Clinical and laboratory profiles of invasive fungal infections in COVID-19 patients Текст научной статьи по специальности «Клиническая медицина»

MIRJournal

MicrobioLogy Independent Research JournaL

DOI: 10.18527/2500-2236-2023-10-1-45-51 RESEARCH PAPER

Clinical and laboratory profiles of invasive fungal infections in COVID-19 patients

Pokkottu M. Sheeba1 , Ali Aysha2, Balachandran Sreeram3, Jose K. Roshni1# , Anila A. Mathews1, Kanapilly F. Magdalene2

1 Department of Microbiology, Government Medical College, Palakkad, India

2 Department of Pathology, Government Medical College, Palakkad, India

3 Department of General Medicine, Government Medical College, Palakkad, India

ABSTRACT

With the COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), many areas in the world witnessed an increase in fungal infections and the corresponding increase in mortality. In our study, we attempted to identify common fungal pathogens encountered in patients with COVID-19 and to find microbiological and histopathological parameters that may help diagnose these etiological agents. The clinical manifestations, demography, and disease outcomes in patients with fungal infections associated with COVID-19 were also studied.

The observational cross-sectional study was performed at the Government Medical College and District Hospital, Palakkad, India. We used case records and laboratory records of patients admitted with COVID-19 and diagnosed with a fungal infection, as well as records of post-COVID-19 patients with fungal infection. The study period covered six months from March to August 2021. The study included 50 patients aged 40-74 years (median: 56 years).

We found that mucormycosis and aspergillosis are important invasive fungal infections that prevail in COVID-19 patients. The most common risk factors associated with these fungal infections include diabetes mellitus and treatment with corticosteroids. KOH (potassium hydroxide) mount test and histopathological examination are very useful methods for etiological diagnostics. The overall mortality rate in the observed patients was 71.4% (85.7% for mucormycosis and 57.1% for aspergillosis). Thus, invasive fungal infections in COVID-19 patients with co-morbidities are associated with a high mortality rate.

Keywords: COVID-19, fungal infection, mucormycosis, aspergillosis, mortality rate

# For correspondence: Dr. Roshni K Jose, Assistant Professor, Department of Microbiology, Government Medical College, Palak kad - 678013, India. Phone number: 8095839642, e-mail: rkjose4665@gmail.com

Citation: Sheeba PM, Aysha A, Sreeram B, Roshni KJ, Mathews AA, Magdalene KF. Clinical and laboratory profiles of invasive fun

gal infections in COVID-19 patients. MIR J 2023; 10(1), 45-51 doi: 10.18527/2500-2236-2023-10-1-45-51.

Received: December 23, 2022

Accepted: March 30, 2023

Published: April 18, 2023

Copyright: © 2023 Sheeba et al. This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International Public License (CC BYNC-SA), which permits the unrestricted use, distribution, and reproduction in any medium, as long as the material is not used for commercial purposes, provided that the original author and source are cited.

Acknowledgements: We thank the staff of the Palakkad Medical Superintendent District Hospital and Palakkad District Hospital, India (Medical Records Department). We are grateful to Mr. K. S. Deepak from the Palakkad Government Medical College for his assistance with statistical analyses.

Conflict of interest: The authors have no conflict of interest.

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INTRODUCTION

The COVID-19 pandemic, which began in Wuhan province of China in December 2019, was caused by a novel coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [1]. The disease caused by this coronavirus was named COVID-19. Besides triggering the respiratory symptoms, SARS-CoV-2 suppresses the patient's immune system resulting in a wide range of secondary bacterial and fungal infections. These secondary infections are usually associated with preexisting conditions, such as diabetes mellitus, bronchial asthma, chronic obstructive pulmonary disease (COPD), and other lung diseases, or develop as a hospital-acquired infection due to corticosteroid treatment of severe COVID-19 [2]. Secondary fungal infections are also very frequent in patients with hematological and other malignancies and in patients undergoing chemotherapy [3]. Although the number of patients with invasive secondary fungal infection is relatively low, the mortality in these patients is extremely high compared to the patients with COVID-19-associated secondary bacterial infections. The incidence of fungal infections caused by pathogens with decreased susceptibility to the currently available antifungals is increasing [4] which makes these infections a severe threat to healthcare worldwide.

We studied common invasive fungal infections, etiological agents that cause these infections, and co-morbidities associated with COVID-19 at the District Medical College Hospital. The clinical manifestations, demographics, treatment, and disease outcomes in patients with COVID-19-associated fungal infections were also analyzed. The presented data on the association of invasive fungal infection with COVID-19 are limited, and further in-depth studies are needed to determine the incidence and outcomes of such infections as well as to establish patient management protocols.

MATERIALS AND METHODS

Study description

An observational cross-sectional study was performed at the Government Medical College and District Hospital in Palakkad, India. The study was executed following the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. The duration of the study was six months from March to August 2021. All documentation has been obtained with permission from Government Medical College and District Hospital in Palakkad, India.

Patients

A total of 50 patients (40 to 74 years old; median 56 years old) were included in the study. Case histories and laboratory records of COVID-19 patients and post-COVID-19 patients with symptoms including facial swelling, headache, and signs of sinusitis or upper air obstruction were analyzed. Data including age, gender, clinical and laboratory data, comorbidities, and duration of hospital stay were registered. A continuous sampling method was used. All measures were taken to keep the confidentiality related to patients' identity while compiling the data.

Sample analysis

Samples received in the microbiology laboratory were divided into three parts. The first part was subjected to a KOH (potassium hydroxide) mount examination under 10x and 40x magnification, while the remaining two parts were cultured on Sabouraud's dextrose agar and kept at 25°C and 37°C, respectively. Colonies on the culture media were identified by a tease mount test using lactophenol cotton blue (LPCB). The slide culture technique was used to study the undisturbed morphology of the fungus. The final identification of the fungus species was based on the morphological characteristics as described in the Larone's Medically Important Fungi: A Guide to Identification [5].

Biopsy samples received for histopathology examination (HPE) were fixed in 10% formalin and processed in tissue processor Histokinette (Leica TP 1020), stained with haema-toxylin and eosin stain (H&E), and observed using Labomed Lx 500 microscope at 40x magnification (Labomed, USA).

For statistical analysis, the data were described as either continuous or categorical variables. The categorical variables were presented as frequency and percentages.

RESULTS

We received a total of 50 samples from COVID-19-posi-tive patients with suspected fungal infections of the respiratory tract. Among these samples, 14 (28%) were positive for fungal pathogens (KOH mount, HPE, or culture). From these 14 samples, seven were positive for Mucorales species and another seven - for Aspergillus species.

Patients diagnosed with mucormycosis were 54.7±9.0 years old on average. The male/female ratio was 3:4. None of the patients was vaccinated with a vaccine against COVID-19. The average interval between COVID-19-positive diagnosis and the onset of mu-cormycosis symptoms reached 10.1 days (from 4 to 20 days). Out of the seven patients with mucormycosis, six (85.7%) had diabetes (Table 1). One of them had diabetic

ketoacidosis, another one was on ayurvedic treatment, and the rest had uncontrolled diabetes. One patient with no prior history of diabetes was detected to have abnormal blood glucose levels during the hospital stay, most probably due to extensive corticosteroid therapy. Finally, three out of seven (42.9%) patients were treated with corticosteroids. Only one patient had received remdesi-vir. None of the patients had a history of admission to the Intensive Care Unit (ICU) for COVID-19 treatment. Four patients had rhino-orbital mucormycosis, three had sinonasal mucormycosis, and all of them had ptosis.

The etiological agent - Rhizopus oryzae (Rhizopus spp.) - was identified in five out of seven (71.4%) samples. One sample demonstrated sporangium along with aseptate hyphae.

Samples obtained from endoscopic sinus surgery were subjected to a KOH mount test and culture analysis. HPE was done in four of these samples. The KOH mount test and HPE of the samples showed aseptate fungal hyphae branching at right angles. In one sample, we noted sporangium along with aseptate hyphae (Fig. 1).

While studying samples obtained from seven patients with mucormycosis, Rhizopus spp. was isolated from five samples, and no fungal pathogens were detected in the other two samples. All these patients were treated with Liposomal Amphotericin B (Bharat Serums, India). Six

Table 1. Patients with symptoms of mucormycosis

patients died, and one 55-year-old male patient with sinonasal mucormycosis recovered. The mortality rate was 85.7%. The clinical data of these patients are summarized in Table 2.

Among patients diagnosed with aspergillosis, none was vaccinated against COVID-19. The mean age of patients was 58.0±7.2 years. The male/female ratio was 4:3. The average interval between COVID-19-positive diagnosis and the onset of symptoms was four days (from 1 to 8 days). All seven patients had diabetes mellitus, five of them had hypertension, and one patient had pulmonary aspergillosis and a history of pulmonary tuberculosis. Five patients had received corticosteroid therapy for COVID-19 treatment: four patients were given remdesivir and one was treated with ivermectin. Cough, breathlessness, sinusitis-like headache, facial pain, and nasal congestion were observed as the common symptoms (Table 3). Some of these patients had other symptoms including chest pain and hemoptysis. None of the patients had a history of prior admission to ICU for COVID-19 treatment. Samples obtained from endoscopic sinus surgery were subjected to the KOH mount test and culture analysis. The KOH mount test and HPE of six samples showed septate fungal hy-phae branching at acute angles. In one sample, both the KOH mount test and H&E staining of the biopsy specimen identified conidia along with septate hyphae (Fig. 2).

Patient No Age / Sex Facial pain Facial numbness Facial abscess Headache Tears from eyes Ptosis Decreased vision Restricted ocular movements Epistaxis

1. 40 / M Yes Yes Yes Yes Yes Yes

2. 45 / F Yes Yes Yes Yes

3. 55 / M Yes Yes

4. 65 / M Yes

5. 58 / F Yes

6. 60 / F Yes Yes Yes Yes

7. 60 / F Yes Yes Yes Yes Yes

Fig. 1. KOH mount test that shows sporangium and aseptate hy- Fig. 2. H&E staining of a biopsy specimen showing conidia of As-phae of Rhizopus spp. (magnification 40x). pergillus flavus (magnification 40x).

Table 2. Clinical data of patients with mucormycosis

Patient No Age/ Sex Comorbidities Corticosteroid therapy KOH mount Culture HPE Treatment Clinical form of mucormycosis Outcome

1 40/M Diabetic on OHA Dexona Aseptate hyphae Rhizopus spp. Broad aseptate hyphae branching at right angles Liposomal amphotericin B Rhino orbital mucormycosis Deceased

2 45/F Diabetic, CKD NA Aseptate hyphae Culture-negative Not performed Liposomal amphotericin B Rhino orbital mucormycosis Deceased

3 55/M Diabetic on Ayurveda treatment NA Aseptate hyphae Rhizopus spp. Broad aseptate hyphae branching at right angles Liposomal amphotericin B Sinonasal mucormycosis Recovered

4 65/M Diabetic, DKA Dexona Aseptate hyphae Rhizopus spp. Broad aseptate hyphae branching at right angles Liposomal amphotericin B Sinonasal mucormycosis Deceased

5 58/F CAD Methylpred-nisolone Aseptate hyphae Rhizopus spp. Broad aseptate hyphae branching at right angles Liposomal amphotericin B Sinonasal mucormycosis Deceased

6 60/F Diabetic, left MCA stroke No Aseptate hyphae Rhizopus spp. Not performed Liposomal amphotericin B Rhino orbital mucormycosis Deceased

7 60/F Uncontrolled diabetes, hypertension NA Aseptate hyphae Culture-negative Not performed Liposomal amphotericin B Rhino orbital mucormycosis Deceased

OHA - oral hypoglycemic agents, CKD - chronic kidney disease, MCA - middle cerebral artery, CAD - coronary artery disease,

DKA - diabetic ketoacidosis, NA - not applicable, F- female, M- male.

Table 3. Clinical data of patients with aspergillosis

Patient No Age/ Sex Comorbidities Corticostero-id therapy KOH mount Culture HPE Treatment Clinical form of aspergillosis Outcome

1 55/M Diabetic, CAD, hypertensive, old pulmonary tuberculosis Dexona Septate hyphae and conidia of Aspergillus Aspergillus flavus Thin septate hy-phae branching at acute angles Liposomal amphoteri-cin B Pulmonary aspergillosis and sphenoid sinusitis Deceased

2 56/M Diabetic No Septate hyphae Aspergillus flavus Thin septate hy-phae branching at acute angles Liposomal amphotericin B Fungal maxillary sinusitis Recovered

3 57/M Diabetic Dexona, Methylpred- nisolone Septate hyphae Aspergil-lus niger Thin septate hy-phae branching at acute angles Liposomal amphotericin B Fungal maxillary sinusitis Deceased

4 56/F Diabetic, hypertensive Methylpred-nisolone Septate hyphae Aspergil-lus fu-migatus Not performed Vorico-nazole Fungal maxillary sinusitis Deceased

5 55/F Diabetic, hypertensive Dexona Septate hyphae Aspergil-lus niger Thin septate hy-phae branching at acute angles Liposomal amphotericin B Fungal maxillary sinusitis Deceased

6 53/F Diabetic, hypertensive Dexona Septate hyphae Aspergil-lus niger Thin septate hy-phae branching at acute angles Vorico-nazole Fungal maxillary sinusitis Recovered

7 74/M Diabetic, CAD hypertensive No Septate hyphae Culture-negative Thin septate hy-phae branching at acute angles Vorico-nazole Fungal maxillary sinusitis Recovered

F - female, M - male.

Six out of the seven samples were fungus-positive according to the culture analysis, of which three were found to have Aspergillus niger, two - Aspergillus flavus, and one - Aspergillus fumigatus. Three of the seven patients were treated with amphotericin B and the rest were given voriconazole. Of these seven patients, three recovered. The final mortality rate was 57.1%.

DISCUSSION

We identified two types of deep fungal infections - mu-cormycosis and aspergillosis - in patients diagnosed with COVID-19. Mucormycosis is caused by the fungi that belong to the order Mucorales, which includes Rhizopus, Mucor, Rhizomucor, Cunninghamella, Lichtheimia, and Apophysomyces spp. [6]. The prevalence of mucormycosis in humans ranges from 0.005 to 1.7 per million worldwide, while in India it ranges from 0.14 to 1000 [7]. In addition, India ranks second in the world in terms of the total number of patients with diabetes mellitus, which is considered a risk factor for mucormycosis [8]. Lionakis et al. [9] showed that a cumulative dose of prednisolone above 600 mg per day or a total dose of 2-7 g of meth-ylprednisolone taken over one month predisposes im-munocompromised individuals to mucormycosis. The unregulated use of corticosteroids for the treatment of COVID-19 contributed to an increased number of cases of mucormycosis during the second COVID-19 wave in India [10]. The probability of mucormycosis in individuals with diabetes treated with corticosteroids is even higher. Over the period from May 5 to July 12, 2021, 41,512 cases of mucormycosis with 3,554 lethal outcomes were reported in India [11]. Most of those cases occurred during SARS-CoV-2 outbreaks in India.

Three theories have been suggested to explain COV-ID-19-associated mucormycosis. The first one suggests that lymphopenia associated with COVID-19 enables opportunistic fungal pathogens to gain entry [11, 12]. Another observation is that increased levels of pro-inflammatory markers in patients with severe disease help fungal pathogens to proliferate in the human body. The third theory suggests that pronounced damage of pulmonary tissues by COVID-19 makes them conducive to invasive fungal pathogens, especially airborne ones [12].

Six clinical forms of mucormycosis defined by the location in the body are described in the literature including rhino-cerebral, pulmonary, cutaneous, gastrointestinal, disseminated, and uncommon presentations [13]. The most common site of invasive mucormycosis is the sinuses (39%), followed by the lungs (24%) and skin (19%). In a study from a tertiary care center in South

India, the rhino-orbito-cerebral mucormycosis was described as the most common type of this disease [14].

Direct microscopy and KOH mount microscopy are invaluable tools for the rapid diagnosis of mucormycosis [15]. Histopathology is important for determining angio-invasion and distinguishing the presence of fungi from culture contamination [16]. In our study of seven patients diagnosed with mucormycosis, 57% had rhino-orbital mucormycosis and 43% had sinonasal mucormy-cosis. Rhizopus oryzae (Rhizopus spp.) was identified as the etiological agent. This is the most common fungus isolated from clinical specimens of patients with mucor-mycosis, followed by Lichtheimia corymbifera and Mucor racemosus [17]. The cases where sporangium along with aseptate hyphae were found in the same sample using the KOH mount test are described in the literature [18].

All patients with mucormycosis that were included in our study were treated according to the Recommendations from the European Conference on Infections in Leukemia (ECIL-6) (2016) and ESCMID/ECMM guidelines, which recommend a lipid formulation of ampho-tericin B (5 mg/kg/day, 10 mg/kg/day for infection of the central nervous system) as the first-line therapy for mucormycosis [15]. A recently developed medication isa-vuconazole also shows good activity against Mucorales [19]. The successful treatment of mucormycosis depends mainly on surgical debridement of the infected tissues. The mortality rate shown for patients with mucormyco-sis in our study (85.7%) corresponds to the literature data (46-96%). The mortality rate depends upon the patient's comorbidities and can reach 90% in case of intracranial involvement [20].

The second infection - aspergillosis - was diagnosed in seven out of 50 patients tested in our study and was caused by Aspergillus niger, Aspergillus flavus, or Aspergillus fumigatus. Aspergillus spp. is one of the most common etiological agents associated with fungal rhinosinusitis; this disease is rare, although it is associated with high mortality [21]. Aspergillus spp. are ubiquitous, and their spores can easily enter the body with airborne droplets. The maxillary sinus is commonly affected by invasive fungal sinusitis. We also noted the involvement of the maxillary sinuses in six of our patients; one patient had aspergillosis of the lungs and sphenoid sinus. The most common Aspergillus species causing invasive infection is A. fumigatus [21]. The main predisposing factors for aspergillosis include uncontrolled diabetes mellitus and human immunodeficiency virus infection, as well as the misuse of antibiotics, immunosuppressants, and cor-ticosteroids [22]. Immune dysregulation associated with acute respiratory distress syndrome (ARDS) and its treatment with tocilizumab or corticosteroids are also factors

contributing to infection. Pulmonary aspergillosis is common in patients with a history of tuberculosis due to extensive structural lung changes [23]. All the patients (7/7) diagnosed with invasive aspergillosis in our study had diabetes. Five of them had received corticosteroids for COVID-19 treatment. One patient had pulmonary as-pergillosis along with sphenoid sinus involvement.

Microscopy is the easiest way to diagnose aspergil-losis, although its specificity is low. Aspergillus species rarely spore in vivo, so the hyphae may represent any of the filamentous fungi. Therefore, the sensitivity of this method varies from 0% to 90% [24]. PCR is the most sensitive method in detecting fungal rhinosinusitis, followed by culture analysis, potassium hydroxide microscopy, and histopathology [25]. Fruiting bodies/conidia are rare in tissue samples, but there are reports of such unusual findings in aspergillosis [26]. In our study, one patient with pulmonary aspergillosis had conidia along with septate hyphae in a direct nasal endoscopy (DNE) specimen. HPE also showed a similar result for this patient. However, diagnosis should not be based on results of a single method. A biopsy can confirm tissue invasion by the fungus [25]. In our study, HPE biopsy analysis and culture analysis confirmed aspergillosis in six out of seven patients, although the latter method is less sensitive for diagnosing aspergillosis [27]. The mortality rate also varies depending on the co-morbidities of the patient. Lin et al. [28] showed that disseminated infection by Aspergillus resulted in 88% mortality rate. The mortality rate in our study was 57.1%. Surgery is an important

element of treatment since it helps to obtain tissue samples for diagnosis and to sanitize necrotic tissues [29]. Isavuconazole and voriconazole are considered as the first-line treatment medications, whereas there is a moderate support for the use of liposomal amphotericin B [30]. Posaconazole is another drug that has been used in the treatment of invasive aspergillosis [30]. Only three patients in our study were treated with voriconazole and four received liposomal amphotericin B.

The small sample size is the main limitation of our study, which makes it difficult to generalize the conclusions.

CONCLUSION

Invasive fungal infections are fatal, especially when seen in patients suffering from severe COVID-19 infections. An early diagnosis and recognition of such infections as well as their prompt treatment are required to reduce the mortality and morbidity in such patients. To achieve a prompt diagnosis of invasive fungal infections, we recommend using the KOH mount examination since both fungal culture analysis and histopatho-logical examination of the endoscopic biopsy samples are more time-consuming, which leads to increased patients' mortality. In conclusion, our data suggest that the risk of invasive fungal infections is increased in COVID-19 patients and their recovery rates are low. Therefore, further research on the association between invasive fungal infections and COVID-19 is needed, especially during the pandemic.

REFERENCES

1. Coronaviridae Study Group of the International Committee on Taxonomy of Viruses. The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2. Nat Microbiol 2020; 5(4), 536-44. doi: 10.1038/ s41564-020-0695-z.

2. Yang W, Cao Q, Oin LE, Wang X, Cheng Z, Pan A et al. Clinical characteristics and imaging manifestations of the 2019 novel coronavirus disease (COVID-19): a multi-center study in Wenzhou city, Zhejiang, China. J Infect 2020; 80(4), 388-93. doi: 10.1016/j. jinf.2020.02.016.

3. Silva DL, Lima CM, Magalhaes VC, Baltazar LM, Peres NT, Caligiorne RB et al. Fungal and bacterial coinfections increase mortality of severely ill COV-ID-19 patients. J Hosp Infect 2021; 113, 145-54. doi: 10.1016/j.jhin.2021.04.001.

4. Zhang G, Hu C, Luo L, Fang F, Chen Y, Li J et al. Clinical features and short-term outcomes of 221 patients

with COVID-19 in Wuhan, China. J Clin Virol 2020; 127, 104364. doi: 10.1016/j.jcv.2020.104364.

5. Walsh TJ, Hayden RT, Larone DH. Larone's medically important fungi: A guide to identification. 6th ed. Washington, DC: ASM Press; 2018. doi: 10.1128/9781555819880.

6. Walther G, Wagner L, Kurzai O. Updates on the taxonomy of Mucorales with an emphasis on clinically important taxa. J Fungi (Basel) 2019; 5(4), 106. doi: 10.3390/jof5040106.

7. Prakash H, Chakrabarti A. Global epidemiology of mucormycosis. J Fungi (Basel) 2019; 5(1), 26. doi: 10.3390/jof5010026.

8. Pradeepa R, Mohan V. Epidemiology of type 2 diabetes in India. Indian J Ophthalmol. 2021; 69(11), 29328. doi: 10.4103/ijo.IJO_1627_21.

9. Lionakis MS, Kontoyiannis DP. Glucocorticoids and invasive fungal infections. Lancet 2003; 362(9398), 1828-38. doi: 10.1016/S0140-6736(03)14904-5.

10. Oruc E, Gulen TA, Turunc T, Ekici NY, Unal N. Comparative Evaluation of COVID-19 Associated Mucormycosis (CAM) and Non-COVID-19-associated Mucormycosis (non-CAM). J Coll Physicians Surg Pak 2023; 33(2), 153-7. doi: 10.29271/jcpsp.2023.02.153.

11. Nayak PS, Goswami P, Kumar AD, Bhaskar P. Quality of Life in Mucormycosis Patients Post Discharge: A Pilot Study Using a New MQOL-36 Questionairre. Indian J Otolaryngol Head Neck Surg 2022, 1-7. doi: 10.1007/s12070-022-03196-w.

12. Bhatt K, Agolli A, Patel MH, Garimella R, Devi M, Garcia E et al. High mortality co-infections of CO-VID-19 patients: mucormycosis and other fungal infections. Discoveries (Craiova) 2021; 9(1), e126. doi: 10.15190/d.2021.5.

13. Petrikkos G, Skiada A, Lortholary O, Roilides E, Walsh TJ, Kontoyiannis DP. Epidemiology and clinical manifestations of mucormycosis. Clin Infect Dis 2012; 54(suppl_1), S23-4. doi: 10.1093/cid/cir866.

14. Priya P, Ganesan V, Rajendran T, Geni VG. Mucormycosis in a tertiary care center in South India: a 4-year experience. Indian J Crit Care Med 2020; 24(3), 16871. doi: 10.5005/jp-journals-10071-23387.

15. Cornely O, Alastruey-Izquierdo A, Arenz D, Chen SCA, Dannaoui E, Hochhegger B et al. Global guideline for the diagnosis and management of mucormycosis: an initiative of the European Confederation of Medical Mycology in cooperation with the Mycoses Study Group Education and Research Consortium. Lancet Infect Dis 2019; 19(12), e405-21. doi: 10.1016/S1473-3099(19)30312-3.

16. Guarner J, Brandt ME. Histopathologic diagnosis of fungal infections in the 21st century. Clin Microbiol Rev 2011; 24(2), 247-80. doi: 10.1128/CMR.00053-10.

17. Chakrabarti A, Chatterjee SS, Das A, Panda N, Shivaprakash MR, Kaur A et al. Invasive zygomyco-sis in India: experience in a tertiary care hospital. Postgrad Med J 2009; 85(1009), 573-81. doi: 10.1136/ pgmj.2008.076463.

18. Vaidya D, Shah P. Coinfection by Aspergillus and Zygomycetes species in a case of acute rhinosinus-itis. Case Rep Otolaryngol 2011; 2011, 382473. doi: 10.1155/2011/382473.

19. Rybak JM, Marx KR, Nishimoto AT, Rogers PD. Isa-vuconazole: pharmacology, pharmacodynamics, and current clinical experience with a new triazole antifungal agent. Pharmacotherapy 2015; 35(11), 103751. doi: 10.1002/phar.1652.

20. Roden MM, Zaoutis TE, Buchanan WL, Knudsen TA, Sarkisova TA, Schaufele RL et al. Epidemiology and outcome of zygomycosis: a review of 929 reported

cases. Clin Infect Dis 2005; 41(5), 634-53. doi: 10.1086/432579.

21. Peral-Cagigal B, Redondo-González LM, Verrier-Hernández A. Invasive maxillary sinus aspergillosis: A case report successfully treated with voriconazole and surgical debridement. J Clin Exp Dent 2014; 6(4), e448-51. doi: 10.4317/jced.51571.

22. Montrucchio G, Lupia T, Lombardo D, Stroffolini G, Corcione S, De Rosa FG, Brazzi L. Risk factors for invasive aspergillosis in ICU patients with COVID-19: current insights and new key elements. Ann Intensive Care 2021; 11(1), 136. doi: 10.1186/s13613-021-00923-4.

23. Romanowski K, Baumann B, Basham CA, Khan FA, Fox GJ, Johnston JC. Long-term all-cause mortality in people treated for tuberculosis: a systematic review and meta-analysis. Lancet Infect Dis 2019; 19(10), 1129-37. doi: 10.1016/S1473-3099(19)30309-3.

24. Ruchel R, Schaffrinski M. Versatile fluorescent staining of fungi in clinical specimens by using the optical brightener Blankophor. J Clin Microbiol 1999; 37(8), 2694-6. doi: 10.1128/JCM.37.8.2694-2696.1999.

25. Singh AK, Gupta P, Verma N, Khare V, Ahamad A, Verma V, Agarwal SP. Fungal rhinosinusitis: microbiological and histopathological perspective. J Clin Diagn Res 2017; 11(7), DC10-12. doi: 10.7860/ JCDR/2017/25842.10167.

26. Anila KR, Somanathan T, Mathews A, Jayasree K. Fruiting bodies of Aspergillus: An unusual finding in histopathology. Lung India 2013; 30(4), 357-9. doi: 10.4103/0970-2113.120623.

27. Fayemiwo S, Moore CB, Foden P, Denning DW, Richardson MD. Comparative performance of Aspergillus galactomannan ELISA and PCR in sputum from patients with ABPA and CPA. J Microbiol Methods 2017; 140, 32-9. doi: 10.1016/j.mimet.2017.06.016.

28. Lin SJ, Schranz J, Teutsch SM. Aspergillosis case-fatality rate: systematic review of the literature. Clin Infect Dis 2001; 32(3), 358-66. doi: 10.1086/318483.

29. Pagella F, De Bernardi F, Dalla Gasperina D, Pusateri A, Matti E, Avato I et al. Invasive fungal rhinosinusitis in adult patients: our experience in diagnosis and management. J Craniomaxillofac Surg 2016; 44(4), 51220. doi: 10.1016/j.jcms.2015.12.016.

30. Ullmann AJ, Aguado JM, Arikan-Akdagli S, Denning DW, Groll AH, Lagrou K et al. Diagnosis and management of Aspergillus diseases: executive summary of the 2017 ESCMID-ECMM-ERS guideline. Clin Microbiol Infect 2018; 24, e1-38. doi: 10.1016/j. cmi.2018.01.002.