New Paradigm for the Roles of Fungi and Eosinophils in Chronic Rhinosinusitis

Jan Sasamaa, David A. Sherrisb, Seung-Heon Shinc, Gail M. Kephartc, Eugene B. Kernb, and Jens U. Ponikaua

Purpose of review
Chronic rhinosinusitis represents a challenge with its poorly understood pathophysiology and limited treatment options. Potential roles of fungi and eosinophils in the etiology and pathophysiology of chronic rhinosinusitis are summarized.

Recent findings
Previously, the fungal role in chronic rhinosinusitis was limited to the rare subgroup, allergic fungal rhinosinusitis. Critical examination of earlier diagnostic criteria for allergic fungal rhinosinusitis reveals limitations. By using updated diagnostic standards and novel sensitive techniques to detect fungi, a higher number of patients can now be diagnosed with fungal rhinosinusitis. A novel non-IgE-mediated immunologic mechanism in chronic rhinosinusitis patients links the predominant eosinophilic inflammation to certain fungi. Overall, these new findings have implications for surgical and medical approaches,
including anti-inflammatory and antifungal medications.

Summary
Several classification schemes and diagnostic criteria describe chronic rhinosinusitis and make comparisons difficult. Preselection of patient groups within the chronic rhinosinusitis population and the lack of sensitive diagnostic techniques have prevented a full understanding of the syndrome complex of chronic rhinosinusitis. New results suggest a broader role for fungi in the pathophysiology of chronic rhinosinusitis, linking the eosinophilic inflammation to the presence of certain molds in the nasal and paranasal cavities. Although fungi are commonly found in nearly everyone, only chronic rhinosinusitis patients respond to them with an eosinophilic inflammation. These findings support a shift in the etiologic understanding of chronic rhinosinusitis away from a bacteriologic infectious pathogenesis to a fungal-driven inflammatory pathophysiology. Herein, the authors review
earlier studies and describe an updated view on an old paradigm.

Keywords
chronic sinusitis, chronic rhinosinusitis, allergic fungal sinusitis, fungi, eosinophils, inflammation

Correspondence to Jan Sasama, MD, Department of Otorhinolaryngology, Mayo Clinic Rochester, 200 First Street SW, Rochester, MN 55905, USA Tel: 507 284 1482; fax: 507 284 8855; e-mail: sasama.jan@mayo.edu Supported by National Institutes of Health grants AI 50494 and AI 49235 and the Mayo Foundation. Dr. Sasama was sponsored by the Max Kade Foundation, New York.

Current Opinion in Otolaryngology & Head and Neck Surgery 2005, 13:2–8

Abbreviations
AFS allergic fungal rhinosinusitis
PBMC peripheral blood mononuclear cells
PCR polymerase chain reaction
ª 2005 Lippincott Williams & Wilkins.

Introduction
Chronic rhinosinusitis is one of the most common diseases, estimated to affect as much as 14.1% (29.2 million) of the adult US population [1,2]. Patients with chronic rhinosinusitis suffer from long-term nasal congestion, thick mucus production, loss of sense of smell, fullness and headaches, and recurrent acute exacerbations secondary to bacterial infections [3]. Chronic rhinosinusitis affects the quality of life for patients more severely than congestive heart failure [4]. Lacking both mutual agreements for the pathophysiology of chronic rhinosinusitis and therapeutic targets, the US Food and Drug Administration
has not approved any drug or treatment for chronic rhinosinusitis.

In chronic rhinosinusitis, symptoms persist longer than 3 months [1]. Chronic rhinosinusitis with or without nasal polyposis is dominated by a heterogeneous inflammatory eosinophilic infiltrate in the mucosa of the nasal cavities and paranasal sinuses [5,6,7•,8]. Eosinophil granule proteins, such as major basic protein, are known to be toxic to sinus epithelium and have been previously colocalized with the epithelial damage found in chronic rhinosinusitis [9]. In contrast with acute rhinosinusitis or an acute exacerbation of chronic rhinosinusitis, in which the neutrophilic inflammation points toward a bacterial etiology, the underlying eosinophilic inflammation in chronic rhinosinusitis suggests a different etiologic mechanism for chronic
rhinosinusitis [3,7•].

First paradigm of allergic fungal sinusitis
In 1981, Millar et al. [10] first described sinus specimens from five patients with chronic rhinosinusitis that showed histologic similarities to allergic bronchopulmonary aspergillosis. Katzenstein et al. [11] independently demonstrated the presence of clusters of necrotic eosinophils, Charcot- Leyden crystals, and noninvasive fungal hyphae in the sinus mucin of patients who had undergone sinus surgery for the treatment of chronic sinusitis and proposed to call this feature allergic mucin. Based on the presence of this predominant eosinophilic inflammation, the authors suggested that the pathophysiology of chronic rhinosinusitis
was allergic and IgE-mediated, because eosinophilia was historically associated with allergy, and named this entity allergic Aspergillus sinusitis. The term allergic fungal rhinosinusitis (AFS) was introduced later, when other investigators cultured other fungi from the sinus mucus of chronic rhinosinusitis patients [12].

Approximately 10 years ago, two groups described empirical diagnostic criteria for AFS; the main difference was the requirement for atopy. In 1994, Bent and Kuhn [13] proposed the following criteria for the diagnosis of AFS: (1) nasal polyposis; (2) allergic mucin as shown by the presence of rubbery, tenacious mucus with sheets of eosinophils and Charcot-Leyden crystals; (3) CTscan findings with characteristic areas of increased attenuation; (4) positive fungal histology or culture; and (5) type I hypersensitivity (atopy) diagnosed by a strong history of allergy, positive skin test, or serology. A critical review reveals that all 15 patients who were included to define these AFStypical criteria were empirically preselected on the presence
of these five clinical criteria and subsequently diagnosed to have ‘‘. AFS based on the current understanding of the disease.’’ The demonstration of all empirically selected features in all 15 patients was taken as a proof for the validity of the suggested diagnostic criteria for AFS. In 1995, deShazo and Swain [14] asked physicians to refer patients meeting the following selected empirical criteria: (1) sinusitis confirmed with imaging methods like CTor radiograph, (2) identification of allergic mucin, (3)
demonstration of fungal elements, (4) absence of immunodeficiency or diabetes, and (5) exclusion of invasive fungal disease. On review of the 13 referred patients, seven patients were diagnosed with AFS based on the authors’ understanding of the disease. Clinically, one of these seven patients with a histologic diagnosis of AFS did not have any evidence of atopy. All findings in these seven newly reviewed patients were compared with the findings from 99 AFS patients reported within the literature available at the time, for whom similar criteria had been applied for diagnosis. Because only six of their seven
newly recruited patients had atopy and because their review of the literature revealed that only two thirds of the 99 AFS patients had positive skin tests to the fungi cultured, they suggested that atopy be excluded as a requirement to diagnose AFS. Nevertheless, like Bent and Kuhn [13], deShazo and Swain [14] based their diagnostic criteria solely on these empirical requirements and did not consider that they had also preselected their patients. Although the sets of criteria were limited by their empirical basis, they were considered to be valid and were used in many subsequent patient reports.

Validity of diagnostic criteria for allergic fungal sinusitis
Features commonly found in chronic rhinosinusitis, such as confirmed mucosal changes or polyposis in the nasal and paranasal sinuses using imaging techniques like CT or radiograph, the absence of immunodeficiency, and the exclusion of invasive fungal disease, are not specific for AFS. The areas of hyperattenuation on CT scans are thought to be representative of the eosinophilic mucin that contains fungi [15], but this feature might not be as obvious in patients who have a smaller fungal and
eosinophilic mucin load.

The remaining diagnostic criteria for AFS were thought to be more disease-specific: (1) the presence of fungi, (2) the presence of allergic mucus containing clusters of eosinophils with Charcot-Leyden crystals, and (3) for Bent and Kuhn [13], the existence of an IgE-mediated allergy. Nevertheless, when newer findings are considered, these three last criteria cannot be seen as specific for AFS.

Presence of fungi
Previously, the presence of fungi in the mucus of the nasal cavity or paranasal sinuses was a required criterion to diagnose AFS [13,14]. Nevertheless, to prove this criterion has been difficult, because several detection methods and culture techniques have been used for many years, resulting in varying rates of fungal detection. Even when fungi could be demonstrated in a histologic specimen, the cultures were often negative [16,17]. Direct microscopic and histopathologic examinations were not highly sensitive or specific, mainly because of their retrospective nature and an inadequate preservation of mucus [11,16]. A recent study by Granville et al. [18] reviewed pathologic slides of sinus tissue from patients who were clinically diagnosed with AFS, revealed a previous 47% failure rate in the diagnostic demonstration of fungi and eosinophilic mucin.

In 1999, another approach was taken to evaluate prospectively an unselected group of consecutive chronic rhinosinusitis
patients undergoing sinus surgery, and fungal elements were found on histology in 82% (81/101) of the patients [19]. In addition, using novel collection and culturing techniques, mucus specimens from 96% of chronic rhinosinusitis patients (202/210) showed cultures positive for fungi, and most demonstrated the presence of multiple organisms. For the first time, specimens from healthy controls were also cultured, demonstrating the presence of fungi in 100% of the specimens (14/14).
In 2003, similar results were reported from Europe, where investigators found fungi by histology in 75.5% (28/37) of consecutive surgical chronic rhinosinusitis patients and after culture in 91.3% (84/92) [20•,21]. In addition, by using these more sensitive techniques, the widespread presence of fungi in specimens from healthy controls was also verified. All three studies had a common approach to fungal detection in nasal and paranasal cavities: they prospectively studied specimens from an unselected, large group of patients with chronic rhinosinusitis or from healthy controls. The novel culture techniques used in these studies highlighted the need to break down the disulfide bridges in the mucin using a solution of dithiothreitol; this procedure liquefied the mucus and allowed direct contact of the fungi with the culture media. Subsequently, a novel
chitin-based staining technique demonstrated the presence of fungi in the mucus in 100% of prospectively collected surgical chronic rhinosinusitis specimens and visually emphasized the limitations and the lack of specificity of previous staining techniques [22].

After the introduction of these new methods, several other groups were able to increase their detection rates of fungi in the paranasal sinuses. For example, Collins et al. [23] demonstrated positive fungal cultures in 65% of a preselected group of 86 patients who were clinically suspected to have fungal sinusitis.Weschta et al. [24] detected fungal elements in 63% of 60 chronic rhinosinusitis patients using both culture and polymerase chain reaction (PCR)methods. Lackner et al. [25••] demonstrated that fungi are present in the nasal cavities of humans very early in life. His group showed positive fungal cultures in six of 30 (20%) nasal secretions from neonates directly after birth, two of 29 (7%) on the second day and four of 26 (15%) positive fungal cultures on the fifth day of life. The number of infants with positive fungal cultures in their nasal mucus increased
with time to 72% in 2-month-old infants and to 94% in 4-month-old infants. This increasing awareness for nearly ubiquitous fungal colonization in the sinus cavities is also reflected by an increasing number of new reports about fungal-associated chronic rhinosinusitis [26–29]. Recently, Gosepath et al. [30••] used highly sensitive PCR methods to demonstrate fungal DNA in polypoid nasal tissues from 27 of 27 patients with chronic rhinosinusitis and in 66.6% of the nasal mucosa derived
from healthy controls. Interestingly, a PCR screening specifically for Alternaria tested positive in 100% of these chronic rhinosinusitis patients, whereas none of the healthy control samples tested positive for Alternaria DNA. In addition, a novel immunoassay for Alternaria detected Alternaria antigens in the mucus of all tested chronic rhinosinusitis patients and all healthy controls [31••]. In view of these newer results based on more sensitive detection techniques, earlier classifications that
stated that chronic rhinosinusitis patients without fungi were a different medical entity must be reconsidered [13,17,32].
Overall, newer collection and detection technologies demonstrate that fungi are present in the mucus of chronic rhinosinusitis and healthy controls. Furthermore, the presence of fungi alone does not indicate disease state, nor should it be used as a diagnostic criterion for AFS or any fungal-associated disease.

Presence of eosinophilic (allergic) mucus By definition, the main histologic features of allergic mucus are clusters of intact and degenerating eosinophils and Charcot-Leyden crystals; these crystals are a product of degenerating eosinophils and are present in eosinophilassociated diseases, including allergic rhinitis. Originally, the term allergic mucus was based on the historic association of eosinophilia and IgE-mediated allergy. It is now recognized that the so-called allergic mucus occurs without
any detectable IgE-mediated allergy. Thus, the terminology has been changed to the more descriptive eosinophilic mucus [33].

In early reports, where retrospective analyses were performed, eosinophilic mucin was described only as an occasional finding when it was discovered in paraffinembedded specimen blocks [11,13]. This created another selection bias, because special histologic examinations for allergic or eosinophilic mucin were performed only if the mucus of the chronic rhinosinusitis patients had a gross macroscopic tenacious appearance. The viscosity of mucus rises significantly with an increasing amount of DNA released from inflammatory cells, as demonstrated in the mucus of patients with cystic fibrosis [34,35]; thus, only the mucus, which contains an abundance of eosinophils and of their degranulation products, shows these typical macroscopic features. Therefore, mucus specimens from chronic rhinosinusitis patients containing smaller numbers of eosinophil clusters, possibly not displaying such a typical tenacious macroscopic appearance, might not be suspected for AFS and might not have been specifically examined histologically for eosinophilic mucin. Prospective studies in which extra care ensured the preservation
of mucus found eosinophilic mucus, containing clusters of eosinophils, by histology in 96% (97/101) and 94% (35/37) of consecutive surgical chronic rhinosinusitis patients [19,20•]. This abundance of eosinophils in the mucus of chronic rhinosinusitis patients suggests that tissue eosinophils in chronic rhinosinusitis are only in transit through the mucosa and migrate into the mucus to form characteristic clusters. Interestingly, this cluster formation seems to occur around fungal elements, suggesting that eosinophils target fungi in the mucin of chronic rhinosinusitis patients and release their toxic granule proteins, like major basic
protein, onto the organisms (Fig. 1A–C) [19,20•]. The presence of eosinophilic mucin is likely a specific feature for chronic rhinosinusitis and is not merely a gross appearance of discolored tenacious mucus.

A, Photomicrograph shows an eosinophil cluster (arrows) in the mucin attached to tissue that was removed during sinus surgery of a patient with chronic rhinosinusitis. Because of the heterogeneous nature of the eosinophilia, where areas with and without eosinophils coexist, these clusters can be easily missed on examination (H&E staining; counterstain of B; original magnification 3400). B, Photomicrograph shows a special eosinophil granule major basic protein (MBP) immunofluorescence staining and reveals that striking amounts of the toxic eosinophil granule protein are released in the area of the eosinophil cluster (MBP immunofluorescence staining; original magnification 3400). C, Photomicrograph shows a special fungal specific chitinase immunofluorescence staining in a serial section to A and B and reveals fungal elements (arrows) in the eosinophil cluster (chitinase immunofluorescence staining; original magnification 3400).

Existence of IgE-mediated allergy
As mentioned, IgE-mediated allergy as a diagnostic criterion for AFS was based on the assumption that an IgEmediated
allergy plays a role in the pathophysiology of AFS. This assumption resulted in a preselection of atopic patients when the diagnosis of AFS was made [13,32,33]. As a notable exception, deShazo and Swain [14] recognized the lack of supportive evidence for a type 1 hypersensitivity and excluded it as a diagnostic criterion. Certainly, local production of IgE might mediate inflammation in the absence of detectable systemic allergy. Recently, Collins et al. [23] preselected 25% (86/342) of
consecutive chronic rhinosinusitis patients undergoing sinus surgery for the macroscopic presence of thick, colored mucus. All 86 patients had eosinophilic mucin by histology, but only 37% of these had fungal specific serum IgE, and an additional six patients had local fungal IgE in the absence of elevated systemic levels. Although some atopic chronic rhinosinusitis patients do produce specific IgE against fungi [36,37], there is no evidence that this IgE production actually results in the disease state of
chronic rhinosinusitis. Furthermore, IgE-mediated allergy and exposure to the relevant allergens does lead to allergic rhinitis; however, the symptom complex of chronic rhinosinusitis develops independently from an elevation of IgE. Thus, patients with chronic rhinosinusitis may or may not have allergic rhinitis to molds as a comorbid disease.

In summary, recent studies suggest that nearly all humans have fungi (eg, Alternaria) present in the mucus from nasal cavities and paranasal sinuses. If the attached mucus is preserved appropriately, eosinophilic mucin can be found histologically in the vast majority of chronic rhinosinusitis patients undergoing sinus surgery. In contrast, there is no evidence to support a role for IgE-mediated allergy to molds in chronic rhinosinusitis patients, although a comorbid allergic rhinitis can occu

.
Broader role for fungi in chronic rhinosinusitis
As mentioned, new histologic studies suggest that eosinophils migrate from the mucosa into the mucus in chronic rhinosinusitis patients [19,20•]. This eosinophilic inflammation is extremely heterogeneous, and areas of eosinophilia in the tissue and mucus might be localized next to areas without eosinophilia; hence, a sampling error could lead to false-negative findings [38•]. Furthermore, as a common presurgical treatment before sinus surgery, the administration of systemic steroids might inhibit
the tissue eosinophilia that is normally present [39]. Thus, eosinophilia can be overlooked during histopathologic evaluations, especially if the collection technique and examination are not focused on this feature.

Eosinophils are generally understood to play a role in host
defense against larger, nonphagocytosable organisms. Thus, the main question arising is whether fungi are the stimulating agents to induce the recruitment, activation, life prolongation, and degranulation of eosinophils. Stated another way: do fungi trigger the production of cytokines regulating the eosinophil inflammation in chronic rhinosinusitis? The main source for eosinophilassociated cytokines is tissue-bound lymphocytes [40]. Interleukin-13 induces the expression of endothelial vascular
cell adhesion molecule 1, which mediates selective eosinophil migration from the vasculature into the tissue [41]. Vascular cell adhesion molecule 1 is increased in tissues from chronic rhinosinusitis patients independently of their allergy status; this may explain why no difference in tissue eosinophilia was found between allergic and nonallergic chronic rhinosinusitis patients [41,42]. Recently, Shin et al. [31••] isolated peripheral blood mononuclear cells (PBMCs), which contain lymphocytes and other cells that can function as antigen-presenting cells. They demonstrated in vitro that stimulation with certain fungal
extracts (mainly Alternaria) induces production of interleukin- 13 in PBMCs from chronic rhinosinusitis patients, but not in those from healthy controls.

To date, interleukin-5 is recognized as the most important cytokine for inducing eosinophilic inflammation; it mediates eosinophilic differentiation, survival, and activation [43,44]. It is significantly elevated in sinus tissues from chronic rhinosinusitis patients compared with tissues from healthy controls, and its presence is independent from the patient’s allergic status [40,45,46•]. T lymphocytes are the main source for interleukin-5 in tissues from chronic rhinosinusitis patients and account for 68% of the interleukin- 5-positive cells, although eosinophils (18%) and mast cells (14%) also show the presence of interleukin-5 [47]. When PBMCs from 18 chronic rhinosinusitis patients were stimulated with an Alternaria extract, cells
from 16 of the 18 chronic rhinosinusitis patients produced elevated interleukin-5, but none of the PBMCs from 15 healthy controls did [31••]. This immune response also occurred when the PBMCs from chronic rhinosinusitis patients were stimulated with Aspergillus (22%) or Cladosporium (33%) antigens, but was not inducible with Penicillium [31••]. In addition, interferon-g, which enhances the killing of parasites by eosinophils [48], is also elevated in nasal polyp tissues of chronic rhinosinusitis
patients, specifically in nonallergic patients [49,50]. PBMCs from chronic rhinosinusitis patients produced 5.5 times more interferon-g when stimulated in vitro with an Alternaria extract than did PBMCs from healthy controls [31••]. These immune responses to Alternaria occurred independently from the allergic status, because only 28% of these chronic rhinosinusitis patients had elevated specific IgE for Alternaria, and no correlation existed between specific IgE for Alternaria and the intensity of the interleukin-5 responses. In addition, the interleukin-5 response by PBMCs to Alternaria showed no statistically
significant difference between cells from allergic and nonallergic patients [31••].

Nasal secretions from nine chronic rhinosinusitis patients and nine healthy controls contained similar amounts of Alternaria proteins, suggesting an equivalent fungal load [31••]. However, compared with 15 healthy controls, 18 chronic rhinosinusitis patients had almost a fivefold excess of Alternaria specific serum IgG, indicating an increased exposure of their immune system to the organisms. In addition, the Alternaria-specific serum IgG levels in the chronic rhinosinusitis patients correlated strongly with the levels of interleukin-5 produced by their PBMCs after stimulation with Alternaria, suggesting a link between the
immunologic exposure and the severity of the immune reaction. More recently, fungal antigens were found to induce
degranulation of eosinophils in vitro. As judged by the release of eosinophil-derived neurotoxin, Alternaria produced a strong stimulus compared with Aspergillus, Cladosporium, Penicillium, and Candida [51]. Overall, chronic rhinosinusitis patients appear to respond vigorously to fungi and produce cytokines that are crucial to drive the eosinophilic inflammation; this response is independent from an IgE-mediated allergy.

Antifungal treatment of chronic rhinosinusitis
The recognition of a potential role for fungus in the pathogenesis of chronic rhinosinusitis opens the way for novel treatment regimens. Whereas earlier approaches in preselected AFS patients failed to demonstrate any benefits for systemic antifungals in the treatment of chronic rhinosinusitis [11,13], a recent study found that revision sinus surgery could be avoided when patients were treated with itraconazole plus steroids [52]. Attempts to alter the prognosis of chronic rhinosinusitis with immunotherapy, which was based on the earlier understanding of an IgE-mediated process, showed no long-term benefit compared with
chronic rhinosinusitis patients not given immunotherapy [53].

Thus, the more recent results suggest a much broader role for fungi in chronic rhinosinusitis patients as noninfectious stimuli and as targets for the eosinophilic inflammation. Intranasal antifungal therapy could reduce the antigenic load and, ultimately, the eosinophilic inflammation. In one pilot study, topical intranasal treatment of 51 chronic rhinosinusitis patients (ie, no preselection criteria except chronic rhinosinusitis) with 20 mL 100 mg/mL amphotericin B solution per nostril twice a day for 11.3 months average (range, 3–17 months) resulted in improvements in both symptoms and endoscopic staging in 75% of patients and in a significant reduction of mucosal thickening on available CT scans [54]. Using intranasal amphotericin B applied as a 20-mL suspension per nostril twice a day with a bulb syringe for 4 weeks, Ricchetti et al. [55] reported disappearance of polyposis on endoscopy in 62% of patients with mild and 42% of patients with moderate chronic rhinosinusitis. However, none of the patients with severe chronic rhinosinusitis and nasal polyps filling the entire nasal cavity showed improvement, potentially
because of limited access of the topical medication to a major surface area of the sinus mucosa or the short duration of the therapy in the study. Likewise, a double-blind, placebo-controlled trial with chronic rhinosinusitis and severe nasal polyposis, using a small amount of amphotericin B in a spray as delivery vehicle (200 mL, four times a day), showed no benefit after 8 weeks [24]. Why this latter study explicitly excluded chronic rhinosinusitis patients with any suspected fungal etiology is unclear.
Also, a spray has been shown to be a vastly inferior delivery vehicle for medications into the sinuses compared with a bulb syringe, even in patients without recurrent nasal polyps after sinus surgery and with open sinus ostia [56••]. Another double-blind, placebo-controlled trial used a bulb syringe as delivery vehicle for intranasal amphotericin B in an unselected chronic rhinosinusitis population [57••]. This protocol found reduced inflammatory mucosal thickening on CT scan and nasal endoscopy and decreased levels of intranasal cytokines and markers for eosinophilic inflammation in chronic rhinosinusitis
patients compared with placebo after 6 months. These results suggest that an antifungal treatment reduces the fungal antigenic load in the nasal and paranasal cavities and subsequently decreases the eosinophilic response. Overall, topical antifungal treatments likely benefit patients with chronic rhinosinusitis; this treatment needs to be long-term; and the dosage, formulation, and application methods still need further optimization, specifically in an obstructive disease like chronic rhinosinusitis.

Conclusion
New data suggest a much broader role for fungi, especially common airborne molds such as Alternaria, in the pathophysiology
of chronic rhinosinusitis, linking the eosinophilic inflammation directly to the presence of certain molds in the nasal and paranasal cavities. The existence of AFS as a distinct entity of chronic rhinosinusitis is questionable because the diagnostic criteria lack validity. Although fungi are commonly found in both chronic rhinosinusitis patients and healthy controls, only chronic rhinosinusitis patients appear to respond to them with an eosinophilic inflammation. Intranasal antifungal therapy shows promise as a novel treatment for chronic rhinosinusitis when used long-term and with appropriate delivery methods and formulations. Overall, these findings support a shift in the etiologic understanding of chronic rhinosinusitis away from a bacteriologic infectious pathogenesis to a fungal-driven inflammatory pathophysiology.

Acknowledgements
The authors thank Hirohito Kita, MD, for his help and support, and Ms. Cheryl R. Adolphson for her support and editorial assistance.

References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest
•• of outstanding interest

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•• 30 Gosepath J, Brieger J, Vlachtsis K, et al. Fungal DNA is present in tissue specimens of patients with chronic rhinosinusitis. Am J Rhinol 2004; 18:9–13. This study demonstrates the existence of fungal DNA in polypoid nasal tissues
of 100% (27/27) patients with chronic rhinosinusitis and in 66.6% (10/15) of the nasal mucosa derived from healthy controls using highly sensitive PCR techniques. Furthermore, PCR screening specifically for Alternaria tested positive in 100% of the chronic rhinosinusitis patients, whereas none of the healthy control samples tested positive for Alternaria DNA. These data provide the previously lacking proof of the existence of fungal antigens possibly reaching the cytokine producing T lymphocytes in the polypoid tissue from chronic rhinosinusitis patients. These findings could link mechanisms driving the eosinophilic inflammation typically found in the polypoid nasal tissue of chronic rhinosinusitis patients to the existence of fungi in the nasal and paranasal cavities.

•• 31 Shin S-H, Ponikau JU, Sherris DA, et al. Rhinosinusitis: an enhanced immune response to ubiquitous airborne fungi. J Allergy Clin Immunol 2004; 114: 1369–1375. This study demonstrates that approximately 90% and 100% of PBMCs derived
from patients with chronic rhinosinusitis produced high levels of interleukin-5 and interleukin-13, respectively, but not PBMCs from healthy controls, on stimulation with Alternaria antigens. Furthermore, the levels of interferon-g released by the PBMCs from chronic rhinosinusitis patients are significantly higher than the levels of interferon-g produced by PBMCs from normal controls after stimulation with Alternaria. These reactions seem to be specific for the fungal species of Alternaria. Furthermore, this study demonstrates higher levels of serum IgG antibodies to Alternaria and Cladosporium in patients with chronic rhinosinusitis compared with healthy controls, indicating an increased immunologic exposure of the chronic rhinosinusitis patient’s immune system to these fungi. Only 28% of these chronic rhinosinusitis patients had elevated specific IgE for Alternaria, and no correlation existed between specific IgE for Alternaria and the intensity of the interleukin-5 responses, which strongly questions the relation of IgE and the eosinophilic inflammation found in chronic rhinosinusitis patients.

32 Ferguson BJ: Eosinophilic mucin rhinosinusitis: a distinct clinicopathological entity. Laryngoscope 2000; 110:799–813.

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37 Feger TA, Rupp NT, Kuhn FA, et al. Local and systemic eosinophil activation in allergic fungal sinusitis. Ann Allergy Asthma Immunol 1997; 79:221– 225.

• 38 Ponikau JU, Sherris DA, Kephart GM, et al. Features of airway remodeling and eosinophilic inflammation in chronic rhinosinusitis: is the histopathology similar to asthma? J Allergy Clin Immunol 2003; 112:877–882. This article describes the heterogeneity of the eosinophilic inflammation found in the polypoid sinus tissue of chronic rhinosinusitis patients. Furthermore, interesting histologic similarities between chronic rhinosinusitis and asthma are presented, suggesting a common pathogenesis for both chronic rhinosinusitis and asthma.

39 Hamelmann E, Schleimer RP: Corticosteroid treatment in bronchial asthma: for better or for worse? J Allergy Clin Immunol 2003; 111:248–250.

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41 Hamilos DL, Leung DY, Wood R, et al. Eosinophil infiltration in nonallergic chronic hyperplastic sinusitis with nasal polyposis (CHS/NP) is associated with endothelial VCAM-1 upregulation and expression of TNF-alpha. Am J Respir
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42 al Ghamdi K, Ghaffar O, Small P, et al. IL-4 and IL-13 expression in chronic sinusitis: relationship with cellular infiltrate and effect of topical corticosteroid treatment. J Otolaryngol 1997; 26:160–166.

43 Durham SR, Ying S, Varney VA, et al. Cytokine messenger RNA expression for IL-3, IL-4, IL-5, and granulocyte/macrophage-colony-stimulating factor in the nasal mucosa after local allergen provocation: relationship to tissue
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44 Lopez AF, Sanderson CJ, Gamble JR, et al. Recombinant human interleukin 5 is a selective activator of human eosinophil function. J Exp Med 1988; 167:219–224.

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• 46 Hamilos DL, Leung DY, Huston DP, et al. GM-CSF, IL-5 and RANTES immunoreactivity and mRNA expression in chronic hyperplastic sinusitis with nasal polyposis (NP). Clin Exp Allergy 1998; 28:1145–1152. This article analyzes the cytokine immunoreactivity for interleukin-5, GM-CSF, and RANTES mRNA in allergic and nonallergic nasal polyps from patients with chronic rhinosinusitis using in situ hybridization. The authors demonstrate that GM-CSF, interleukin-5, and RANTES are produced in increased amounts in the polypoid sinus tissue of chronic rhinosinusitis patients compared with healthy controls independently of the patients’ allergy status. The authors hypothesize that mechanisms independent from allergic reactions might be responsible for eosinophil accumulation and activation in allergic vs nonallergic chronic rhinosinusitis
patients.

47 Hamilos DL: Chronic sinusitis. J Allergy Clin Immunol 2000; 106:213–227.

48 Oliveira SH, Fonseca SG, Romao PR, et al. Microbicidal activity of eosinophils is associated with activation of the arginine-NO pathway. Parasite Immunol 1998; 20:405–412.

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50 Sanchez-Segura A, Brieva JA, Rodriguez C: T lymphocytes that infiltrate nasal polyps have a specialized phenotype and produce a mixed TH1/TH2 pattern of cytokines. J Allergy Clin Immunol 1998; 102:953–960.

51 Inoue Y, Shin SH, Ponikau JU, et al. The fungus, Alternaria, induces activation and degranulation of human eosinophils [abstract]. J Allergy Clin Immunol 2000; 109(suppl):S165.

52 Rains BM 3rd, Mineck CW: Treatment of allergic fungal sinusitis with highdose itraconazole. Am J Rhinol 2003; 17:1–8.

53 Marple B, Newcomer M, Schwade N, et al. Natural history of allergic fungal rhinosinusitis: a 4- to 10-year follow-up. Otolaryngol Head Neck Surg 2002; 127:361–366.

54 Ponikau JU, Sherris DA, Kita H, et al. Intranasal antifungal treatment in 51 patients with chronic rhinosinusitis. J Allergy Clin Immunol 2002; 110:862–866.

55 Ricchetti A, Landis BN, Maffioli A, et al. Effect of anti-fungal nasal lavage with amphotericin B on nasal polyposis. J Laryngol Otol 2002; 116:261–263.

•• 56 Miller TR, Muntz HR, Gilbert ME, et al. Comparison of topical medication delivery systems after sinus surgery. Laryngoscope 2004; 114:201–204. In this article, different delivery vehicles for medications into the sinus cavities of patients with chronic rhinosinusitis are investigated. The authors demonstrate that a spray is a vastly inferior delivery vehicle for medications into the sinuses compared with a bulb syringe, even in patients without recurrent nasal polyps after sinus surgery and with open sinus ostia.

•• 57 Ponikau JU, Sherris DA, Weaver A, et al. Treatment of chronic rhinosinusitis with intranasal amphotericin B: a randomized, placebo-controlled, doubleblinded pilot trial. J Allergy Clin Immunol 2005; in press. This is the first double-blind, placebo-controlled trial of consecutive and unselected patients with chronic rhinosinusitis using a bulb syringe as a delivery vehicle. This study demonstrates a reduced inflammatory mucosal thickening on CT scan and nasal endoscopy and decreased levels of intranasal cytokines and markers for eosinophilic inflammation in chronic rhinosinusitis patients compared with placebo after 6 months. These results suggest a beneficial effect of topical antifungal treatment of patients with chronic rhinosinusitis when appropriate delivery methods of the medication and a sufficient duration of the treatment are considered.