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BMC Infectious Diseases

, 18:323 | Cite as

Primary invasive laryngeal mycosis in an immunocompetent patient: a case report and clinico-epidemiological update

  • Supram Hosuru Subramanya
  • Joseph Jillwin
  • Shivaprakash Mandya Rudramurthy
  • Krishna Chandra Rijal
  • Niranjan Nayak
  • Arunaloke Chakrabarti
  • Arnab Ghosh
Open Access
Case report
  • 602 Downloads
Part of the following topical collections:
  1. Bacterial and fungal diseases

Abstract

Background

Laryngeal aspergillosis is uncommon and is usually secondary to pulmonary involvement in immunocompromised patients. Primary laryngeal aspergillosis in immunocompetent individuals is extremely rare, with a few cases documented over the last five decades.

Case presentation

We report a case of primary localised laryngeal aspergillosis in a 21-year-old apparently immunocompetent student. Septate hyphae were observed on histopathology of the laryngeal lesion, which was further confirmed as Aspergillus fumigatus after extraction of fungal DNA from formalin fixed paraffin embedded tissue (FFPET) and sequencing. The patient responded well to oral itraconazole therapy over a month.

Conclusions

Since last few decades, cases of primary laryngeal aspergillosis in immunocompetent individuals are on the rise, globally. This is the first case of invasive laryngeal aspergillosis reported in Nepal. The extraction of DNA from tissue and sequencing helps to identify the etiological agent, when culture fails to isolate the fungus.

Keywords

Primary laryngeal aspergillosis Aspergillus fumigatus FFPE-PCR 

Abbreviations

AMP-B

Amphotericin B

ATCC

American Type Culture Collection

COPD

Chronic obstructive pulmonary disease

DM

Diabetes mellitus

DNA

Deoxyribonucleic acid

FFPET

Formalin fixed paraffin embedded tissue

GERD

Gastroesophageal reflux disease

HE

Histopathological examination

HIV

Human immunodeficiency virus

ITCZ

Itraconazole

ITS

Internal transcribed spacer

PCR

Polymerase chain reaction

SEM

Scanning electron microscopy

VCZ

Voriconazole

VDRL

Venereal disease research laboratory test

Background

Primary fungal laryngitis is commonly attributable to yeasts such as Candida, and Cryptococcus or fungi are known to cause endemic mycoses like Blastomyces, Paracoccidioides, and Coccidioides. The mold forms, such as Aspergillus and Mucor, may involve larynx as secondary pulmonary invasion [1, 2]. Immunocompromisation due to leukaemia, AIDS, severe aplastic anaemia, lymphoreticular neoplasms, or immunosuppressive therapy predispose person to invasive fungal infection [1, 2]. Primary laryngeal aspergillosis in immunocompetent individuals is extremely rare. It often mimics the pre-malignant and malignant conditions (squamous cell carcinoma) of larynx. Fungal laryngitis is usually characterized by sore throat, earache, hoarseness of voice, cough, odynophagia, formation of endolaryngeal and perilaryngeal white plaques, granulation tissue, ulcerations, erythema and edema [3]. Diagnosis and prompt treatment are essential to prevent complications like scarring of the vocal folds, compromised airway due to glottic edema and dissemination of the pathogen. First case of aspergillosis of larynx was reported in 1969 from Pondicherry, South India [4]. Globally, less than 50 cases over the period of last 50 years have been documented. Herein, a case of primary laryngeal aspergillosis in an apparently immunocompetent young adult is reported. To the best of our knowledge, this is the first such case report from Nepal. In this endeavour, we conducted a comprehensive review of literature and analysed all previously reported cases.

Case presentation

A 21-year young male presented to Manipal Teaching Hospital, Pokhara, with progressive hoarseness of voice for two months and frequent cough with expectoration since one month. He had no history of phonotrauma, apparent immune deficiency, leukaemia, malignant disease, diabetes mellitus, broad-spectrum antibiotics or immunosuppressive therapy, including corticosteroids. He was not habituated to tobacco or alcohol. He did not have any previous history of laryngeal trauma, allergies or mycosis. A general physical examination did not reveal lymphadenopathy or organomegaly. There were no visible lesions or masses in the oral cavity, oropharynx or nasopharyngeal mucosa. His paranasal sinuses and chest X-rays were clear. Routine blood test report was within normal limits. Serological markers for Hepatitis B, C, and HIV were negative and VDRL test was non-reactive.

Clinical examination and laboratory findings

A direct laryngoscopic examination was performed under general anaesthesia. Videostroboscopy revealed a smooth, diffused whitish spheroid submucosal mass on the anterior surface of the left vocal cord. Vocal cord mobility was normal bilaterally, the airway was adequate, and both subglottis and supraglottis showed normal mucosa.

In order to exclude, glottic carcinoma the patient was subjected to punch biopsy from the lesion by the micro-laryngeal procedure under general anaesthesia. The histopathological examination showed conidia and broad septate hyphae, most of them showing acute angle branching without any evidence of malignant cells (Fig. 1). Repeat biopsy specimen processed for fungal culture did not yield any growth. For the identity of fungi, sections from paraffin-embedded tissue block were analysed by Polymerase Chain Reaction (PCR).
Fig. 1

a Histopathological features of biopsy sample showing numerous septate hyphae, “spaghetti-like” fungal filaments branching at an angle of approximate 450, interspersed with shreds of vocal cord squamous epithelium (Haematoxylin and Eosin stained, magnification X400. b Calcofluor white staining of tissue section observed under fluorescent microscopy showing numerous branched septate hyphal forms X1000

Molecular identification by PCR

Extraction of DNA from formalin fixed paraffin embedded (FFPE) tissue

To avoid environmental fungal DNA (or) amplicon contamination, all steps were performed inside laminar air flow cabinets in separate closed cabins equipped with a dedicated set of micropipettes and instruments. A 50 μm thick FFPE tissue section was cut using a clean blade by microtomy and transferred to a 1.5 ml microcentrifuge tube. Deparaffinization and DNA extraction was performed as per Anna Lau et al. [5] with minor modifications where phenol-CHCl3-isoamyl alcohol extraction protocol was performed. The pellet was dissolved in 50 μm of nuclease-free water and stored at − 200 C until further testing.

Amplification of 28 s region of rDNA

PCR was performed in a 45 μl mixture consisting of 1× PCR buffer without MgCl2 (Genei, Bangalore), 2.5 mM MgCl2, 0.25 mM deoxyribonucleotide blend (Fermentas), 0.4 μM primers 12F 5’GTTGATAGAAYAATGTAGATAAGG3’ and 13R 5’GACAGTAAGATTCCCCTTG3’ (1) (Eurofins), 1.2 U of Taq Polymerase (Bangalore Genie) and 5 μl (~ 80 ng) of template DNA. Thermal cycling was performed in an Eppendorf Mastercycler Gradient thermal cycler (Eppendorf AG, Hamburg) with the following conditions: denaturation at 95 °C for 10 mins followed by 60 cycles of 94 °C for 15 secs, annealing at 53 °C for 20 secs, and extension 72 °C for 25 secs and finally once at 72 °C for 5 mins. Positive and negative controls were included. Amplification was confirmed by electrophoresis on 2% agarose gel with ethidium bromide, and the amplicon was purified using Qiagen gel extraction kit according to manufacturer’s instructions.

Sequencing

Bidirectional Sanger sequencing of purified amplicons was performed with the primers mentioned above, using the BigDye Terminator sequencing ready reaction kit (v 3.1) and the products was capillary electrophoresed in an ABI Prism 3130 genetic analyser (Applied Biosystems). Sequences were analysed using Bionumerics software version 7.1 (Applied Maths, Ghent, Belgium and identified through BLASTn (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch). On the basis of comparing the sequences from the specimen with those in the GenBank databases, the fungus in the biopsy was identified as Aspergillus fumigatus. Our isolate had 100% matches with the standard strain of ATCC 1022. The sequence data have been deposited in the GenBank database (http://www.ncbi.nlm.nih.gov/Genbank/index.html) with the accession number MH465665.

Treatment and follow-up of the patient

According to the clinical features and the mycological data, diagnosis of vocal cord aspergillosis was made and the patient was treated with oral itraconazole 200 mg BD for four weeks. Clinical improvement was noticed after a month of antifungal therapy. At the third months’ follow up, his voice had returned to normal, and no residual lesion was seen on laryngoscopy. At the end of 6 months follow up, there was no recurrence.

Literature review

Extensive search in PubMed/MEDLINE and Google Scholar by two investigators independently, revealed only 38 peer-reviewed reported cases of primary laryngeal aspergillosis in immunocompetent subjects (Table 1).
Table 1

Details of 38 cases of primary laryngeal Aspergillosis in immunocompetent patients reported over the period of 50 years, (English literature)

Case

Reference

Age/gender

Geographical area

Clinical presentation

Initial diagnosis

Associated factors

Diagnosis method

Fungal culture

Aspergillus species involved

treatment

Follow up period and Outcome

1

Rao PB. 1969

48/M

Pondicherry, S. India

Hoarseness of voice

NA

None

HE

No

NA

No treatment

2 M-asymptomati

2

Ferlito A. et al. 1974

76/M

Verona, Italy

Hoarseness of voice

NA

None

HE

No

NA

No treatment

2 M- asymptomatic

3

Kheir SM et al. 1983

50/M

Birmingham, UK

Hoarseness of voice

Malignancy

COPD

HE and Immunofluorescence studies

No

NA

Topical nystatin powder

24 M- asymptomatic

4

Benson-Mitchell R et al. 1994

62/M

London, UK

Hoarseness of voice

Malignancy

None

HE

No

NA

No treatment

2 M- asymptomatic

5

Nong D, et al. 1997

30–40 4 M + 4F

China

Hoarseness of voice leading to aphonia, sore throat,

Acute laryn giti, TB, malignancy

None

NA

NA

NA

NA

NA

6

Beust L, et al.. 1998

53/M

Rennes, France

Hoarseness of voice, respiratory distress

None

None

HE

No

NA

Laryngectomy

3 M- asymptomatic

7

Fairfax AJ, et al. 1999

75/M

Staffor, UK

Hoarseness of voice, aphonia

None

None

HE and culture

Yes

A. fumigatus

AMP-lozenges, 10 mg-4 W

1 M- Improved

8

Dean CM, et al. 2001

17/F

Philadelphia, USA.

Hoarseness of voice, vocal fatigue

None

NG

NA

NA

NA

NA

NA

9

Ogawa Y, et al. 2002

73/M

Tokyo, Japan

Hoarseness of voice (History of Radiotherapy and DB)

Malignancy

None

HE, surgery

NO

NA

Oral ITCZ- 8 W and AMP-B gargle

2 M- No recurrence

10

Wittkopf J, et al. 2006

62/F

Iowa, USA

Fluctuating hoarseness

True vocal fold cyst (? aspergilloma)

Type II DB (well-controlled), hypertension, and GERD

HE

No

NA

Surgery

NA- No recurrence

11

Ran Y, et al. 2008

36/F

China

Hoarseness of voice, vocal fatigue

None

Dexamethasone therapy for rhinitis and asthma

HE, KOH, SEM and Culture

Yes

a A fumigatus

Oral ITCZ (200 mg bd-4 W)

1 M- asymptomatic

12

Liu YC, et al. 2010

30/F

32/F

Hangzhou, China

Hoarseness of voice

True vocal cord cyst

Vocal abuse, broad spectrum antibiotic therapy

HE and Culture and FFPE-PCR

Yes (no growth)

a A. fumigatus

Oral ITCZ (200 mg bd- 4 W)

1 M- asymptomatic

13

Ran Y, et al. 2011

30/F

Chengdu, China

Hoarseness of voice, vocal fatigue, expectoration, and occasional vomiting

Laryngitis

Vocal abuse, oral Antibiotics and dexamethasone use

HE, KOH, SEM and Culture

Yes

a A fumigatus

Oral ITCZ (200 mg bd- first 2 W, 200 mg qd next 2 W

1 M- asymptomatic

14

Sundarray C et al. 2011

NA

Cuttack, India

NA

NA

NA

NA

NA

NA

NA

NA

15

Ran Y, et al. 2013

23/F

China

Hoarseness of voice, severe paroxysmal cough, tachypnea

None

Oral sex

HE, SEM and Culture

Yes

a A. fumigatus

Oral ITCZ (200 mg bd- 4 W

1 M- asymptomatic

16

Doloi PK, et al. 2014

35/F

Assam, India

Hoarseness of voice, cough

None

Keratosis of the larynx

HE, KOH and Culture

Yes

A. fumigatus

Oral ITCZ (100 mg qd- 3 W)

3 W- asymptomatic

17

Al-Ogaili Z, et al. 2014

77/F

Australia

Dysphagia and hoarseness

Lymphoma

Smoking,inhaled corticosteroids

HE and Culture

Yes

Not speciated

NA

NA

18

Gangopadhyay M, et al. 2014

42/M

West Bengal, India.

Hoarsenes, fever, cough with expectoration

Malignancy

Smoking, vocal abuse

HE, and Culture

Yes

A. niger

Oral ITCZ

18 M- asymptomatic

19

Ravikumar et al. 2014

34/F

52/F

38/M

Tamil Nadu, India

Hoarseness, cough, Dysphagia, vocal fatigue

None

NA GERD

HE and KOH mount

No

NA

Oral ITCZ (100 mg bd- 3 W)

3 W- asymptomatic

20

David et al. 2014

59/F

Sydney, Australia

Hoarseness of voice

None

Asthma- fluticasone therapy

HE

No

NA

Oral ITCZ

NA: No recurrence

21

M Dutta, et al. 2015

45/F

WB, India

Hoarseness of voice

Malignancy

None

HE, KOH and Culture

Yes

A. fumigatus

Oral ITCZ (300 mg qd-3 W)

6 M- asymptomatic

22

JCR Villanueva, et al. 2015

28/F

Philippines

Hoarseness of voice

Antibiotics and steroids

None

HE

No

No

Oral VCZ (400 mg qd– 4 W)

1 M- asymptomatic

23

Arpita Saha, et al. 2015

28/F

Odisha, India

severe dysphonia

None

Asthma, long-term steroid inhaler, vocal abuse, broadspectrum antibiotics

HE, and Culture

Yes

A. fumigatus

VCZ (200 mg bd-8 days)

2 W- asymptomatic

24

Santosh Kumar Swain et al. 2016

35/M

Orissa, India

Hoarseness of voice

Flute player

Malignancy

HE, KOH and Culture

Yes

A. fumigatus

Oral ITCZ- 100 mg bd- 3 W

6 M- asymptomatic

25

Richard H. et al.. 2016

73/F

USA

persistent hoarseness

None

Inhaled and oral corticosteroids, and nebulized tobramycin

HE

NO

NA

Oral ITCZ-20 W

5 M- asymptomatic

26

Santosh Kumar et al. 2017

12/M

India

Hoarseness of voice

None

Asthma, inhaled corticosteroids, microlaryngeal surgery with stripping of the vocal cords

HE, and Culture

Yes

A. fumigatus

Oral ITCZ-50 mg bd- 3 W

3 W- asymptomatic

27

Soumen Chatterjee et.al. 2017

43/F

India West Bengal

Hoarseness of voice

None

None

HE, KOH and Culture

Yes

A. fumigatus

Oral ITC -100 mg bd- 8 W

1 M- asymptomatic

28

Present study

22/M

Nepal

hoarseness and frequent expectoration

None

None

HE, KOH, Culture and FFPET-PCR assay

Yes

a A. fumigatus

Oral ITCZ- 4 W

1 M- asymptomatic

No recurrence

Legend: aSpecies identified via sequencing; HE Histopathological examination, COPD Chronic obstructive pulmonary disease, ITCZ Itraconazole, W Weeks, M Month, AMP-B Amphotericin B, DM Diabetes mellitus, GERD Gastroesophageal reflux disease, SEM scanning electron microscopy, VCZ Voriconazole

Discussion

Aetiology of laryngeal aspergillosis

Primary invasion of the larynx by Aspergillus is uncommon and is very rare in immunocompetent individuals. As per the literature review, till date, 38 cases of primary laryngeal aspergillosis in immunocompetent patients have been documented over 50 years. Aspergillus fumigatus was reported to be the underlying causative mould in the majority of cases, documented so far; except for two cases. A. niger infection reported by Gangopadhyay et al. from India [6] and Gallo and colleagues from Italy reported Aspergillus flavus as the etiologic agent in a patient with Felty’s syndrome [7]. In the present case a immunocompetent student had Aspergillus fumigatus responsible for the laryngeal pathology.

Disease pathogenesis

Aspergillus is a well-known opportunistic fungus causing allergic and invasive disease in immunocompromised hosts [8]. The pathogenesis of laryngeal aspergillosis in an immunocompetent host is not well understood. The Aspergillus conidia are ubiquitous in nature as the fungus grows in a saprophytic environment (soil and decaying matter), it could well be possible that exposure of heavy fungal load in air may allow the fungus colonise the dark airway cavities [3] which could favour their slow germination without any symptoms. Such colonisation of the paranasal sinuses leads to fungal ball formation [9]. Hoarseness of voice was the only symptom in this case without any other predisposing conditions like corticosteroid or systemic antibiotic therapy. This is in contrast with other reports where patients developed symptoms after being treated with corticosteroid or systemic antibiotic therapy or after vocal abuse for many years (Table 1).

Epidemiology and risk factors for developing primary laryngeal aspergillosis

Amongst the reviewed 38 reported cases 16/37 (44%) were males, and 21/37 (57%) were females. Age group ranged from 12 to 74 years. Dutta M et al. [3] reported in 2015 that 50% of immunocompetent subjects with primary laryngeal aspergillosis had no identifiable contributory factors, but 14.29% had vocal abuse and steroid intake. Smoking, broad-spectrum antibiotics and exposure to radiation was detected in 10.7% of cases. Of the cases, 7.1% had vocal fold cyst, whereas 3.6% had a history of COPD, oral sex and diabetes. Six cases have been reported between 2015 and 2017 (Table 1); three of these cases were without any identifiable risk factors. Remaining three cases (and a few documented prior to 2015) had a history of using corticosteroid inhalers for bronchial asthma, which could have led to abrogation of the local immunity in the throat or could have altered the flora of the laryngeal mucosa, allowing the overgrowth of Aspergillus [10]. The exact predisposing conditions contributing towards the disease progression in the present case remains obscure but could be multifactorial with a complex interplay between host and the environment.

Time trend and geographical distribution of primary laryngeal aspergillosis

Laryngeal aspergillosis in the immunocompetent individual, though infrequently reported, seems to be an emerging condition. Lack of definite guidelines for clinical diagnosis due to the rarity of the disease might have resulted in under-reporting in the past. As shown in Fig. 2, the incidence of primary laryngeal aspergillosis in immunocompetent patients has been steadily rising over the past ten years. It seems that there has been a higher rate of reporting of the cases, especially after the 1990s, with a steady rise in the number of cases in the past seven years (Fig. 2). As depicted in the map, (Fig. 2) most of the new cases reported (11/20,) between 2010 and 2017, were from the Indian subcontinent, followed by China (4/20). This emphasizes that possibility of primary laryngeal aspergillosis must be entertained in all cases, presenting with typical features of laryngeal inflammation along with hoarseness of voice.
Fig. 2

Time trend and geographical distribution of 38 cases of primary laryngeal aspergillosis in immunocompetent patients. Legend: https://commons.wikimedia.org/wiki/Maps_of_the_world

Challenges in the diagnosis of laryngeal aspergillosis and utility of molecular diagnostics methods

As per the literature review, most of the laryngeal aspergillosis cases were diagnosed by the characteristic morphological features of the fungus in the biopsied material. However, result on species identification was lacking in majority of reported cases. Detection of hyphae, simulating those of Aspergillus in a biopsy specimen can be suggestive of fungal invasion but, is not necessarily pathognomonic of aspergillosis. Therefore, it becomes mandatory that the organism be isolated in pure culture and accurately identified. Few authors identified Aspergillus up to species level based on morphology and a couple of reports provided evidences of identification based on molecular methods [11, 12]. Moreover it is well known that fungal identification with conventional culture technique has its own limitations. As per the studies [13] conducted earlier, as well as in the present case, we could not successfully culture the fungus from the laryngeal biopsy. In these situations, etiological identification directly from clinical specimen via extraction of DNA and sequencing is advantageous. In this study, PCR on DNA extracted from paraffin-embedded tissue confirmed the aetiology. The extended region of the gene encoding the large ribosomal subunit (28S) of fungi was used for PCR amplification and sequencing. This region was previously explored for designing of broad range PCR primers and showed generation of successful amplicons and sequences from yeasts and filamentous fungi [14]. Because of the paucity of sequences of this extended region of fungal 28S rDNA in the public databases, the non D1/D2 region was rarely utilized for sequence-based detection and identification of fungi directly from clinical specimens. A recent study [15] showed the utility of the non D1/D2 region as a favorable target for the genus, and to a limited extent, species-level identification of pathogenic fungi in various fresh and FFPE samples. In the present study, attempt to amplify the internal transcribed spacer 1 (ITS1) region from the DNA extracted from the sample was not successful. One possible explanation might be due to the relatively larger size of the ITS1 region (~ 250–350 bp) than this non D1/D2 region (198 + _25 bp). Although accurate species identification required sequencing of at least a partial ITS region such as ITS 1 or ITS2, the non D1/D2 multicopy gene could give a satisfying genus level identification. In our study, this region could identify the genus and species of the pathogen with clear discrimination from other species of Aspergilli (with less % similarity scores) as evidenced from the BLAST hits. Therefore, this non D1/D2 region must be considered for PCR-sequencing from direct clinical specimens in those cases where partial ITS genes fail to amplify.

Treatment of cases

In majority of the reported cases, including the present one, itraconazole was used as an empiric treatment, though voriconzole is the treatment of choice against invasive apsergillosis [16]. Possibly cost of the antifungal agent is an important limiting factor during treatment of fungal infections in developing countries. The critical condition of the patient, arising out of the acute laryngeal pathology may be a compelling reason for the empiric treatment on an emergency basis, yielding invariably positive outcome following therapy. Recent reports of the global emergence of azole resistance in A. fumigatus [17] may be of concern in the management of such patients in future. Prompt species identification and detection of resistance are of paramount importance in the management of laryngeal mycosis.

Therapeutic outcome and relapse

In all 38 cases reviewed (Table 1), there was complete resolution of symptoms without any relapse, irrespective of the therapeutic modality adopted. There was not much difference in the time period between administration of antifungal drugs and relief of symptoms, regardless of whether the drug administered was itraconazole or voriconazole. Thus, considering the toxicity of conventional amphotericin B, and the cost of liposomal amphotericin B; empiric therapy with either itraconazole or voriconazole may be strongly advocated as better therapeutic options.

Conclusion

Since last few decades cases of primary laryngeal aspergillosis in immunocompetent individuals are on the rise, globally. Patients responded to azoles with good prognosis. This is the first case of invasive laryngeal mycosis reported in Nepal. The extraction of DNA from tissue and sequencing helps to identify the etiological agent, when culture fails to isolate the fungus.

Notes

Acknowledgements

The authors gratefully acknowledge the Dr. Narasimha Rao, professor of pathology for expertise opinion and Dr. Shishir Gokhale, professor of microbiology, Dr. Deependra Hamal, faculty of microbiology, Manipal College of Medical Sciences, Nepal for support in drafting the manuscript. We also extended special thanks to Dr. Peralam Yegneswaran Prakash, In-charge medical mycology laboratory, Kasturba Medical College and Dr. Indira Bairy, Professor and Head, Department of Microbiology, Melaka Manipal Medical College, Manipal University, India for valuable suggestions. We extend our special thanks to Manipal Teaching Hospital, Pokhara, Nepal, for providing the facility to carry out the study.

Availability of data and materials

All data generated or analyzed during this study are included in this published article. The sequence data have been deposited in the GenBank database (http://www.ncbi.nlm.nih.gov/Genbank/index.html) with the accession number MH465665.

Authors’ contributions

SHS: observed the incidence and case, performed the laboratory tests, followed the case, literature review and wrote the manuscript; KR: observed the incidence and case, collected specimens, case follow-up, and treatment, JJ: molecular studies and manuscript preparation, SMR: molecular analysis and manuscript drafting, NN & AC: Contributed toward providing clinical relevance, manuscript drafting, and critically reviewed the manuscript, AG: contributed toward histopathological diagnosis. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Due approval from the Institutional Research and Ethics Committee of the Manipal College of Medical Sciences, Pokhara, Nepal was obtained for analysing the case study, for writing the manuscript. Consent was obtained from patient to publish the data.

Consent for publication

Authors would like to acknowledge the patient, who gave written, informed consent for the publication of this case report.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  1. 1.
    Athanassiadou F, Kourti M, Papageorgiou T, Danielidis J. Invasive aspergillosis of the larynx in a child with acute lymphoblastic leukemia. Pediatr Infect Dis J. 2005;24:190–1.CrossRefPubMedGoogle Scholar
  2. 2.
    Vrabec DP. Fungal infections of the larynx. Otolaryngol Clin N Am. 1993;26(6):1091–114.Google Scholar
  3. 3.
    Dutta M, Jotdar A, Kundu S, Ghosh B, Mukhopadhyay S. Primary laryngeal aspergillosis in the immunocompetent state: a clinical update. Braz J Otorhinolaryngol. 2017;83(2):228–34.  https://doi.org/10.1016/j.bjorl.2015.06.002.CrossRefPubMedGoogle Scholar
  4. 4.
    Rao PB. Aspergillosis of larynx. J Laryngol Otol. 1969;83(4):377–9.CrossRefPubMedGoogle Scholar
  5. 5.
    Lau A, Chen S, Sorrell T, Carter D, Malik R, Martin P, Halliday C. Development and clinical application of a panfungal PCR assay to detect and identify fungal DNA in tissue specimens. J Clin Microbiol. 2007;45(2):380–5.CrossRefPubMedGoogle Scholar
  6. 6.
    Gangopadhyay M, Majumdar K, Bandyopadhyay A, Ghosh A. Invasive primary aspergillosis of the larynx presenting as hoarseness and a chronic nonhealing laryngeal ulcer in an immunocompetent host: a rare entity. Ear Nose Throat J. 2014;93:265–8.PubMedGoogle Scholar
  7. 7.
    Gallo A, Manciocco V, Simonelli M, Minni A, De Vincentiis M. Clinical findings of laryngeal aspergillosis. Otolaryngol Head Neck Surg. 2000;123(5):661–2.CrossRefPubMedGoogle Scholar
  8. 8.
    Barnes RA, Rogers TR. Aspergillosis in immunocompromised patients part I: the problem of diagnosis. In: Drouhet E, Cole GT, de Repentigny L, Latgé JP, Dupont B, editors. Fungal Antigens. Boston: Springer; 1988.Google Scholar
  9. 9.
    Vennewald I, Henker M, Klemm E, Seebacher C. Fungal colonization of the paranasal sinuses. Mycoses. 1999;42(Suppl 2):33–6.Google Scholar
  10. 10.
    Darley D, Lowinger D, Plit M. Laryngeal aspergilloma: a complication of inhaled fluticasone therapy for asthma. Respirology Case Reports. 2014;2(4):123–5.  https://doi.org/10.1002/rcr2.70.
  11. 11.
    Ran Y, Yang B, Liu S, Dai Y, Pang Z, Fan J, Bai H, Liu S. Primary vocal cord aspergillosis caused by aspergillus fumigatus and molecular identification of the isolate. Med Mycol. 2008 Aug;46(5):475–9.  https://doi.org/10.1080/13693780701851703.CrossRefPubMedGoogle Scholar
  12. 12.
    Ran Y, Li L, Cao L, Dai Y, Wei B, Zhao Y, Liu Y, Bai H, Zhang C. Primary vocal cord aspergillosis and scanning electron microscopical observation of the focus of infection. Mycoses. 2011;54(5):e634–7.  https://doi.org/10.1111/j.1439-0507.2010.01954.x.CrossRefPubMedGoogle Scholar
  13. 13.
    Liu YC, Zhou SH, Ling L. Aetiological factors contributing to the development of primary laryngeal aspergillosis in immunocompetent patients. J Med Microbiol. 2010;59:1250–3.  https://doi.org/10.1099/jmm.0.021634-0. CrossRefPubMedGoogle Scholar
  14. 14.
    Khot PD, Ko DL, Fredricks DN. Sequencing and analysis of fungal rRNA operons for development of broad-range fungal PCR assays. Appl Environ Microbiol. 2009;75(6):1559–65.  https://doi.org/10.1128/AEM.02383-08.CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Gade L, Hurst S, Balajee SA, Lockhart SR, Litvintseva AP. Detection of mucormycetes and other pathogenic fungi in formalin fixed paraffin embedded and fresh tissues using the extended region of 28S rDNA. Med Mycol. 2017;55(4):385–95.  https://doi.org/10.1093/mmy/myw083.
  16. 16.
    Patterson TF, Thompson GR, Denning DW, et al. Practice guidelines for the diagnosis and Management of Aspergillosis: 2016 update by the Infectious Diseases Society of America. Clin Infect Dis. 2016;63(4):e1–60.CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Jacques F. Meis, Anuradha Chowdhary, Johanna L. Rhodes, Matthew C. Fisher, Paul E. Verweij. Clinical implications of globally emerging azole resistance in Aspergillus fumigatus. Phil Trans R Soc B. 2016;371:20150460.  https://doi.org/10.1098/rstb.2015.0460. Published 24 October 2016.

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© The Author(s). 2018

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Authors and Affiliations

  1. 1.Department of Medical MicrobiologyManipal College of Medical SciencesPokharaNepal
  2. 2.Department of Medical MicrobiologyPostgraduate Institute of Medical Education and ResearchChandigarhIndia

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