A study on the fungal rhinosinusitis: Causative agents, symptoms, and predisposing factors
Ebrahim Taghian1, Sayed Hamidreza Abtahi2, Abdolrasoul Mohammadi1, Seyed Mostafa Hashemi2, Kazem Ahmadikia3, Somayeh Dolatabadi4, Rasoul Mohammadi5
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 Public Health, Tehran University of Medical Sciences, Tehran, Iran
4 Cellular and Molecular Research Center, Sabzevar University of Medical Sciences, Sabzevar, Iran
5 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 Submission||10-Apr-2022|
|Date of Decision||01-Sep-2022|
|Date of Acceptance||26-Sep-2022|
|Date of Web Publication||16-Mar-2023|
Dr. Rasoul Mohammadi
Department of Medical Parasitology and Mycology, School of Medicine, Infectious Diseases and Tropical Medicine Research Center, Isfahan University of Medical Sciences, Isfahan
Source of Support: None, Conflict of Interest: None
Background: In natural conditions, inhaled fungi are considered a part of the microflora of nasal cavities and sinuses. However, subsequent to the protracted use of corticosteroids and antibacterial agents, suppression of the immune system by chemotherapy, and poor ventilation, these fungi can become pathogens. Fungal colonization in the nose and paranasal sinuses is a prevalent medical issue in immunocompetent and immunosuppressed patients. In this study, we aimed to categorize fungal rhinosinusitis (FRS) among immunocompetent and immunosuppressed patients and identified the etiologic agents of disease by molecular methods. Materials and Methods: A total of 74 cases were evaluated for FRS. Functional endoscopic sinus surgery was performed for sampling. The clinical samples were examined by direct microscopy with potassium hydroxide 20% and subcultured on Sabouraud Dextrose Agar with chloramphenicol. Polymerase chain reaction sequencing was applied to identify causative agents. Results: Thirty-three patients (44.6%) had FRS. Principal predisposing factors were antibiotic consumption (n = 31, 93.9%), corticosteroid therapy (n = 22, 66.6%), and diabetes mellitus (n = 21, 63.6%). Eyesore (n = 22, 66.6%), proptosis (n = 16, 48.5%), and headache (n = 15, 45.4%) were the most common clinical manifestations among patients. Rhizopus oryzae (n = 15, 45.4%) and Aspergillus flavus (n = 10, 30.3%) were the most prevalent fungal species. Conclusion: Diagnosis and classification of FRS are crucial, and a lack of early precise diagnosis can lead to a delay in any surgical or medical management. Since there are a variety of treatments for FRS, accurate identification of etiologic agents should be performed based on phenotypic and molecular methods.
Keywords: Clinical signs, etiologic agents, fungal rhinosinusitis, invasive, noninvasive, predisposing factors
|How to cite this article:|
Taghian E, Abtahi SH, Mohammadi A, Hashemi SM, Ahmadikia K, Dolatabadi S, Mohammadi R. A study on the fungal rhinosinusitis: Causative agents, symptoms, and predisposing factors. J Res Med Sci 2023;28:12
|How to cite this URL:|
Taghian E, Abtahi SH, Mohammadi A, Hashemi SM, Ahmadikia K, Dolatabadi S, Mohammadi R. A study on the fungal rhinosinusitis: Causative agents, symptoms, and predisposing factors. J Res Med Sci [serial online] 2023 [cited 2023 Mar 20];28:12. Available from: https://www.jmsjournal.net/text.asp?2023/28/1/12/371842
| Introduction|| |
Fungal colonization in the nose and paranasal sinuses is a prevalent medical issue in immunocompetent and immunosuppressed patients. Fungal sinusitis can be subdivided into noninvasive and invasive. The invasive type is a destructive infection with intracranial and orbital implications and usually affects immunocompromised individuals; however, the noninvasive type is typically more chronic. Owing to an elevating number of diabetes mellitus, human immunodeficiency virus infections, bone marrow transplant recipients, and chemotherapy, the number of vulnerable immunosuppressed hosts is increasing. Aspergillus species and the Mucorales are the main causative agents of fungal rhinosinusitis (FRS), nevertheless, other genera such as Alternaria, Bipolaris, Schizophyllum, Ulocladium, and Curvularia are also not infrequent. Facial pain, nasal obstruction, headache, rhinorrhea, and epistaxis are common nonspecific clinical signs that are often seen in patients with FRS. This study was planned to determine the prevalence of FRS in the university hospitals in Isfahan, Iran, and to identify the etiologic agents using polymerase chain reaction (PCR)-sequencing.
| Materials and Methods|| |
The protocol of the study was approved by the Ethics Committee of Isfahan University of Medical Science (no.IR.MUI.MED.REC.1399.384). From February 2020 to October 2021, a total of 74 cases were referred to the University hospitals (Al-Zahra and Kashani) of Isfahan University of Medical Sciences, Isfahan, Iran, due to microbial sinusitis. Thirty-five out of 74 cases were immunocompromised (47.3%). Nineteen patients were hospitalized in the intensive care unit (ICU) (25.7%), 16 patients (21.6%) in the Ear, Nose and Throat (ENT) ward, eight patients (10.8%) in the infectious disease ward, three patients (4%) in the surgical ward, two patients (2.7%) in the oncology ward, and the rest (35%) were outpatients.
Patients with persistent fever despite antibiotic therapy, patients with facial swelling, patients with necrotic lesions in the mouth, patients with ptosis, and patients with nasal discharge and prolonged congestion were included in the study.
Patients who did not agree to participate in the survey or sign the informed consent form were excluded from the study.
Functional endoscopic sinus surgery was carried out using the Messerklinger technique using KARL STORZ Endoscope (Germany). It is a diagnostic method demonstrating that the maxillary and frontal sinuses are subordinate cavities. All clinical specimens were transferred to the mycology reference laboratory for direct microscopic examination (DME) with potassium hydroxide 20%, culture on Sabouraud Dextrose Agar (Merck, Germany) with chloramphenicol, and molecular characterization. The periodic acid–Schiff was used to stain histological sections.
Molecular techniques for species identification
Polymerase chain reaction
Genomic DNA was extracted from fresh colonies by DNA Isolation Kit (MoBio Inc. Solana Beach, CA, USA), according to the manufacturer's instructions. For Aspergillus and Penicillium spp., the beta-tubulin protein-coding gene (BT2) and for other molds, internal transcribed spacer regions were used for species identification.,, PCR reactions were carried out by Corbett Research thermal cycler (mod. CG1-96) (Sydney, Australia) in a final volume of 25 μL including 2.5 μL of 10 × reaction buffer (0.1 M Tris-HCl, pH 8.0, 0.5 M KCl, 15 mM MgCl2, 0.1% gelatin, and 1% Triton X-100), 1.5 mM MgCl2, 0.2 mM of each dNTP, 30 pmol of forward and reverse primers, 1.25 U of Taq polymerase (ITK diagnostics, Leiden, The Netherlands), and 2 μL of extracted genomic DNA. Amplification was started with one cycle of 5 min at 94°C for primary denaturation, followed by 32 cycles of 95°C for 40 s, 55°C for 60 s, and 72°C for 60 s, with a final extension of 72°C for 7 min. Five microliters of amplicons were run on 1.5% agarose gel and stained with SYBR Safe DNA gel stain (1:10,000 dilution in Tris/Borate/EDTA buffer), and in the end, visualized by gel documentation system (UVITEC, UK).
All PCR products were purified using QIAquick PCR Purification Kit (Hilden, Germany), and used for Sanger sequencing in a forward direction (Bioneer, South Korea). The products were analyzed with Chromas 2.4 (https://chromas.software.informer.com/2.4/) and then compared with fungal sequences existing in DNA databases (https://blast.ncbi.nlm.nih.gov/Blast.cgi) using the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool.
SPSS software version 23 (IBM, Chicago, Illinois, USA) was used for data analysis. The correlation between fungal species and type of sinusitis was adjusted using Chi-square, Fisher's exact test, and Mann–Whitney U-test. A P < 0.05 was considered statistically significant.
| Results|| |
FRS was diagnosed in 33 patients (44.6%) according to clinical manifestation, DME [Figure 1], culture, and histopathological findings [Figure 2]. The age range of the patients was between 6 and 74 years with a median age of 47.1 years. The age ranges of 50–59 (27.3%) and 70–79 (6%) years had the highest and lowest frequency, respectively. The male-to-female ratio of the present study was 19/14. Thirty-one out of 33 patients (93.9%) used antibiotics. Meropenem, cephalexin, and tazocin were the most antibacterial agents consumed by patients. Eight patients (24.2%) had chronic sinusitis with an average time of 3.5 years. [Figure 3] shows predisposing factors among patients. Eyesore (n = 22, 66.6%), proptosis (n = 16, 48.5%), headache (n = 15, 45.4%), ptosis (n = 14, 42.4%), and blurred vision (n = 11, 33.3%) were the most common clinical manifestations among patients [Table 1]. Twenty patients revealed invasive FRS [Table 2]. Rhizopus oryzae was the most prevalent fungal species (n = 15, 45.4%), followed by Aspergillus flavus (n = 10, 30.3%), Alternaria multiformis (n = 2, 6.1%), Neoscytalidium novaehollandiae (n = 2, 6.1%), Uncinocarpus reesii (n = 1, 3%), Curvularia hawaiiensis (n = 1, 3%), Aspergillus tubingensis (n = 1, 3%), and Penicillium polonicum (n = 1, 3%). The mortality rate was 39.4% in the present study. The causative agent was R. oryzae in nine patients who died, A. flavus in three patients, and A. tubingensis in one patient. Maxillary sinuses were the most common involved sinuses among patients [Figure 4]. Fisher's exact test showed that the association between the type of FRS and fungal species was statistically significant in the invasive group (P = 0.037), but it was not statistically significant in the noninvasive FRS (P = 0.128). Statistically, there was no significant relationship between the etiologic agents and suppressed immune system (P = 0.113).
|Figure 1: Direct microscopic examination with potassium hydroxide 20%, aseptate ribbon-like hyphae in Rhizopus oryzae (a) and septate hyphae with dichotomous branching in Aspergillus flavus (b)|
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|Figure 2: Black arrows show nonseptate hyaline hyphae of Rhizopus oryzae (a) and septate hyphae of Aspergillus flavus (b) in histopathological findings, periodic acid–Schiff staining, original × 40|
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|Figure 3: Predisposing factors among suspected cases of FRS. FRS: Fungal rhinosinusitis|
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|Table 1: Clinical signs of fungal rhinosinusitis among patients in the present investigation|
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|Table 2: Causative agents of fungal rhinosinusitis depending on the invasive or noninvasive type|
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| Discussion|| |
Fungal spores are ubiquitous in the environment, and human vulnerability to fungi is inevitable. The spectrum of infections of the nose and paranasal sinuses is characterized by the presence or absence of fungi, and the immune system status and may range from saprophytic colonization to invasion of the orbit and cerebrum with often serious effects. Hora reported two wide categories of FRS containing invasive and noninvasive types, depending on being restricted to the superficial epithelium in noninvasive form, and the possible invasion of the fungal hyphae to the tissues through the mucosa, neurovascular structures, bones, and surrounding organs. Noninvasive FRS is categorized into saprophytic fungal infestation, fungus ball, and allergic FRS (AFRS)., Fungal ball is designated as a thick fungal hypha causing partial mucosal inflammation. In the present study, no cases of fungus balls were found in patients. AFRS is a type I hypersensitivity reaction to fungal components and the most prevalent type of fungal sinusitis. The disease is mostly asymmetric and bilateral implicating several sinuses. Patients usually have a history of atopies such as asthma, an intact immune system, or facial dysmorphia. Hyaline molds (Aspergillus and Fusarium) and dematiaceous fungi (Alternaria, Curvularia, and Bipolaris) are considered etiologic agents., According to Bent and Kuhn criteria, we detected 13 cases of AFRS in the present investigation (27.3%). Causative agents of AFRS were A. flavus (66.6%), A. multiformis (22.2%), and P. polonicum (11.1%). AFRS commonly is found among younger individuals (21–33 years) with a competent immune system; however, the age range of patients with AFRS in the present study was 39–68 years. In agreement with Glass and Amedee, the most commonly affected sinuses in patients with AFRS were the ethmoids, and the disease was mostly asymmetrical. In our study, R. oryzae was found to be the most prevalent fungal species causing FRS. This contradicts the results of studies conducted in India and the United States, where Aspergillus spp. were the most common cause of the infection.,, This difference can be related to geographical regions and climate. In the present investigation, all clinical specimens revealed positive mycological and histopathological findings; nevertheless, one of the major limitations of our survey was the lack of direct PCR on the samples for diagnosis of FRS. Noninvasive FRS (NFRS) mainly affects immunocompetent patients of all age groups; however, 8 out of 13 patients (61.5%) with NFRS had risk factors including diabetes mellitus and cortone therapy. Invasive fungal sinusitis can be more frequently found in immunosuppressed patients and remarkably may lead to consequential morbidity and mortality. Suppression of the immune system in these patients can be a result of different sources containing diabetes mellitus, bone marrow transplantation, hematologic malignancies, and cytotoxic chemotherapy-induced neutropenia. Infection is commonly appears in the nasal cavity and develops in the paranasal sinuses. It can be caused by various fungal agents, such as Mucorales (commonly in patients with diabetes), Aspergillus species (mostly in neutropenic patients), and dematiaceous fungi. In the present study, most cases of the invasive type were caused by R. oryzae (42.4%); however, Aspergillus species (12.1%) and C. hawaiiensis (as a dematiaceous fungus) (3%) were also isolated from patients. In agreement with our findings, Bakhshaee et al. reported R. oryzae as the most prevalent etiologic agent of invasive rhinosinusitis, nevertheless, A. flavus was the leading agent of invasive fungal sinusitis in a study conducted by Alotaibi et al. Coronavirus disease-2019 (COVID-19) is a primary respiratory infection which may lead to secondary bacterial or fungal infections owing to the use of corticosteroids and immune system impairment. In the present survey, 20 out of 33 patients (60.6%) had coinfection with COVID-19 of which 18 cases (90%) had the invasive type of FRS; however, not known whether these patients were in the wards or in the ICU is another limitation of the present study.
| Conclusion|| |
FRS is a wide spectrum and challenging disease seen in immunocompetent or immunodeficient patients. With the excess knowledge about this spectrum of conditions, we can use proper medical management strategies to control the disease. In this regard, accurate criteria to classify invasive and noninvasive disease are essential to reduce the mortality rate of invasive FRS.
The authors would like to thank the personnel of Al-Zahra and Kashani hospitals for helping to collect clinical specimens.
Financial support and sponsorship
This research was financially supported by Isfahan University of Medical Sciences (no. 399275) and approved by the Ethics Committee of Isfahan University of Medical Science (no. IR.MUI.MED.REC.1399.384).
Conflicts of interest
There are no conflicts of interest.
| References|| |
Gavito-Higuera J, Mullins CB, Ramos-Duran L, Sandoval H, Akle N, Figueroa R. Sinonasal fungal infections and complications: A pictorial review. J Clin Imaging Sci 2016;6:23.
Soler ZM, Schlosser RJ. The role of fungi in diseases of the nose and sinuses. Am J Rhinol Allergy 2012;26:351-8.
Badiee P, Gandomi B, Sabz G, Khodami B, Choopanizadeh M, Jafarian H. Evaluation of nested PCR in diagnosis of fungal rhinosinusitis. Iran J Microbiol 2015;7:62-6.
Nasri T, Hedayati MT, Abastabar M, Pasqualotto AC, Armaki MT, Hoseinnejad A, et al.
PCR-RFLP on β-tubulin gene for rapid identification of the most clinically important species of Aspergillus
. J Microbiol Methods 2015;117:144-7.
Barreto M, Houbraken J, Samson R, Frisvad JC, San-Romão M. Taxonomic studies of the Penicillium glabrum
complex and the description of a new species P. subericola
. Fungal Divers 2011;49:23-33.
White TJ, Bruns T, Taylor J. Amplifi cation and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, editors. PCR Protocols: A Guide to Methods and Applications. London, UK: Academic Press; 1990. p. 315-22.
Hora JF. Primary aspergillosis of the paranasal sinuses and associated areas. Laryngoscope 1965;75:768-73.
Aribandi M, McCoy VA, Bazan C 3rd
. Imaging features of invasive and noninvasive fungal sinusitis: A review. Radiographics 2007;27:1283-96.
Thompson GR 3rd
, Patterson TF. Fungal disease of the nose and paranasal sinuses. J Allergy Clin Immunol 2012;129:321-6.
Jiang RS, Huang WC, Liang KL. Characteristics of sinus fungus ball: A unique form of rhinosinusitis. Clin Med Insights Ear Nose Throat 2018;11:1179550618792254.
Hoxworth JM, Glastonbury CM. Orbital and intracranial complications of acute sinusitis. Neuroimaging Clin N Am 2010;20:511-26.
Glass D, Amedee RG. Allergic fungal rhinosinusitis: A review. Ochsner J 2011;11:271-5.
Bent JP 3rd
, Kuhn FA. Diagnosis of allergic fungal sinusitis. Otolaryngol Head Neck Surg 1994;111:580-8.
Watkinson JC, Clarke RW. Scott-Brown's Otorhinolaryngology and Head and Neck Surgery: 3 Volume Set. Florida, USA: CRC Press; 2018.
Ashraf MJ, Azarpira N, Badiee P, Khademi B, Shishegar M. Fungal characterization using polymerase chain reaction in patients with fungal sinusitis. Indian J Pathol Microbiol 2011;54:415-7.
] [Full text]
Singh AK, Gupta P, Verma N, Khare V, Ahamad A, Verma V, et al
. Fungal rhinosinusitis: Microbiological and histopathological perspective. J Clin Diagn Res 2017;11:DC10-2.
deShazo RD, Swain RE. Diagnostic criteria for allergic fungal sinusitis. J Allergy Clin Immunol 1995;96:24-35.
Ragab A, Samaka RM, Salem M. Impact of fungal load on diagnosis and outcome of allergic fungal rhinosinusitis. Eur Arch Otorhinolaryngol 2014;271:93-101.
Bakhshaee M, Bojdi A, Allahyari A, Majidi MR, Tavakol S, Najafzadeh MJ, et al
. Acute invasive fungal rhinosinusitis: Our experience with 18 cases. Eur Arch Otorhinolaryngol 2016;273:4281-7.
Ismaiel WF, Abdelazim MH, Eldsoky I, Ibrahim AA, Alsobky ME, Zafan E, et al
. The impact of COVID-19 outbreak on the incidence of acute invasive fungal rhinosinusitis. Am J Otolaryngol 2021;42:103080.
Alotaibi NH, Omar OA, Altahan M, Alsheikh H, Al Mana F, Mahasin Z, et al
. Chronic invasive fungal rhinosinusitis in immunocompetent patients: A retrospective chart review. Front Surg 2020;7:608342.
Mehta S, Pandey A. Rhino-Orbital mucormycosis associated with COVID-19. Cureus 2020;12:e10726.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]