Abstract
Objective and hypothesis
The objective of the study was to define the true incidence of fungal elements in the nasal and sinus mucous in cases of chronic rhinosinusitis (CRS) with bilateral polyposis compared with normal controls—in an Egyptian African population—via mycological and histologic techniques.
Study design
This study was conducted prospectively on 100 patients with the clinical diagnosis of CRS with bilateral nasal polyposis. Fifty volunteers with no history of nasal or paranasal sinus disease served as a control group.
Results and conclusion
The postulated criteria for the diagnosis of allergic fungal sinusitis were present in 92% of CRS with polyposis, suggesting that fungi are involved in the disease process of most CRS patients.
1
Introduction
The etiology of nasal polyposis was a subject of great conflict between the authors; some of the most commonly reported causes of nasal polyps include infection, allergy, immunologic factors, metabolic diseases, hereditary diseases, and autonomic dysfunction .
A new etiologic factor was introduced by Safirstein in 1976 who noticed the combination of nasal polyposis, thick tenacious mucin, crust formation, and sinus cultures yielding Aspergillus ; and this started the era of fungal condemnation and gave rise to several descriptive nomenclatures such as allergic aspergillosis of the paranasal sinus , allergic aspergillus sinusitis , and allergic fungal rhinosinusitis .
In 1999, Ponikau et al presented their Mayo clinic experience that showed that most patients with chronic rhinosinusitis (CRS) actually meet their verified criteria for allergic fungal sinusitis (AFS); and they stated a new theory for the pathogenesis that gave rise to the term eosinophilic fungal rhinosinusitis (EFS).
This new theory was presented during the 1999 fall meeting of the American Rhinologic Society (New Orleans); and it was discussed widely during the XVIIth World Congress of the International Federation of Oto-Rhino-Laryngological Societies held in Cairo in 2002, although this theory did not gain the acceptance of all the polyposis-minded rhinologists. However, it provided a new hope for those who are suffering from this dilemma.
The aim of this work is to define the true incidence of fungal elements in the nasal and sinus mucous in cases of CRS with bilateral polyposis compared with normal controls—in an Egyptian African population—via mycological and histologic identification of the various fungal species colonizing the viscid allergic mucin using recent handling techniques in collecting the mucin, and to compare the fungal yield quantitatively and qualitatively with the 2 other reports in the literature studying the fungi in American and European population.
2
Material and methods
2.1
Patients and controls
2.1.1
Inclusion criteria
This study was conducted on 100 patients with the clinical diagnosis of CRS with bilateral nasal polyposis.
Fifty volunteers with no history of nasal or paranasal sinus disease, with no symptoms of inhalant allergy, with no history of smoking, and with normal-appearing mucosa confirmed by nasal endoscopy served as a control group.
Patients and controls were instructed not to use any steroidal medications 1 month before the study to prevent diminishing the eosinophilic inflammation.
2.1.2
Exclusion criteria
All patients with history of diabetes mellitus or with local or systemic corticosteroids medication 1 month before the study were excluded from this study.
All patients studied presented to the Kasr El Aini hospital outpatient clinic during the period from August 2002 to July 2004. Their ages ranged between 14 and 56 years. The study included 66 males and 34 females, and all were subjected to the following:
- □
Full ENT examination.
- □
Nasal endoscopic examination.
- □
Computed tomographic scanning of the nose and paranasal sinuses (coronal and axial 5-mm–thick cuts) in both soft tissue and bone windows.
Informed consents were signed by all the patients for the surgery and for sharing in the scientific research, and the study design was approved by the Cairo University Ethics Committee.
2.2
Collection and culture techniques of nasal lavage
The collection, handling, and culturing techniques were carried out using the novel method described by Ponikau et al in 1999. They found that this method allows collecting as much mucus as possible, improving the fungal yield in the cultures.
Two puffs of phenylephrine hydrochloride 1% are sprayed into each nostril to produce vasoconstriction. After approximately 2 minutes, each nostril is flushed with 20 mL of sterile saline using a sterile syringe with a sterile curved blunt needle. The patient takes a deep inspiratory breath and holds it before the injection of saline. The patient then forcefully exhales through the nose during the flushing. The return is collected in a sterile pan.
The collected fluid was placed into centrifuge tubes and sent directly to the mycology laboratory where the specimen is processed under a laminar flow hood to prevent contamination. One vial (10 mL) of sterile dithiothreitol (Sputolysin; Oxoid, England) was diluted with 90 mL of sterile water. The collected specimen is suspended with an equal volume of diluted dithiothreitol and vortexed for 30 seconds. The mixture was allowed to stand at room temperature for 15 minutes while the dithiothreitol breaks apart the disulfide bonds, thus liquefying the mucus and allowing the fungal elements to be released from the viscid mucus and contact the growth media adequate for fungal growth.
The mixture is then centrifuged at 3000 rpm in a 50-mL tube for 10 minutes. The supernatant was discarded, and the sediment was vortexed for 30 seconds. One-half milliliter of the prepared sediment was inoculated onto each of 3 types of cultures: (1) Sabouraud dextrose agar (SDA) plate, (2) SDA plate containing gentamicin (5 μ g/mL) and chloramphenicol (15 μ g/mL), and (3) inhibitory mould agar containing 5% chloramphenicol (15 μ g/mL) and cycloheximide (5 mg/mL). The plates were incubated at 30°C and allowed to grow for 30 days. The plates were examined at 2-day intervals, and all cultures were identified. The resulting growth was subcultured on corn meal agar for proper identification and isolation of the different species harvested.
2.3
Collection of surgical specimens and histologic examination
The patients were subjected to endoscopic sinus surgery under general anesthesia. The principle of maximum mucus preservation was adhered to during the acquisition of surgical specimens. This enabled the pathologist to find allergic mucin and fungal elements within the mucus. The use of suction devices was limited. The mucus was manually removed together with inflamed tissue and placed on a saline-moistened nonstick sheet. Specimens were not placed directly on a surgical towel or on gauze because these carriers absorb a large amount of the mucus.
The paraffin-embedded blocks were cut into several serial sections of 5 mm each and baked onto positively charged glass slides. The sections were stained with hematoxylin and eosin (H&E), Gomori methenamine silver (GMS), periodic acid-Schiff stain (PAS), and chitinase stain.
Each of the H&E-, GMS-, and PAS-stained specimens was thoroughly examined under light microscopy for the presence of fungal elements. A specimen was considered positive if at least one fungal hypha could be clearly identified in an entire slide.
The serial section slides immediately adjacent to the PAS slides were used for chitinase staining following the technique stated by Taylor et al in 2002. The specimens were deparaffinized and rehydrated using xylene, ethanol, and distilled water. Because the eosinophil-rich environment increases the background staining of fluorescein, the sections were incubated for 30 minutes in 1% chromotrope 2R to decrease nonspecific binding. The specimens were then washed in phosphate-buffered saline for 15 minutes and incubated in 5% periodic acid for 10 minutes. The slides were then rinsed with distilled water and incubated in 5% bovine serum albumin containing 0.1% azide for 10 minutes. One drop of the fluorescein-labeled chitinase stain (Fungalase-F; Anomeric, Inc, Baton Rouge, LA) was then added; and the slides were incubated in a dark, humidified chamber for 30 minutes. Once the staining was completed, the slides were rinsed and washed in phosphate-buffered saline solution for 2 minutes. The specimens were then mounted with 1 drop of Vectashield mounting medium (Oxoid, Hampshire, United Kingdom), and a coverslip was applied. All stained specimens were stored in the darkness at 4°C .
Each slide was then examined with a Zeiss microscope equipped with Zeiss UV FL vertical illumination for epifluorescence and a fluorescein filter system (Carl Zeiss, Inc, Oberkochen, Germany) (magnification range: ×100, ×200, ×400, and ×1000) within 1 week to avoid fading. Fluorescence photomicrographs were obtained through both the immunofluorescence microscope and the confocal laser microscope.
A specimen was considered positive if all of the 3 following criteria were met: (1) the element in question enhanced brightly; (2) it had the characteristic morphology of fungal hyphae; and (3) it traveled through the mucin indicating the tubular structure, not merely overlying the specimen. Because chitinase binds to the fungal cell wall only, specimens were examined for the fluorescent outline of fungal hyphae.
These outlines vary in shape, ranging from circular to oblique to longitudinal depending on the angle at which the specimen was cut. The outlined rims of the fungal hyphae could be followed as it traveled through the full thickness of the specimen, ensuring that the slides were not secondarily contaminated by environmental fungi.
2
Material and methods
2.1
Patients and controls
2.1.1
Inclusion criteria
This study was conducted on 100 patients with the clinical diagnosis of CRS with bilateral nasal polyposis.
Fifty volunteers with no history of nasal or paranasal sinus disease, with no symptoms of inhalant allergy, with no history of smoking, and with normal-appearing mucosa confirmed by nasal endoscopy served as a control group.
Patients and controls were instructed not to use any steroidal medications 1 month before the study to prevent diminishing the eosinophilic inflammation.
2.1.2
Exclusion criteria
All patients with history of diabetes mellitus or with local or systemic corticosteroids medication 1 month before the study were excluded from this study.
All patients studied presented to the Kasr El Aini hospital outpatient clinic during the period from August 2002 to July 2004. Their ages ranged between 14 and 56 years. The study included 66 males and 34 females, and all were subjected to the following:
- □
Full ENT examination.
- □
Nasal endoscopic examination.
- □
Computed tomographic scanning of the nose and paranasal sinuses (coronal and axial 5-mm–thick cuts) in both soft tissue and bone windows.
Informed consents were signed by all the patients for the surgery and for sharing in the scientific research, and the study design was approved by the Cairo University Ethics Committee.
2.2
Collection and culture techniques of nasal lavage
The collection, handling, and culturing techniques were carried out using the novel method described by Ponikau et al in 1999. They found that this method allows collecting as much mucus as possible, improving the fungal yield in the cultures.
Two puffs of phenylephrine hydrochloride 1% are sprayed into each nostril to produce vasoconstriction. After approximately 2 minutes, each nostril is flushed with 20 mL of sterile saline using a sterile syringe with a sterile curved blunt needle. The patient takes a deep inspiratory breath and holds it before the injection of saline. The patient then forcefully exhales through the nose during the flushing. The return is collected in a sterile pan.
The collected fluid was placed into centrifuge tubes and sent directly to the mycology laboratory where the specimen is processed under a laminar flow hood to prevent contamination. One vial (10 mL) of sterile dithiothreitol (Sputolysin; Oxoid, England) was diluted with 90 mL of sterile water. The collected specimen is suspended with an equal volume of diluted dithiothreitol and vortexed for 30 seconds. The mixture was allowed to stand at room temperature for 15 minutes while the dithiothreitol breaks apart the disulfide bonds, thus liquefying the mucus and allowing the fungal elements to be released from the viscid mucus and contact the growth media adequate for fungal growth.
The mixture is then centrifuged at 3000 rpm in a 50-mL tube for 10 minutes. The supernatant was discarded, and the sediment was vortexed for 30 seconds. One-half milliliter of the prepared sediment was inoculated onto each of 3 types of cultures: (1) Sabouraud dextrose agar (SDA) plate, (2) SDA plate containing gentamicin (5 μ g/mL) and chloramphenicol (15 μ g/mL), and (3) inhibitory mould agar containing 5% chloramphenicol (15 μ g/mL) and cycloheximide (5 mg/mL). The plates were incubated at 30°C and allowed to grow for 30 days. The plates were examined at 2-day intervals, and all cultures were identified. The resulting growth was subcultured on corn meal agar for proper identification and isolation of the different species harvested.
2.3
Collection of surgical specimens and histologic examination
The patients were subjected to endoscopic sinus surgery under general anesthesia. The principle of maximum mucus preservation was adhered to during the acquisition of surgical specimens. This enabled the pathologist to find allergic mucin and fungal elements within the mucus. The use of suction devices was limited. The mucus was manually removed together with inflamed tissue and placed on a saline-moistened nonstick sheet. Specimens were not placed directly on a surgical towel or on gauze because these carriers absorb a large amount of the mucus.
The paraffin-embedded blocks were cut into several serial sections of 5 mm each and baked onto positively charged glass slides. The sections were stained with hematoxylin and eosin (H&E), Gomori methenamine silver (GMS), periodic acid-Schiff stain (PAS), and chitinase stain.
Each of the H&E-, GMS-, and PAS-stained specimens was thoroughly examined under light microscopy for the presence of fungal elements. A specimen was considered positive if at least one fungal hypha could be clearly identified in an entire slide.
The serial section slides immediately adjacent to the PAS slides were used for chitinase staining following the technique stated by Taylor et al in 2002. The specimens were deparaffinized and rehydrated using xylene, ethanol, and distilled water. Because the eosinophil-rich environment increases the background staining of fluorescein, the sections were incubated for 30 minutes in 1% chromotrope 2R to decrease nonspecific binding. The specimens were then washed in phosphate-buffered saline for 15 minutes and incubated in 5% periodic acid for 10 minutes. The slides were then rinsed with distilled water and incubated in 5% bovine serum albumin containing 0.1% azide for 10 minutes. One drop of the fluorescein-labeled chitinase stain (Fungalase-F; Anomeric, Inc, Baton Rouge, LA) was then added; and the slides were incubated in a dark, humidified chamber for 30 minutes. Once the staining was completed, the slides were rinsed and washed in phosphate-buffered saline solution for 2 minutes. The specimens were then mounted with 1 drop of Vectashield mounting medium (Oxoid, Hampshire, United Kingdom), and a coverslip was applied. All stained specimens were stored in the darkness at 4°C .
Each slide was then examined with a Zeiss microscope equipped with Zeiss UV FL vertical illumination for epifluorescence and a fluorescein filter system (Carl Zeiss, Inc, Oberkochen, Germany) (magnification range: ×100, ×200, ×400, and ×1000) within 1 week to avoid fading. Fluorescence photomicrographs were obtained through both the immunofluorescence microscope and the confocal laser microscope.
A specimen was considered positive if all of the 3 following criteria were met: (1) the element in question enhanced brightly; (2) it had the characteristic morphology of fungal hyphae; and (3) it traveled through the mucin indicating the tubular structure, not merely overlying the specimen. Because chitinase binds to the fungal cell wall only, specimens were examined for the fluorescent outline of fungal hyphae.
These outlines vary in shape, ranging from circular to oblique to longitudinal depending on the angle at which the specimen was cut. The outlined rims of the fungal hyphae could be followed as it traveled through the full thickness of the specimen, ensuring that the slides were not secondarily contaminated by environmental fungi.
3
Results
In this study, the age and sex distribution of cases is seen in Table 1 . The average period of the patient complaint was 1.2 years, 58 patients did not have any previous surgical intervention, 22 patients had a single previous nasal operation to relief symptoms, 15 patients had 2 operations, and 5 patients had 3 or more previous operations.
| Sex | n | Minimum age | Maximum age | Mean age | SD |
|---|---|---|---|---|---|
| Female | 34 | 19 | 60 | 36.04 | 9.787 |
| Male | 66 | 17 | 55 | 27.65 | 8.447 |
All patients had a broad spectrum of inflammatory mucosal thickening ranging from minimal polypoid changes (n = 32) to massive polyposis (n = 68).
3.1
Mycological results
The new collection and culturing method stated by Ponikau et al (1999) that was used in this study resulted in positive cultures for fungi in 100 (100%) of the 100 consecutive patients with CRS using the SDA medium and in 50 (100%) of the control group of normal, healthy volunteers using the same medium.
The cultures using the SDA, chloramphenicol, and gentamicin medium resulted in 98 (98%) positive cultures of the same 100 consecutive patients and 49 (98%) positive cultures of the control group of normal, healthy volunteers using the same medium.
The cultures using the SDA and cycloheximide medium resulted in 83 (83%) positive cultures of the same 100 consecutive patients and 41 (82%) positive cultures of the control group of normal, healthy volunteers using the same medium.
In the patients group, an average of 2.8 organisms per patient and a maximum of 5 different organisms were found on cultures. A total of 29 different genera or species of fungi were identified. Interestingly, in the control group of normal, healthy volunteers, an average of 2.8 different organisms per volunteer also and a maximum of 4 different organisms per subject were grown. The organisms grown from the controls were not markedly different than those from the CRS patients ( Tables 2-4 ).
| Category of cases studied | No. of organisms | Frequency | Percentage |
|---|---|---|---|
| Controls | 0 | 0 | 0.00 |
| 1 | 7 | 14.00 | |
| 2 | 19 | 38.00 | |
| 3 | 19 | 38.00 | |
| 4 | 5 | 10.00 | |
| 5 | 0 | 0.00 | |
| Total | 50 | 100.00 | |
| Patients | 0 | 0 | 0.00 |
| 1 | 15 | 15.00 | |
| 2 | 34 | 34.00 | |
| 3 | 38 | 38.00 | |
| 4 | 12 | 12.00 | |
| 5 | 1 | 1.00 | |
| Total | 100 | 100.00 |
| Category of cases studied | No. of organisms | Frequency | Percentage |
|---|---|---|---|
| Controls | 0 | 1 | 2.00 |
| 1 | 11 | 22.00 | |
| 2 | 21 | 42.00 | |
| 3 | 14 | 28.00 | |
| 4 | 3 | 6.00 | |
| 5 | 0 | 0.00 | |
| Total | 50 | 100.00 | |
| Patients | 0 | 2 | 2.00 |
| 1 | 20 | 20.00 | |
| 2 | 39 | 39.00 | |
| 3 | 28 | 28.00 | |
| 4 | 9 | 9.00 | |
| 5 | 2 | 2.00 | |
| Total | 100 | 100.00 |
| Category of cases studied | No. of organisms | Frequency | Percentage |
|---|---|---|---|
| Controls | 0 | 9 | 18.00 |
| 1 | 15 | 30.00 | |
| 2 | 21 | 42.00 | |
| 3 | 3 | 6.00 | |
| 4 | 2 | 4.00 | |
| 5 | 0 | 0.00 | |
| Total | 50 | 100.00 | |
| Patients | 0 | 17 | 17.00 |
| 1 | 38 | 38.00 | |
| 2 | 30 | 30.00 | |
| 3 | 9 | 9.00 | |
| 4 | 5 | 5.00 | |
| 5 | 1 | 1.00 | |
| Total | 100 | 100.00 |
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