OtolaryngologyHead and Neck Surgery (2005) 133, 27-31

Prevalence of Allergic Fungal Sinusitis in Refractory Chronic Rhinosinusitis in Adult Malaysians

Bee-See Goh, MS,a Balwant Singh Gendeh, MS,a Isa Mohamed Rose, DCP,b Sabiha Pit, FRCPath,c Shamim Abdul Samad, PhDc
a b

From the Department of Otorhinolaryngology, Faculty of Medicine; Department of Pathology; and c Department of Microbiology, National University of Malaysia.
OBJECTIVE: To determine the prevalence of allergic fungal sinusitis (AFS) in refractory chronic rhinosinusitis (CRS) in adult Malaysians. STUDY DESIGN AND SETTING: This cross-sectional study involved 30 immunocompetent CRS patients who underwent surgery. Specimens were sent for mycology and histopathologic analysis for identication of fungi. Clinical and immunological workup was performed for atopy in all patients and controls. RESULTS: Fungal cultures were positive in 5 (16.7%) and 11 (36.7%) of 30 patients from nasal secretions and surgical specimens, respectively. Allergic mucin was found in 8 surgical specimens (26.7%). Hence, prevalence of AFS was 26.7%. The most common causative agent was Aspergillus sp. (54.5%). In 3 (37.5%) of 8 patients, AFS was found to be associated with asthma. Twenty-ve percent (2/8 patients) had aspirin intolerance, and 62.5% (5/8 patients) had elevated total immunoglobulin E levels. All patients had positive skin test reactivity to fungal allergen. CONCLUSIONS: This preliminary study suggests that AFS does exist in Malaysia. Proper handling of surgical specimens and accurate diagnosis by the pathologist and mycologist are essential. 2005 American Academy of OtolaryngologyHead and Neck Surgery Foundation, Inc. All rights reserved.

etrospective studies have estimated that 5% to 10% of cases of chronic rhinosinusitis (CRS) requiring surgical intervention are caused by allergic fungal sinusitis (AFS).1 Katzenstein et al2 and Cody et al3reported that the

incidence of AFS in cases of CRS treated surgically has been approximately 6% to 7%. Cody et al3 noted that AFS is more commonly found among adolescents and young adults (mean age at diagnosis, 21.9 years). The disease is noted to be more common in warmer and humid climates.4,5 Ferguson et al6 reported that the most commonly encountered region includes the Mississippi basin, the Southeast, and the Southwest of the United States. The reason for this geographic difference remains unexplained. Atopy is characteristic in AFS. Approximately 50% of the patients in a series by Manning et al7 had a history of asthma. Cody et al reported that 27% of AFS patients were aspirin sensitive and that 54% were asthmatic.3 Ferguson8 proposed eosinophilic mucin rhinosinusitis (EMRS) as a distinct clinicopathological entity in sinusitis patients histologically similar to AFS except for the absence of fungal hyphae. This investigator further stressed that there is an association between EMRS and aspirin-sensitive asthma (ASA), which is also called triad syndrome, ASA disease, ASA triad syndrome, or Samters triad.8 This triad is composed of aspirintriggered asthma, which is associated with nasal polyps. On the basis of their clinical ndings in 16 patients, Bent and Kuhn9 proposed 5 criteria for the diagnosis of AFS: (1) nasal polyposis; (2) allergic mucin; (3) CT scan ndings consistent with CRS; (4) positive fungal histology or culture; and (5) type I hypersensitivity (atopy) diagnosed by
Reprint requests: Bee-See Goh, MS, Department of Otorhinolaryngology, Faculty of Medicine, National University of Malaysia (UKM), Jalan Yaacob Latif, Bandar Tun Razak, 56000 Kuala Lumpur, Malaysia.

Supported by National University of Malaysia (UKM) research grant FF/12/2001. Presented as a poster at the 10th Congress of International Rhinology Society, Seoul, Korea, October 23-26, 2003.

0194-5998/$30.00 2005 American Academy of OtolaryngologyHead and Neck Surgery Foundation, Inc. All rights reserved. doi:10.1016/j.otohns.2005.03.028

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OtolaryngologyHead and Neck Surgery, Vol 133, No 1, July 2005 sterile kidney dish. The collected uid was placed into a sterile bottle and sent on the same day directly to the mycology laboratory for culture.

history, positive skin test, or serology. Recently, Deshazo and Swain10 described 7 patients with AFS in whom they applied similar diagnostic criteria, with the exception of atopy. They found that only two thirds of patients had positive skin test reactivity to the fungi cultured. Cody et al3 had proposed the criteria for diagnosis of AFS as the presence of characteristic allergic mucin and one of the following: (1) fungal hyphae within allergic mucin with no evidence of tissue invasion and (2) positive results of culture for fungi. In the most recent study by the Mayo Clinic in September 1999, Ponikau et al11 suggested that diagnostic criteria for AFS are (1) CRS; (2) presence of allergic mucin; and (3) the presence of fungal organisms within that mucin conrmed by histology, culture, or both. The atopic patient who lacks polyps, has a diagnostic CT, or positive cultures but who has the characteristic histopathology of allergic mucin with hyphal elements is diagnosed as having AFS.12 In the United States, the incidence of AFS varies geographically. There are reports of AFS from England, Saudi Arabia, Israel, and India. However, the incidence in Malaysia is not known, which may very well be because of the difcult histopathologic identication. We sincerely hope that this study will help in the detection and establishment of the prevalence of AFS in refractory CRS in Malaysian patients.

Collection of Surgical Specimens

The surgical procedure was performed initially without powered instrumentation (microdebrider) to ensure maximal mucin collection. The mucus was removed using suction apparatus together with inamed tissue and polyps and was placed in a saline-moistened sterile bottle. Specimens were not placed on a surgical towel so that absorption would not be problematic. Specimens were processed for histologic analysis and were stained with hematoxylin and eosin (H & E), periodic acidSchiff (PAS), and Gomori methenamine silver. The role of the histopathologist was to screen for presence of mucin. All mucus removed intraoperatively was inoculated into Sabouraud dextrose agar (SDA) plates in the operating room and sent for mycology cultures and identication of fungus the same day.

Procedure for Treatment of Specimens

Specimens from nasal lavage were vortexed for 30 seconds, placed at room temperature for 15 minutes, and subsequently processed by centrifuge at 5,000 rpm for 10 minutes using sterile universal bottles. The pellets were cultured onto SDA medium. The culture plates were incubated at 30C and allowed to grow for 4 weeks before discard. The plates were examined daily for fungal growth identication. For lamentous fungi, identication was performed under microscopic examination of lactophenol cotton blue preparation. In the absence of conidia, the fungi were further inoculated into nutrient deciency medium to facilitate growth and hence identication. For yeast identication, the analytical prole index (API 20C) method was used. The culture plates from the operating room were incubated immediately and allowed to grow, and identication of fungi was performed as mentioned above.

MATERIAL AND METHODS

Patients
A total of 30 adult patients clinically diagnosed with immunocompetent CRS and who underwent sinonasal surgery performed between April 2001 and August 2002 in the National University of Malaysia Hospital were included in the study. Twenty volunteers with no history of allergy or nasal or paranasal disease and with normal-appearing mucosa on nasal endoscopy served as controls. Informed consent was obtained from each patient. This study was approved by the Research and Ethics Committee, National University of Malaysia. Immunocompromised or immunosuppressed patients were excluded; this included patients with diabetes mellitus, who had been diagnosed with malignant disease, who were on long-term oral steroids or cytotoxic drugs, who were AIDS or HIV positive, who had a chronic debilitating disease, or who had received an organ transplant.

Immunological Workup
Blood collected from patients and control groups was analyzed for total immunoglobulin E (IgE) titer. Skin test reactivity was used to screen for IgE-mediated hypersensitivity. Eight commercially available fungal extracts were used to perform skin test by using Multi-Test II system.

Technique of Collecting Specimens

Two puffs of oxymetazoline 0.05% nasal spray were used as decongestant to increase the nasal lumen and consequently the yield from nasal lavage. After about 2 minutes, each nostril was ushed with 10 mL of sterile saline using a sterile syringe. The patient was advised to take a deep inspiratory breath and hold it before the injection of saline. The patient then forcefully exhaled through the nose immediately after the ushing. The return was collected in a

RESULTS
Prevalence of Allergic Fungal Sinusitis
On the basis of Bent and Kuhn criteria,9 8 of the 30 CRS patients were diagnosed as having allergic fungal sinusitis (26.7%). Histologically fungal hyphae was demonstrated in only 3 patients (Figs 1 and 2). The most common fungi cultured from AFS patients was Aspergillus spp. (54.5%, or 6/11 positive cultures from surgical specimens).

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Prevalence of Allergic Fungal Sinusitis in Refractory . . .

Table 1 Number of organisms (in alphabetical order) recovered from patient with CRS (n 30) and percentage colonized with the species Nasal washout, n (%) Aspergillus A. niger

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Surgical specimens, n (%) 6 2 4 1 1 2 4 (42.9) (7.1) (7.1) (14.3) (28.6)

1 (16.7) Aspergillus 1 A. avus A. niger Candida albicans 1 (16.7) Epicoccum sp. Rhizopus sp. 4 (66.7) Paecilomyces sp. Penicillium sp. Rhizopus sp.

Figure 1 Allergic mucin showing fungal hyphae (arrow) surrounded by acellular debris. Hematoxylin and eosin stain (40).

Culture Results
Nasal washout grew fungi in 5 CRS patients (16.7%). Rhizopus species was found to be the most common organism (66.7%). Intraoperative specimens were positive for fungi in 11 patients (36.7%), with Aspergillus species outnumbering other organisms (42.9%; Table 1). Cultures from control groups were obtained by nasal washout returns. Three patients (15%) were cultured positive for fungi with 3 different organisms identied (Table 2). However, positive fungal cultures from nasal washout returns among the CRS and control group showed no signicant difference statistically (P 0.05).

100% had positive skin test reactivity (Table 3). Total IgE level was considered raised when the level was 100 ku/L (UNICAP method). The majority of CRS patients demonstrated raised total IgE (17/30). Two patients had levels 1000 kU/L (1924 kU/L and 1923 kU/L, respectively). The mean level of total IgE in the control group was 130.7 kU/L, and the mean levels in AFS and non-AFS groups were 573.6 kU/L and 270.5 kU/L, respectively. The ratio of total IgE in control group, non-AFS, and AFS patients was 1:2:3. Statistically (2 test), only skin test reactivity showed a significant difference among the AFS and non-AFS group regarding atopy (P 0.05).

DISCUSSION
Association With Atopy
Atopy was assessed by the history of asthma, aspirin hypersensitivity and intolerance, level of serum total IgE, and skin test reactivity. Among the AFS group, 37.5% were asthmatic, and 25% had aspirin hypersensitivity. Two of the AFS patients with aspirin hypersensitivity fullled the criteria of Samters triad, that is, nasal polyposis, asthma, and aspirin intolerance; 62.5% had elevated total IgE level and Several decades ago, fungal disease of the nose and paranasal sinuses represented an invasive deadly disease, and management consisted of extensive surgical debridement, followed by systemic and topical antifungal therapy. For the past 2 decades, AFS identication has become more dened. AFS is now believed to be an allergic reaction to aerosolized environmental fungi in an immunocompetent host. With heightened awareness and sophisticated laboratory diagnostic techniques, an increased number of reports have been published.3,4,13 A full consensus among rhinologists worldwide concerning diagnostic criteria for AFS is much awaited. The traditional gold-standard criteria are the demonstration of allergic mucin and the presence of noninvasive fungi from patients with CRS. It is believed that the incidence of AFS

Table 2 Number of organisms (in alphabetical order) recovered from healthy control subjects (n 20) Fungi cultured Figure 2 Allergic mucin showing scattered inammatory cells that are mainly eosinophils. Several Charcot-Leyden crystals are shown (arrow) with hematoxylin and eosin stain (100). Penicillium sp. Rhizopus sp. Trichosporon sp. Number (%) 1 (33.3) 1 (33.3) 1 (33.3)

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OtolaryngologyHead and Neck Surgery, Vol 133, No 1, July 2005 cillium sp. and Claudosporium sp. Fadl et al16 from Saudi Arabia reported 4 cases of AFS, and all grew Aspergillus. Even though the reports from Asia and the Middle East were small as compared with the Western literature review, the main fungi causing AFS was probably inuenced by geographic location. Nasal washout was performed for all the 30 CRS patients and 20 individuals from the control group. Five CRS patients had positive growth (16.7%) with Rhizopus species being the most common fungi, and 3 individuals cultured positive for fungi (15%) for 3 different species, namely Penicillium sp., Rhizopus sp., and Tricosporum sp. each individually (Tables 1 and 2). These results revealed no statistical difference for cultures obtained from nasal washouts from CRS patients and control group. All 8 AFS patients cultured positive from mucin collected intraoperatively, but only 3 had positive cultures from nasal washouts. Literature review showed that most investigators did not perform nasal washouts but cultured mucin from the specimens collected intraoperatively, which correlated with the histology ndings. Rhizopus sp. is a common mycology laboratory contaminant; isolation of this organism must be interpreted with caution except in diabetic or immunosuppressed patients. Penicillium sp. is also common of all laboratory contaminants. Isolation of Penicillium sp. does not necessarily indicate an etiologic role unless the isolate is accompanied by typical fungal elements in tissue or smears of lesional exudate. The incidence of saprophytic fungal infestation is unknown, but investigators generally agree that it is best separated from asymptomatic individuals with positive fungal cultures. The investigators believed that nasal washouts may not be helpful in investigating patients suspected of AFS. In our study, only 3 patients showed obvious fungal elements on special staining, although 8 patients demonstrated allergic mucin histologically. It is possible that the other 5 patients represent the variant of AFS, allergic mucin sinusitis without fungus, as reported by Allphin et al.17 Similarly, Cody et al3 reported allergic fungal sinusitis-like syndrome in 20 of 51 cases of rhinosinusitis with polyposis in whom fungus could not be isolated. Three years later, Ramadan and Quraishi18 described 12 patients with AFS in which 4 patients had allergic mucin on histopathology, but fungal hyphae could not be identied on silver stain, and fungal cultures were negative. Our study actually grew fungi on culture from mucin, but the negative fungal staining may be a result of the delay in processing and examination secondary to human factors in delivering the specimens from the operating room to the laboratory. In general, AFS is related to atopy. In our study, association with asthma, aspirin sensitivity, skin, and serology tests using total IgE were analyzed in AFS and non-AFS group in CRS patients. The study showed that there was no signicant difference statistically among these 2 groups in relation to asthma, aspirin sensitivity, and total IgE (Table 3). The only signicant issue was the positive skin test

Table 3 Statistical analysis on association between atopy among AFS and non-AFS groups in 30 CRS patients Variables Asthmatic Aspirin hypersensitivity Elevated total immunoglobulin E Positive skin test AFS, Non-AFS, P values n 8 (%) n 8 (%) 0.05 3 (37.5) 2 (25.0) 5 (62.5) 8 (100.0) 8 (36.4) 0 12 (54.5) 2 (9) 1.000 0.064 1.000 0.001

appears to be inuenced by geographic factors. Literature review reveals that the majority of regions reporting cases of AFS are sited in temperate regions of relatively high humidity. However, the incidence varied remarkably on the basis of the location of reporting sites. Retrospective studies estimated that the incidence of AFS was 5% to 10% of CRS patients requiring surgical intervention.1 A higher incidence of 93% was reported by a prospective study performed at the Mayo Clinic.11 In our study, the prevalence of AFS among the CRS patients was 26.7%. This percentage was slightly higher than the incidence reported elsewhere. All of the 8 patients did not state a history of travel to India for at least 1 year before the study. Therefore, we strongly believe that AFS does exist in Malaysia. It is notable that a positive fungal culture does not conrm the diagnosis of AFS, nor does a negative culture rule it out. Fungi may proliferate as saprophytic growth in diseased sinuses. Furthermore, mycology laboratories vary in capability and specimen handling, which signicantly inuences the rate of positive fungal cultures reporting. Hence, allergic mucin remains the most reliable indicator of AFS. The causative fungi in AFS usually are dematiaceous fungi and, less frequently, Aspergillus. In the literature review performed by Manning and Holman,5 263 cases of AFS were identied, of which 168 cases yielded positive cultures, 87% were from the dematiaceous genera, and only 13% yielded Aspergillus. In our study, cultures from surgical specimens grew 42.9% of Aspergillus (6/14) in which 4 grew Aspergillus niger and 2 were Aspergillus avus, followed by 28.6% of Rhizopus species, 14.3% of Penicillium species, and Epicoccum species and Paecilomyces species in 1 patient each (Table 1). Among these, only 8 patients were diagnosed as having AFS after histologic conrmation of allergic mucin with or without the presence of fungal element on special staining. Aspergillus species was also found to be the most common fungi cultured in AFS patients (54.5%), followed by Rhizopus species (18.1%). However, the results correlate well with the results reported by investigators from Asia and Middle Eastern countries, unlike that of the West. Rupa et al14 from India reported that Aspergillus species were the most common fungi isolated (95.8%) in a series of 24 patients with AFS. In Japan, Matsuwaki et al15 reported a case of AFS caused by Peni-

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Prevalence of Allergic Fungal Sinusitis in Refractory . . .

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reactivity. All of the AFS patients reacted positively to at least 1 fungal allergen, whereas only 2 patients in the nonAFS group had a positive reaction toward fungal allergen. Among the AFS patients, 3 (37.5%) were asthmatic, and only 2 patients had a history of aspirin intolerance (25%). These ndings correlate with the literature, which reported that one third to one half of AFS patients have asthma.3-5 Cody et al3 described a 27% incidence of aspirin sensitivity in AFS patients. However, Ferguson8 described EMRS as a distinct clinicopathological entity in which asthma and aspirin sensitivity are signicantly higher compared with the AFS group. Are these 2 groups different clinically, or is EMRS a variant of AFS? Further studies and research with larger samples and a standard criteria are essential to take into account to conclude that atopy is a characteristic of AFS. Our study revealed that 2 AFS patients were identied as having Samters triad or as having ASA disease, which is a distinct association in CRS. Immunological workup is essential in the investigations of AFS patients. Total IgE levels are essential for clinical indicators of allergic bronchopulmonary fungal disease or AFS activity. Total IgE values are generally elevated in AFS, often to more than 1000 U/mL, but a level of 100 U/mL is an accepted value of raised IgE. In our study, 5 of the AFS patients had raised total IgE (62.5%) with the mean level of 573.6 KU/L compared with a mean level of 130 KU/L in the control group. All 8 AFS patients (100%) had positive skin test reactivity toward fungal antigens, compared with 2 non-AFS patients (9.0%) with positive skin test reactivity toward 1 fungal antigen (Table 3). This study did encounter some limitations. The sample study was small, and the time taken to transport the specimen to the histopathology and mycology laboratories was not strictly observed. Because of budget constraints, fungalspecic IgE and specic IgG, which are good screening tools, were not performed.

further study should be recommended involving a larger number of samples.

SIGNIFICANCE
It is important to differentiate AFS from other forms of fungal sinusitis for appropriate management of the patient.
I wish to thank Dr Shanaz Murad from Allergy and Immunology Research Centre, Institute For Medical Research, Kuala Lumpur for her support in this study. I also acknowledge with gratitude the cooperation of staff nurses in the operating room, staffs in the mycology, histopathology laboratory and ENT clinic; and Dr. Mohd Nizam for his assistance in the statistical analysis.

REFERENCES
1. Schubert MS. Medical treatment of allergic fungal sinusitis. Ann Allergy Asthma Immunol 2000;85:90 101. 2. Katzenstein AA, Sale SR, Greenberger PA. Allergic Aspergillus sinusitis: a newly recognized form of sinusitis. J Allergy Clin Immunol 1983;72:89 93. 3. Cody DT II, Neel HB III, Ferreiro JA, et al. Allergic fungal sinusitis: the Mayo Clinic experience. Laryngoscope 1994;104:1074 9. 4. Manning SC, Merkel M, Kriesel K, et al. Computed tomography and magnetic resonance diagnosis of allergic fungal sinusitis. Laryngoscope 1997;107:170 6. 5. Manning SC, Holman M. Further evidence for allergic pathophysiology in allergic fungal sinusitis. Laryngoscope 1998;108:148596. 6. Ferguson BJ, Barnes L, Bernstein JM, et al. Geographic distribution of AFS. Otolaryngol Clin N Am 2000;33:4419. 7. Manning S, Vuitch F, Weinberg A. Allergic aspergillosis: a newly recognized form of sinusitis in the paediatric population. Laryngoscope 1989;99:6815. 8. Ferguson BJ. Eosinophilic mucin rhinosinusitis: a distinct clinicopathological entity. Laryngoscope 2000;110:799 813. 9. Bent JP III, Kuhn FA. Diagnosis of allergic fungal sinusitis. Otolaryngol Head Neck Surg 1994;111:580 8. 10. DeShazo RD, Swain RE. Diagnostic criteria for allergic fungal sinusitis. J Allergy Clin Immunol 1995;96:24 35. 11. Ponikau JU, Sherris DA, Kern EB, et al. The diagnosis and incidence of allergic fungal sinusitis. Mayo Clinic Proc 1999;74:877 84. 12. Houser SM, Corey JP. Allergic fungal sinusitis: pathology, epidemiology, and diagnosis. Otolaryngol Clin N Am 2000;33:399 408. 13. Bent JP III, Kuhn FA. Allergic fungal sinusitis/polyposis. Allergy Asthma Proc 1996;17:259 68. 14. Rupa V, Jacob M, Mathews MS, et al. Clinicopathological and mycological spectrum of allergic fungal sinusitis in South India. Mycoses 2002;45:364 7. 15. Matsuwaki Y, Nakajima T, Iida M, et al. A case report of allergic fungal sinusitis caused by Penicillium sp. and Claudosporium sp. Nippon Jibiinkoka Gakkai Kaiho 2001;104:114750. 16. Fadl FA, Hassan KM, Faizuddin M. Allergic fungal rhinosinusitis: report of 4 cases from Saudi Arabia. Saudi Med J 2000;21:581 4. 17. Allphin AL, Strauss M, Abdul-Karim FW. Allergic fungal sinusitis; problems in diagnosis and treatment. Laryngoscope 1991;101:315-20. 18. Ramadan HH, Quraishi HA. Allergic mucin sinusitis without fungus. Am J Rhinol 1997;11:1457.

CONCLUSION
AFS is a relatively new, only characterized disease entity that commands a great deal of interest. Although controversy exists, our study revealed that the prevalence of AFS in refractory chronic rhinosinusitis in adult Malaysians was 26.7%. The most common causative fungus was found to be Aspergillus sp (54.5%). AFS was also found to be associated with atopy. Removed surgical specimens rather than nasal washouts are recommended for fungal identication by culture and histologically. Handling laboratory specimens and frequent communication with the pathologist and mycologist are essential for an accurate diagnosis of AFS. A