Home About us Editorial board Ahead of print Browse Articles Search Submit article Instructions Subscribe Contacts Login 
  • Users Online: 468
  • Home
  • Print this page
  • Email this page


 
Previous article Browse articles Next article 
ORIGINAL ARTICLE
J Res Med Sci 2017,  22:67

An investigation on non-invasive fungal sinusitis; Molecular identification of etiologic agents


1 Department of Medical Parasitology and Mycology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
2 Department of Otolaryngology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
3 Department of Medical Parasitology and Mycology, School of Medicine; Infectious Diseases and Tropical Medicine Research Center, Isfahan University of Medical Sciences, Isfahan, Iran

Date of Submission13-Feb-2017
Date of Decision22-Feb-2017
Date of Acceptance25-Feb-2017
Date of Web Publication30-May-2017

Correspondence Address:
Rasoul Mohammadi
Department of Medical Parasitology and Mycology, School of Medicine, Infectious Diseases and Tropical Medicine Research Center, Isfahan University of Medical Sciences, Isfahan
Iran
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jrms.JRMS_166_17

Rights and Permissions
  Abstract 

Background: Fungal sinusitis is increasing worldwide in the past two decades. It is divided into two types including invasive and noninvasive. Noninvasive types contain allergic fungal sinusitis (AFS) and fungus ball. AFS is a hypersensitivity reaction to fungal allergens in the mucosa of the sinonasal tract in atopic individuals. The fungus ball is a different type of noninvasive fungal rhinosinusitis which is delineated as an accumulation of debris and fungal elements inside a paranasal sinus. Fungal sinusitis caused by various fungi such as Aspergillus species, Penicillium, Mucor, Rhizopus, and phaeohyphomycetes. The aim of the present study is to identify fungal species isolated from noninvasive fungal sinusitis by molecular methods. Materials and Methods: During 2015–2016, a total of 100 suspected patients were examined for fungal sinusitis. Functional endoscopic sinus surgery was performed using the Messerklinger technique. Clinical samples were identified by phenotypic and molecular methods. Polymerase chain reaction (PCR) sequencing of ITS1-5.8S-ITS2 region and PCR-restriction fragment length polymorphism with Msp I restriction enzyme was performed for molecular identification of molds and yeasts, respectively. Results: Twenty-seven out of 100 suspected cases (27%) had fungal sinusitis. Nasal congestion (59%) and headache (19%) were the most common clinical signs among patients. Fifteen patients (55.5%) were male and 12 patients (44.5%) were female. Aspergillus flavus was the most prevalent fungal species (26%), followed by Penicillium chrysogenum (18.5%) and Candida glabrata species complex (15%). Conclusion: Since clinical manifestations, computed tomography scan, endoscopy, and histopathological findings are very nonspecific in AFS and fungus ball; therefore, molecular investigations are compulsory for precise identification of etiologic agents and appropriate management of these fungal infections.

Keywords: Fungal agents, molecular identification, non-invasive, sinusitis


How to cite this article:
Mohammadi A, Hashemi SM, Abtahi SH, Lajevardi SM, Kianipour S, Mohammadi R. An investigation on non-invasive fungal sinusitis; Molecular identification of etiologic agents. J Res Med Sci 2017;22:67

How to cite this URL:
Mohammadi A, Hashemi SM, Abtahi SH, Lajevardi SM, Kianipour S, Mohammadi R. An investigation on non-invasive fungal sinusitis; Molecular identification of etiologic agents. J Res Med Sci [serial online] 2017 [cited 2018 Jul 20];22:67. Available from: http://www.jmsjournal.net/text.asp?2017/22/1/67/207290


  Introduction Top


Allergic fungal sinusitis (AFS) is a hypersensitivity reaction to fungal allergens including surface antigens and fungal metabolites in the mucosa of the sinonasal tract in atopic individuals. It was first reported by Katzenstein et al. in 1983.[1] Clinical manifestations present with different signs containing nasal congestion, facial deformity, diplopia, osteolytic impairment, and entanglement of the skull base.[2] The incidence of disease is unknown and seems account for up to 10% of surgical cases;[3] however, mixed bacterial and fungal infections represent 13%–28.5% of all maxillary sinusitis cases.[4] The diagnosis of AFS is supported by the presence of thickened allergic mucin that is coarsely evident at surgery and a positive histopathological staining for fungal hyphae.[5] Fungus ball is a type of noninvasive chronic fungal sinusitis without thickened allergic mucin. It is composed of fungal hypha, fibrin, inflammatory cell, shapeless debris, and mucous in pulmonary cavities. It usually appears in the maxillary and sphenoid sinuses.[4] Fungus ball is commonly found in older individuals and females.[6],[7] A variety of fungal agents such as yeasts, dematiaceous fungi, and hyaline fungi have been involved in fungal sinusitis.[8],[9] The aim of the present study is to identify the etiologic agents of fungal sinusitis isolated from AFS and fungus ball using molecular methods.


  Materials and Methods Top


Clinical samples

During December 2015 to December 2016, a total of 100 cases referred to the two university hospitals (Alzahra and Kashani Hospitals) of Isfahan University of Medical Sciences, Isfahan, Iran, due to the acute or chronic sinusitis. All patients were nominated for computed tomography (CT) scan of the paranasal sinuses (PNS). Boney erosion, inspissated mucus in sinuses that cause heterogeneous soft tissue density and calcification were clinical symptoms among patients. Functional endoscopic sinus surgery was performed using the Messerklinger technique by means of Karl Storz Endoskope (Germany). The Messerklinger technique is a mainly diagnostic endoscopic concept presenting that the maxillary and frontal sinuses are subordinate cavities. It can be used for various scopes of indications, excluding nasal polyposis. Sufficient experience and training are needed for the surgical approach.[10] All samples were sent to the medical mycology laboratory in serum saline for direct microscopy and culture. Potassium hydroxide 20% and Sabouraud Dextrose Agar with chloramphenicol (BD, USA) were used for direct microscopic examination and culture, respectively. Periodic acid-Schiff (PAS) (Sigma-Aldrich, USA) was applied for histopathological staining.

Molecular identification

DNA extraction

DNA of fungi was extracted using glass beads (Sigma-Aldrich, USA) and boiling method.[11] Briefly, a bit of fresh colonies were suspended in 50 μl of double distilled water and simmered for 10 min, afterward centrifuged for 5 min at 5000 rpm, and supernatant was separated for polymerase chain reaction (PCR). ITS1-5.8S-ITS2 rDNA region of fungi was amplified using ITS1 (5'-TCC GTA GGT GAA CCT GCG G-3') and ITS4 (5'-TCC TCC GCT TAT TGA TAT GC-3') primers.[12]

Polymerase chain reaction

The PCR cycling was performed as follows: initial denaturation phase at 95°C for 5 min, followed by 32 cycles of denaturation at 94°C for 35 s, annealing at 55°C for 45 s, and extension at 72°C for 1 min, with a final extension phase at 72°C for 7 min. PCR products were purified by QIAquick PCR Purification Kit (Qiagen, USA). Five microliters of each amplicon was run onto 1.5% agarose gel, electrophoresed in TBE buffer (90 mM boric acid, 2 mM 127 ethylenediaminetetraacetic acid, and 90 mM Tris) at 10 V/cm for 90 min, and stained with 0.5 μg/ml ethidium bromide (Sigma-Aldrich, USA).

Sequencing

Purified PCR products were employed for cycle sequencing reactions in forward direction (Bioneer, Korea). Sequence products were analyzed with Chromas Software 2.4 (http://www.chromas.software.informer.com/2.4/) and evaluated using of NCBI BLAST searches against fungal sequences existing in DNA databases (http://www.blast.ncbi.nlm.nih.gov/Blast.cgi).

Restriction fragment length polymorphism

All clinical yeasts were identified by restriction fragment length polymorphism patterns of PCR products.[13],[14]Msp I (Hpa II) restriction enzyme (Fermentas, Vilnius, Lithuania) was used in this step of identification.


  Results Top


Fungal sinusitis was diagnosed for 27 out of 100 suspected cases (27%). The most patients were house keeper (41%) [Table 1]. Nasal congestion (n = 16; 59%) and headache (n = 5; 19%) were the most common clinical signs among patients [Table 1]. Fifteen patients (55.5%) were male and 12 patients (44.5%) were female. Polyps (33%), diabetes mellitus (30%), and hypothyroidism (15%) were the most risk factors among patients. Age range of patients was between 8 and 88 years, with a mean age of 46 years. Aspergillus flavus was the most prevalent fungal species (26%), followed by Penicillium chrysogenum (18.5%) and Candida glabrata species complex (15%). [Table 1] summarizes characteristics of patients of the present study.
Table 1: Details of patients with allergic fungal sinusitis and fungus ball in the present study

Click here to view



  Discussion Top


Fungal sinusitis is increasing worldwide in the past two decades. It is divided into two types including invasive and non-invasive. Invasive fungal sinusitis (IFS) represents acute, chronic, and granulomatous forms of infection. Many fungi are implicated containing: Zygomycetes (usually in diabetic patients), Aspergillus species (in neutropenic patients), Mucor spp., Rhizopus spp., Rhizomucor spp., and Lichtheimia spp. Noninvasive types contain AFS and fungus ball.[9] AFS is pathologically and clinically a specific form of rhinosinusitis in atopic immunocompetent people. It is most frequent among young individuals; the mean age of occurrence is about 21 years, whereas the mean age of patients in the present study was 46 years. Many studies reported that the male-to-female sex ratio is same;[15],[16] however, in the present investigation, the sex ratio of infected individuals was nearly the same (15/12). Hypersensitivity reaction in the mucosa of the sinonasal tract, tissue edema, and reproduction of organisms intensifies inflammation that cause polyposis and chronic rhinosinusitis;[16] here, we found nine patients (33%) with polyposis in this study, too. This condition occurs with a wide variety of fungi such as Aspergillus species, Penicillium, Mucor, Rhizopus, and many phaeohyphomycetes.[17] Patients with AFS usually show signs of chronic sinusitis and allergic rhinitis containing purulent rhinorrhea, nasal obstruction, bony erosion, postnasal drainage, and headache. Pain is a rare sign; however, seven patients (26%) had made a complaint of headache, eyesore, and facial pain, in the present survey. Mucin in the AFS is plentiful, thick, firm, and adhesive. It has better appearance leading to the nonspecific radiographic findings of AFS on CT scans, i.e. heterogeneous areas within the PNS that are seen due to the collection of heavy metals such as manganese, iron, accompanied by calcium crystals in the inspissated mucin.[18] All PNS and nasal cavity are usually involved, with the maxillary and ethmoid sinuses being the most common.[15] In the present investigation, 15% of cases were diagnosed with the involvement of maxillary and ethmoid sinuses. In terms of histology, AFS has a typical appearance of “tree rings” or “tide lines” of mucin interchanging with inflammatory fragments, which reveals as alternating blue and pink lines. The debris is composed of aggregated neutrophils, eosinophils, and deteriorated eosinophils forming Charcot–Leyden crystals that are bipyramidal, refractile, and needle shaped.[3] Identification of fungal elements is complicated on hematoxylin and eosin staining and may be seen with PAS and Grocott's methenamine silver. For this reason, all clinical samples in this study were stained with PAS, and fungal hyphae were diagnosed clearly. If fungal elements are not seen using of culture and staining, the entity of AFS may be diagnosed as eosinophilic mucin rhinosinusitis.[19] Treatment of AFS is efficient with debridement of involved tissue, complete evacuation of inspissated mucus, and polypectomy. [Table 1] show the different types of surgery were performed for each patient in the present study. Postoperative steroids' therapy is usually recommended to decrease allergy, inflammation, and sensitization for long-term control, but recurrences are usual.[15],[16],[20] All patients are usually treated with intranasal steroid sprays, similar to the patients of this investigation. AFS is treated much differently than IFS and must be marked with discerning histopathologic features of IFS that is inflammatory cells within the tissue instead of free-floating in mucin, presence of fungal elements within the vascular spaces and tissues.[16],[21] The fungus ball (mycetoma) is a different type of noninvasive fungal rhinosinusitis which is delineated as an accumulation of debris and fungal elements inside a PNS. Fungus ball of the PNS is characterized by a mass of fungal elements and fruiting heads of fungi and minimal host immune responses. It is always located in the maxillary sinus and sphenoid sinus, respectively.[22] Various fungal species have been isolated from fungus ball such as Aspergillus, Alternaria, Mucor, Penicillium, and Bipolaris.[22],[23] Some risk factors have been suggested for fungus ball such as diabetes mellitus,[24] previous surgery on the sinonasal area,[25] estrogens,[26] and living in a rural area.[25] In the present study, we recognized a patient with fungus ball (patient no. 24) [Table 1]; however, she did not live in rural regions, and also she did not have any predisposing factor. The etiologic agent of fungus ball was Geotrichum candidum, which is yeast-like fungus with worldwide distribution. The pathogenicity of G. candidum in humans has not been obviously determined.[27] To the best of our knowledge, this is the first case of fungus ball owing to G. candidum reported in Iran. A. flavus was the most common species in the present study, whereas Marglani and Shaikh [9] reported Aspergillus terreus and Aspergillus fumigatus as the etiologic agent of AFS by phenotypic methods. Cavanna et al.[28] isolated a dematiaceous fungus as the Curvularia lunata from an immunocompetent male with allergic fungal rhinosinusitis. Similar to the present study, the fungus was identified by sequencing of internal transcribed spacer regions of fungal ribosomal DNA. Gupta et al.[29] reported a case of chronic rhinosinusitis with the involvement of orbit by Exserohilum rostratum in an immunocompetent child. In opposition to the present study, the fungus was only identified on the basis of morphological features such as direct microscopy, culture, and histopathology. Azar et al.[30] reported the frequency of various fungi using histological assays and culture from biopsy samples of patients with rhinosinusitis, in Khorramabad. Candida albicans, A. flavus, and A. fumigatus were the most common fungal species in their study. Nazeri et al.[31] showed A. flavus as the most prevalent causative agent of allergic fungal rhinosinusitis, chronic invasive type, and fungus ball. They also isolated a few infrequent fungal species such as Fusarium proliferatum and Schizophyllum commune from clinical samples based on PCR sequencing. Phenotypic methods were used in Jahromi and Khaksar in a retrospective investigation.[32] They reported A. flavus and C. albicans as predominant fungal species isolated from PNS.


  Conclusion Top


Gene sequencing can be helpful for identification of etiologic agents of AFS and fungus ball and may be functional for discriminating of phaeohyphomycetes from other filamentous fungi, especially if nonsporulating molds that are fast growing at 37°C; fails to identify on general media with actidione. Since clinical manifestations, CT scan, endoscopy, and histopathological reactions are very nonspecific in AFS and fungus ball; therefore, molecular investigations are compulsory for precise identification of etiologic agents and appropriate management of these fungal infections.

Acknowledgments

The authors thank Alzahra and Kashani Hospitals personnel for their cooperation.

Financial support and sponsorship

This investigation has been sponsored by Isfahan University of Medical Sciences, Isfahan, Iran (Thesis No. 395196).

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Katzenstein AL, Sale SR, Greenberger PA. Allergic Aspergillus sinusitis: A newly recognized form of sinusitis. J Allergy Clin Immunol 1983;72:89-93.  Back to cited text no. 1
    
2.
Manning SC, Merkel M, Kriesel K, Vuitch F, Marple B. Computed tomography and magnetic resonance diagnosis of allergic fungal sinusitis. Laryngoscope 1997;107:170-6.  Back to cited text no. 2
    
3.
Marple BF. Allergic fungal rhinosinusitis: Current theories and management strategies. Laryngoscope 2001;111:1006-19.  Back to cited text no. 3
    
4.
Grosjean P, Weber R. Fungus balls of the paranasal sinuses: A review. Eur Arch Otorhinolaryngol 2007;264:461-70.  Back to cited text no. 4
    
5.
Schubert MS. Allergic fungal sinusitis: Pathophysiology, diagnosis and management. Med Mycol 2009;47 Suppl 1:S324-30.  Back to cited text no. 5
    
6.
Lai JC, Lee HS, Chen MK, Tsai YL. Patient satisfaction and treatment outcome of fungus ball rhinosinusitis treated by functional endoscopic sinus surgery. Eur Arch Otorhinolaryngol 2011;268:227-30.  Back to cited text no. 6
    
7.
Nicolai P, Lombardi D, Tomenzoli D, Villaret AB, Piccioni M, Mensi M, et al. Fungus ball of the paranasal sinuses: Experience in 160 patients treated with endoscopic surgery. Laryngoscope 2009;119:2275-9.  Back to cited text no. 7
    
8.
Al-Dousary SH. Allergic fungal sinusitis: Radiological and microbiological features of 59 cases. Ann Saudi Med 2008;28:17-21.  Back to cited text no. 8
[PUBMED]  [Full text]  
9.
Marglani O, Shaikh AM. Allergic fungal sinusitis eroding the pterygoid plates: A rare case series. Braz J Otorhinolaryngol 2015;81:109-12.  Back to cited text no. 9
    
10.
Stammberger H, Posawetz W. Functional endoscopic sinus surgery. Concept, indications and results of the Messerklinger technique. Eur Arch Otorhinolaryngol 1990;247:63-76.  Back to cited text no. 10
    
11.
Silva GA, Bernardi TL, Schaker PD, Menegotto M, Valente P. Rapid yeast DNA extraction by boiling and freeze-thawing without using chemical reagents and DNA purification. Braz Arch Biol Technol 2012;55:319-27.  Back to cited text no. 11
    
12.
Mohammadi R, Abastabar M, Mirhendi H, Badali H, Shadzi S, Chadeganipour M, et al. Use of restriction fragment length polymorphism to rapidly identify dermatophyte species related to dermatophytosis. Jundishapur J Microbiol 2015;8:e17296.  Back to cited text no. 12
    
13.
Chadeganipour M, Mohammadi R. Causative agents of onychomycosis: A 7-year study. J Clin Lab Anal 2016;30:1013-20.  Back to cited text no. 13
    
14.
Yazdani MR, Foroughifar E, Mohammadi R. Identification of Candida species isolated from renal transplant recipients with candiduria. Int J Organ Transplant Med 2016;7:206-11.  Back to cited text no. 14
    
15.
Correll DP, Luzi SA, Nelson BL. Allergic fungal sinusitis. Head Neck Pathol 2015;9:488-91.  Back to cited text no. 15
    
16.
Huchton DM, editor. Allergic fungal sinusitis: An otorhinolaryngologic perspective. Allergy and Asthma Proceedings. USA: OceanSide Publications, Inc.; 2003.  Back to cited text no. 16
    
17.
Arsenijevic VA, Barac A, Pekmezovic M, Stosovic R, Pender I. Allergic fungal sinusitis – New aspects of clinical features, laboratory diagnosis and therapy. Srp Arh Celok Lek 2013;141:698-704.  Back to cited text no. 17
    
18.
deShazo RD, O'Brien M, Chapin K, Soto-Aguilar M, Swain R, Lyons M, et al. Criteria for the diagnosis of sinus mycetoma. J Allergy Clin Immunol 1997;99:475-85.  Back to cited text no. 18
    
19.
Thompson LD, Wenig BM. Diagnostic Pathology: Head and Neck. Amirsys: Elsevier Health Sciences; 2016.  Back to cited text no. 19
    
20.
Zeid NG, Mohammed AS, El-Fouly ME, El-Essawy MS. The relationship between systemic strioid therapy and the level of serum IgE in allergic fungal rhinosinusitis. Zagazig Univ Med J 2016;22:250-7.  Back to cited text no. 20
    
21.
Driemel O, Wagner C, Hurrass S, Müller-Richter U, Kühnel T, Reichert TE, et al. Allergic fungal sinusitis, fungus ball and invasive sinonasal mycosis – Three fungal-related diseases. Mund Kiefer Gesichtschir 2007;11:153-9.  Back to cited text no. 21
    
22.
Lop-Gros J, Gras-Cabrerizo JR, Bothe-González C, Montserrat-Gili JR, Sumarroca-Trouboul A, Massegur-Solench H. Fungus ball of the paranasal sinuses: Analysis of our serie of patients. Acta Otorrinolaringol Esp 2016;67:220-5.  Back to cited text no. 22
    
23.
Welsh O, Vera-Cabrera L, Salinas-Carmona MC. Mycetoma. Clin Dermatol 2007;25:195-202.  Back to cited text no. 23
    
24.
Chusakul S. Is high blood sugar a risk factor for paranasal sinus fungus ball? Otolaryngol Head Neck Surg 2013;149 2 Suppl: P261.  Back to cited text no. 24
    
25.
Nomura K, Asaka D, Nakayama T, Okushi T, Matsuwaki Y, Yoshimura T, et al. Sinus fungus ball in the Japanese population: Clinical and imaging characteristics of 104 cases. Int J Otolaryngol 2013;2013:731640.  Back to cited text no. 25
    
26.
Chobillon MA, Jankowski R. What are the advantages of the endoscopic canine fossa approach in treating maxillary sinus aspergillomas? Rhinology 2004;42:230-5.  Back to cited text no. 26
    
27.
Yegneswaran Prakash P, Seetaramaiah VK, Thomas J, Khanna V, Rao SP. Renal fungal bezoar owing to Geotrichum candidum. Med Mycol Case Rep 2012;1:63-5.  Back to cited text no. 27
    
28.
Cavanna C, Seminari E, Pusateri A, Mangione F, Lallitto F, Esposto MC, et al. Allergic fungal rhinosinusitis due to Curvularia lunata. New Microbiol 2014;37:241-5.  Back to cited text no. 28
    
29.
Gupta A, Xess I, Sharma SC, Mallick S. Invasive rhinosinusitis by Exserohilum rostratum in an immunocompetent child. BMJ Case Rep 2014;2014. pii: Bcr2013202380.  Back to cited text no. 29
    
30.
Azar S, Mansour B, Parivash K, Babak B. Fungal rhinosinusitis in hospitalized patients in Khorramabad, Iran. Middle East J Sci Res 2011;7:387-91.  Back to cited text no. 30
    
31.
Nazeri M, Hashemi SJ, Ardehali M, Rezaei S, Seyedmousavi S, Zareei M, et al. Fungal rhino sinusitisin in Tehran, Iran. Iran J Public Health 2015;44:374-9.  Back to cited text no. 31
    
32.
Jahromi SB, Khaksar AA. Paranasal sinus mycosis in suspected fungal sinusitis. Arch Clin Infect Dis 2006;1:25-9.  Back to cited text no. 32
    



 
 
    Tables

  [Table 1]


This article has been cited by
1 Isolation and molecular characterization of Cryptococcus species isolated from pigeon nests and Eucalyptus trees
Abuzar Kamari,Asghar Sepahvand,Rasoul Mohammadi
Current Medical Mycology. 2017; 3(2): 20
[Pubmed] | [DOI]



 

Top
Previous article  Next article
 
  Search
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Access Statistics
    Email Alert *
    Add to My List *
* Registration required (free)  

 
  In this article
Abstract
Introduction
Materials and Me...
Results
Discussion
Conclusion
References
Article Tables

 Article Access Statistics
    Viewed964    
    Printed5    
    Emailed0    
    PDF Downloaded132    
    Comments [Add]    
    Cited by others 1    

Recommend this journal