fungal elements have been frequently identified within sinus mucosal bacterial biofilms by fluorescent in situ hybridisation.Reference Healy, Leid, Sanderson and Hunsaker14 Common saprophytic and commensal fungi can promote the release of proinflammatory cytokines through orchestrating the innate and acquired immune responses.Reference Inoue, Matsuwaki, Shin, Ponikau and Kita15 Fungi and bacteria may interact symbiotically as protection against host defences and antimicrobials.Reference Healy, Leid, Sanderson and Hunsaker14 In addition, fungal products were reported to cause epithelial cell desquamation in a cell culture model.Reference Kauffman, Tomee, van de Riet, Timmerman and Borger16 Recently, Louw reviewed the role of microorganism-induced alterations in lipid metabolism in matrix cell apoptosis and cholesteatoma deterioration.Reference Louw17 It is therefore likely that fungal elements associated with cholesteatoma play a significant role in the disease progression.
The major goal of cholesteatoma surgery is to completely remove keratin and matrix debris from the middle-ear cleft to achieve a safe, stable ear.Reference Nyrop and Bonding18 However, fungal diseases have been detected upon operating on a contaminated ear. Invasive fungal disease causing intracranial complications have been described following surgery on an ear with chronic suppurative otitis media.Reference Bhatt, Pahade and Nair19 This flare-up was described as a ‘forest fire phenomenon’, in which the manipulation of previously quiescent fungi leads to activation and invasiveness.Reference Anwar, Enam, Jooma, Shah, Shamim and Siddiqui20 In addition, a non-invasive fungal-related entity was recently reported following cholesteatoma surgery. This condition, termed ‘allergic fungal otomastoiditis’, was attributed to a hypersensitivity reaction to fungal elements in the middle-ear cleft.Reference Chen and Chiang21 With increasing numbers of patients receiving immunosuppressants for a variety of illnesses, including the acquired immunodeficiency syndrome pandemic, otologists should be alert to the possibility of fungal diseases in the differential diagnosis of unusual presentations of middle-ear cleft disorders.Reference Yates, Upile, Axon and de Carpentier22, Reference Kuruvilla, Job, Mathew, Ayyappan and Jacob23
Conclusion
The current study identified fungal isolates in 89 per cent of samples of cholesteatoma keratin obtained during primary mastoid surgery. Fungal colonisation in cholesteatoma probably plays a significant role in disease progression through maintaining an inflammatory response in the peri-matrix region. Moreover, saprophytic fungal colonisation in cholesteatoma keratin may be responsible for the fetor associated with ear discharge. Otologists should consider the possibility of fungal infections associated with cholesteatoma and surgery in a contaminated ear.
