Hostname: page-component-74656f9884-6grmk Total loading time: 0 Render date: 2026-11-06T03:45:16.012Z Has data issue: false hasContentIssue false
  • English
  • Français

Identification of pathogens causing invasive fungal rhinosinusitis in surgical biopsies using polymerase chain reaction

Published online by Cambridge University Press:  20 July 2020

S Chaturantabut ,
N Kitkumthorn ,
A Mutirangura ,
N Praditphol ,
A Chindamporn ,
P S Thorner  and
S Keelawat
S Chaturantabut
Affiliation:
Medical Science Program, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
N Kitkumthorn
Affiliation:
Department of Oral Biology, Faculty of Dentistry, Mahidol University, Bangkok, Thailand
A Mutirangura
Affiliation:
Department of Anatomy, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
N Praditphol
Affiliation:
Department of Pathology, Rajavithi Hospital, Bangkok, Thailand
A Chindamporn
Affiliation:
Department of Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
P S Thorner
Affiliation:
Department of Laboratory Medicine and Pathobiology, University of Toronto, Canada Department of Pathology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
S Keelawat*
Affiliation:
Department of Pathology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
*
Author for correspondence: Dr Somboon Keelawat, Department of Pathology, Faculty of Medicine, Chulalongkorn University, 1873 King Rama IV Street, Bangkok, 10330 Thailand E-mail: trcskl@gmail.com
Rights & Permissions [Opens in a new window]

Abstract

Background

Invasive fungal rhinosinusitis is associated with high morbidity and mortality. Rapid pathogen identification is mandatory, but fresh tissue is not always available. A polymerase chain reaction method was designed in order to detect fungi in formalin-fixed paraffin-embedded samples. This was applied to a retrospective series of tissue biopsies from Thai patients with invasive fungal rhinosinusitis.

Methods

Tissue blocks from 64 cases yielded adequate DNA. Three sequential polymerase chain reaction were performed: ZP3 (housekeeping gene) and panfungal polymerase chain reactions, and a differentiating polymerase chain reaction based on the 5.8s ribosomal RNA and internal transcribed spacer 2 regions. The polymerase chain reaction products were then sequenced.

Results

Polymerase chain reaction identified a fungal pathogen in 20 of 64 cases (31 per cent). Aspergillus species was the most common cause of invasive fungal rhinosinusitis (nine cases). Other causes included candida (n = 4), cladosporium (n = 4), mucor (n = 1), alternaria (n = 1) and dendryphiella (n = 1) species.

Conclusion

Polymerase chain reaction can provide rapid identification of fungal pathogens in paraffin-embedded tissue, enabling prompt treatment of invasive fungal rhinosinusitis.

Keywords

Invasive Fungal RhinosinusitisPolymerase Chain ReactionAspergillusCandidaCladosporium

Information

Type
Main Articles
Information
The Journal of Laryngology & Otology , Volume 134 , Issue 7 , July 2020 , pp. 632 - 635
Copyright
Copyright © The Author(s), 2020. Published by Cambridge University Press

Introduction

Invasive fungal infection is a rare cause of rhinosinusitis, and is mostly found in immunocompromised patients.Reference Das, Bal, Chakrabarti, Panda and Joshi1,Reference Taxy2 It is usually related to neutropenia, especially if the neutrophil count is less than 500 cells/μl.Reference Chen, Sheng, Cheng, Chen, Tsay and Tang3,Reference Iwen, Rupp and Hinrichs4 Invasive fungal rhinosinusitis may be acute or chronic. In the acute form, the infection typically progresses rapidly, and frequently has a fatal outcome, particularly in the form of rhizopus infection, in which the organism usually invades into blood vessels and along nerves, leading to intracerebral extension.Reference Schell5 In the chronic form of infection, the clinical course is less aggressive, with a slower rate of progress.Reference Schell5 Management includes surgical debridement and anti-fungal drug therapy,Reference Gillespie and O'Malley6 as well as the correction of any pre-existing diseases or conditions such as neutropenia or diabetes mellitus.Reference Groppo, El-Sayed, Aiken and Glastonbury7 Without early surgical and proper medical treatment, the mortality rate can be extremely high.Reference Schell5

In patients with invasive fungal rhinosinusitis, the identification of the causative fungal organism helps to determine the correct drug(s) for treatment.Reference Lai, Tan, Huang, Shao and Hsueh8 For example, treatment for aspergillus is often voriconazole, whereas fusarium is treated by a combination of amphotericin B and voriconazole.Reference Stergiopoulou and Walsh9 Fungal infection is often diagnosed by histological examination, but specific identification of the fungus depends greatly on the quality of the biopsy and the expertise of the observer. Furthermore, some fungi cannot reliably be distinguished based on morphology alone. For example, fusarium may be confused with aspergillus when using only light microscopy.Reference Ruangritchankul, Chindamporn, Worasilchai, Poumsuk, Keelawat and Bychkov10 Culture testing is a more specific way to identify fungal organisms; however, this can require up to four weeks, needs fresh tissue that is not always available, and has sensitivity in the range of 40–65 per cent.Reference Babouee Flury, Weisser, Prince, Bubendorf, Battegay and Frei11–Reference Rickerts, Mousset, Lambrecht, Tintelnot, Schwerdtfeger and Presterl13

More recently, molecular methods have been used to identify the causative fungal organism, such as the polymerase chain reaction, which is more sensitive and specific.Reference Babouee Flury, Weisser, Prince, Bubendorf, Battegay and Frei11,Reference Rickerts, Mousset, Lambrecht, Tintelnot, Schwerdtfeger and Presterl13–Reference Salehi, Hedayati, Zoll, Rafati, Ghasemi and Doroudinia25 The two fungal internal transcribed spacer regions 1 (‘ITS1’) and 2 (‘ITS2’) are highly variable and can be amplified by polymerase chain reaction using panfungal primers. Sequencing of these regions allows high specificity for the identification of fungi.Reference Munoz-Cadavid, Rudd, Zaki, Patel, Moser and Brandt24

The present study aimed to devise a polymerase chain reaction based method that would allow the specific identification of fungal pathogens using paraffin-embedded tissue biopsies, rather than requiring fresh tissue. Novel primers focusing on the 5.8s ribosomal RNA and internal transcribed spacer 2 regions were designed to detect specific types of fungal organisms known to be common causative agents for invasive fungal rhinosinusitis. The method was then tested in a series of biopsy-proven cases of invasive fungal rhinosinusitis.

Materials and methods

Study sample

This was a retrospective study of specimens received between 2005 and 2014 from three pathology laboratories in Bangkok, Thailand (King Chulalongkorn Memorial Hospital, Rajavithi Hospital and the Hi-Tech Lab (private pathological laboratory)).

A total of 81 patients with biopsy-proven invasive fungal rhinosinusitis were identified. All specimens had been formalin-fixed and paraffin-embedded at the time of the original procedure, and all cases had tissue blocks available. The study was approved by the Institutional Review Board of the Faculty of Medicine, Chulalongkorn University (institutional review board number: 426/57).

Polymerase chain reaction

Tissue slices of 10 μM thickness were cut from each formalin-fixed and paraffin-embedded block for DNA extraction. Extraction was performed according to the following steps: (1) deparaffinisation of the tissue with xylene followed with 100 per cent alcohol; (2) lysis of cell membranes using lysis buffer II, 10 per cent sodium dodecyl sulphate and proteinase K (20 mg/ml); and (3) DNA extraction using a standard phenol-chloroform technique.Reference Sambrook and Russell26

The polymerase chain reaction test was designed as sequential set of reactions to identify specific fungi. First, the quality of extracted DNA was assessed by amplifying the human housekeeping gene ZP3 as an internal control. Specimens with acceptable DNA quality were then subjected to the first amplification, designed to detect any fungus, using primers for a conserved sequence in the 18s ribosomal RNA conserved sequence (panfungal primer). Positive cases were then subjected to a second amplification procedure designed to distinguish specific fungi.

For primers that would differentiate fungal species, we designed novel primers focusing on the 5.8s ribosomal RNA and internal transcribed spacer 2 regions, based on data obtained from GenBank (the National Institutes of Health genetic sequence database). We compared the sequences for 10 common fungal species causing rhinosinusitis, including: Aspergillus clavatus (GenBank code: AB 244778.1), Aspergillus fischerianus (AB 185254.1), Aspergillus flavus (AB 008415.1), Aspergillus fumigatus (AB 055971.1), Rhizopus oryzae (AB 181327.1), Mucor indicus (AB 113027.1), Fusarium solani (AM 412639.1), Fusarium chlamydosporum (HG 798770.1), Candida albicans (HE 860439.1) and Pseudallescheria boydii (AJ 888438.1). The sequences, annealing temperatures and product sizes for the primers are detailed in Table 1.

Table 1.

Primers used in PCR for ZP3 housekeeping gene, panfungal detection and differentiating specific fungi

PCR = polymerase chain reaction

Polymerase chain reaction conditions consisted of an initial denaturation of DNA at 95°C for 15 minutes, and 40 cycles of amplification performed as follows: denaturation at 95°C for 45 seconds, annealing for 45 seconds, and extension at 72°C for 45 seconds with a final extension for 7 minutes. Polymerase chain reaction products were visualised by 2 per cent agarose gel electrophoresis. Depending on the fungus, the expected size of the final polymerase chain reaction product ranged from 250 to 300 bp. Polymerase chain reaction product bands were extracted and sequenced. Each polymerase chain reaction test was performed together with negative (distilled water) and positive controls. Positive controls consisted of culture samples of aspergillus sp. (TISTR Culture Collection (Bangkok, Thailand) code: TISTR3464) and fusarium sp. (TISTR3436) obtained from the Thailand Institute of Scientific and Technological Research. Before undergoing DNA extraction, the live micro-organisms from these samples were inactivated by heat.

Results

Before analysing patient samples, the specificity and sensitivity of the polymerase chain reaction were determined. For specificity, samples of aspergillus sp. and fusarium sp. (obtained from the Thailand Institute of Scientific and Technological Research) were subjected to polymerase chain reaction and sequenced, and the expected results were obtained. The sensitivity of the reaction was determined by performing polymerase chain reaction on serial dilutions of positive DNA control template. The lowest DNA template concentration that resulted in visible DNA band product on an agarose gel was 0.1 ng/μl (data not shown).

The tissue blocks in the study ranged from less than one year to nine years old. Of the 81 samples, 64 (79 per cent) contained DNA of adequate quality for further analysis. Of these 64 samples, 20 (31 per cent) yielded polymerase chain reaction products for sequencing. For the samples that were sequenced, the results were compared to GenBank submissions.

None of the polymerase chain reaction products were from human DNA, confirming the specificity of the reaction for fungal DNA. The negative controls were always negative, indicating that environmental fungi were not being amplified in the polymerase chain reaction reactions. The most common fungus identified was aspergillus spp., which was detected in 9 out of 20 cases (45 per cent). For the remaining 11 cases, the fungi detected were: candida sp., in 4 cases (20 per cent); cladosporium sp., in 4 cases (20 per cent); mucor sp., in 1 case (5 per cent); alternaria sp., in 1 case (5 per cent); and dendryphiella sp., in 1 case (5 per cent).

Discussion

The diagnosis of invasive fungal rhinosinusitis traditionally relies on histopathological examination and fungal culture testing, both of which have limitations in terms of specificity (with respect to morphological diagnosis) and sensitivity (with respect to culture). A polymerase chain reaction based diagnosis of fungal disease provides higher specificity and sensitivity, and a faster turnaround time, than culture testing.Reference Babouee Flury, Weisser, Prince, Bubendorf, Battegay and Frei11,Reference Rickerts, Mousset, Lambrecht, Tintelnot, Schwerdtfeger and Presterl13–Reference Salehi, Hedayati, Zoll, Rafati, Ghasemi and Doroudinia25 While culture testing takes about four weeks to get results, our polymerase chain reaction based method requires only one week for the whole process. The cost of the latter test is in the range of $40 USD. Although fungal culture testing is less expensive, the cost of additional patient days in hospital while awaiting the results makes a polymerase chain reaction based test the cheaper option.

Furthermore, under the correct conditions, polymerase chain reaction can be applied to paraffin-embedded tissue, which has the advantages of: (1) avoiding the need for triaging fresh tissue at the time of biopsy, which reduces the material available for microscopic examination; (2) allowing selection of the most involved area of the biopsy sample for further molecular testing; (3) enabling retesting as needed, without requiring a repeat biopsy; and (4) facilitating retrospective studies for epidemiological and quality control purposes.

In Thailand, there has been no study to date on samples from patients with invasive fungal rhinosinusitis using a polymerase chain reaction based technique. All previous studies have relied on histomorphological examination and fungal culture testing for diagnosis.Reference Praweswararat27–Reference Soontrapa, Larbcharoensub, Luxameechanporn, Cheewaruangroj, Prakunhungsit and Sathapatayavong29

  • • Adapting polymerase chain reaction to paraffin-embedded material allows entire biopsy tissue testing and avoids the need for fresh tissue for culture

  • • Positive results can lead to earlier and more specific anti-fungal treatment of invasive fungal rhinosinusitis

  • • Our differentiating polymerase chain reaction method is based on fungal 5.8s ribosomal RNA and internal transcribed spacer 2 regions

  • • Aspergillus species was confirmed as the most common organism causing invasive fungal rhinosinusitis in Thai patients

  • • A polymerase chain reaction based approach can identify unsuspected or previously unrecognised fungal pathogens

  • • Dendryphiella species is described for the first time as a cause of invasive fungal rhinosinusitis

In our study, no human genes were amplified by our protocol, confirming the specificity for fungal DNA. The most common causative organism was aspergillus sp. (45 per cent of positive samples). This is in line with all the previous case series from Thailand and some nearby regions such as Taiwan.Reference Chen, Sheng, Cheng, Chen, Tsay and Tang3 However, it is not in agreement with other nearby regions, such as India where rhizopus sp. was found to be the most common fungi causing acute invasive fungal rhinosinusitis.Reference Das, Bal, Chakrabarti, Panda and Joshi1,Reference Michael, Michael, Ashbee and Mathews30 The second most common causative organism in our series was candida sp., detected in 20 per cent of positive samples.

We also identified some unusual causative fungal species, namely cladosporium sp., alternaria sp. and dendryphiella sp. These organisms were not in the list of common fungi that we selected for designing our primers, but happened to share the same sequences used in our primers. Cladosporium and alternaria have been described in allergic rhinitis, but only rarely in invasive fungal rhinosinusitis.Reference Bhatt, Viola and Ferrajoli31,Reference Pastor and Guarro32 In our study, the proportion of cladosporium was relatively high (20 per cent). Both fungi are commonly found in the atmosphere.Reference Hamilos33 Whether the high prevalence of cladosporium in our samples is associated with a high concentration of these fungi in the environment requires further investigation. However, as our negative controls were consistently negative, the detection of this fungus is not a reflection of atmospheric contamination in the laboratory. Dendryphiella sp. occurs naturally on aquatic plants,Reference Lee and Baker34 but there has been no reported case causing invasive fungal rhinosinusitis in humans. This may indicate a new fungal pathogen and confirmation is needed.

While our polymerase chain reaction method was able to provide a diagnosis of fungal organisms in patients with invasive fungal rhinosinusitis, the overall detection rate was low, with only 31 per cent of specimens (20 out of 64) giving a molecular diagnosis. The detection rates from other reports using formalin-fixed and paraffin-embedded samples are higher than in our study (at 54–94 per cent), with some using a similar polymerase chain reaction protocol to ours and others using different ones.Reference Babouee Flury, Weisser, Prince, Bubendorf, Battegay and Frei11,Reference Rickerts, Mousset, Lambrecht, Tintelnot, Schwerdtfeger and Presterl13,Reference Bialek, Konrad, Kern, Aepinus, Cecenas and Gonzalez17,Reference Buitrago, Bernal-Martinez, Castelli, Rodriguez-Tudela and Cuenca-Estrella19,Reference Gholinejad-Ghadi, Shokohi, Seifi, Aghili, Roilides and Nikkhah21,Reference Munoz-Cadavid, Rudd, Zaki, Patel, Moser and Brandt24,Reference Salehi, Hedayati, Zoll, Rafati, Ghasemi and Doroudinia25,Reference Lau, Chen, Sorrell, Carter, Malik and Martin35

Moreover, our detection rate of 31 per cent is in contrast with our previous study based on autopsy material, in which the detection rate for fungal organisms by polymerase chain reaction was 100 per cent.Reference Ruangritchankul, Chindamporn, Worasilchai, Poumsuk, Keelawat and Bychkov10 While both studies were based on formalin-fixed and paraffin-embedded material, the autopsy study allowed selection of large tissue blocks containing the most abundant fungus, whereas the surgical biopsy samples in the present series were small, with a lower amount of fungi per block.

A failure to detect fungus using polymerase chain reaction could also be related to degradation associated with formalin fixation, or inhibitors to the polymerase chain reaction that are present in the block.Reference Babouee Flury, Weisser, Prince, Bubendorf, Battegay and Frei11,Reference Munoz-Cadavid, Rudd, Zaki, Patel, Moser and Brandt24 While we analysed only specimens with adequate quality human DNA, we did not test the quality of fungal DNA specifically, and we did not attempt to remove any inhibitors. The age of the tissue block was not found to be associated with polymerase chain reaction failure in one study.Reference Babouee Flury, Weisser, Prince, Bubendorf, Battegay and Frei11 In our study, the age of the tissue blocks in which fungus was detected by polymerase chain reaction ranged from less than one year to nine years. While these are not particularly old samples, the ambient storage temperature may have affected the quality of DNA in our tissue blocks. This is a concern for tropical countries such as Thailand, where the ambient temperature is usually over 30°C. Most of the previous studies with higher detection rates are from countries with more temperate climates.

In summary, polymerase chain reaction testing for fungal pathogens using formalin-fixed and paraffin-embedded tissue has several advantages over fungal culture testing using fresh tissue. A polymerase chain reaction based assay avoids the need for dividing up fresh tissue, only part of which is rerouted to a microbiology laboratory and therefore not representative of the entire biopsy tissue, and this submitted portion may not even contain fungal organisms. Instead, if the entire biopsy tissue is retained in the pathology laboratory and embedded in paraffin, then polymerase chain reaction performed on material from these tissue blocks reflects the entire sample, instead of only a portion. This approach may avoid the need for a larger initial biopsy or a repeat biopsy.

A polymerase chain reaction based approach may lead to the identification of unsuspected or previously unrecognised fungal pathogens that might have gone unrecognised using standard morphology and culture testing. In addition, as the turnaround time for polymerase chain reaction testing is faster than that for culture testing, positive results can lead to earlier and more specific anti-fungal treatment of invasive fungal rhinosinusitis, which is a potentially lethal infection. While not all centres have a laboratory equipped to carry out polymerase chain reaction, rapid referral to a testing centre with this capability can still achieve an acceptable turnaround time, translating to improved patient care.

Acknowledgements

We would like to thank the Thailand Institute of Scientific and Technological Research for providing the cultured samples of the fungi used in this study. This study was financially supported by grants from the Ratchadaphiseksompot Fund, Faculty of Medicine, Chulalongkorn University (grant number: 58/60) and the National Science and Technology Development Agency, Thailand (grant number: FDA-CO-2561-8477-TH).

Competing interests

None declared

Footnotes

Dr S Keelawat takes responsibility for the integrity of the content of the paper

References

Das, A, Bal, A, Chakrabarti, A, Panda, N, Joshi, K. Spectrum of fungal rhinosinusitis; histopathologist's perspective. Histopathology 2009;54:854–9CrossRefGoogle ScholarPubMed
Taxy, JB. Paranasal fungal sinusitis: contributions of histopathology to diagnosis: a report of 60 cases and literature review. Am J Surg Pathol 2006;30:713–20CrossRefGoogle ScholarPubMed
Chen, CY, Sheng, WH, Cheng, A, Chen, YC, Tsay, W, Tang, JL et al. Invasive fungal sinusitis in patients with hematological malignancy: 15 years experience in a single university hospital in Taiwan. BMC Infect Dis 2011;11:250CrossRefGoogle Scholar
Iwen, PC, Rupp, ME, Hinrichs, SH. Invasive mold sinusitis: 17 cases in immunocompromised patients and review of the literature. Clin Infect Dis 1997;24:1178–84CrossRefGoogle ScholarPubMed
Schell, WA. Histopathology of fungal rhinosinusitis. Otolaryngol Clin North Am 2000;33:251–76CrossRefGoogle ScholarPubMed
Gillespie, MB, O'Malley, BW. An algorithmic approach to the diagnosis and management of invasive fungal rhinosinusitis in the immunocompromised patient. Otolaryngol Clin North Am 2000;33:323–34CrossRefGoogle ScholarPubMed
Groppo, ER, El-Sayed, IH, Aiken, AH, Glastonbury, CM. Computed tomography and magnetic resonance imaging characteristics of acute invasive fungal sinusitis. Arch Otolaryngol Head Neck Surg 2011;137:1005–10CrossRefGoogle ScholarPubMed
Lai, CC, Tan, CK, Huang, YT, Shao, PL, Hsueh, PR. Current challenges in the management of invasive fungal infections. J Infect Chemother 2008;14:77–85CrossRefGoogle ScholarPubMed
Stergiopoulou, T, Walsh, TJ. Clinical pharmacology of antifungal agents to overcome drug resistance in pediatric patients. Expert Opin Pharmacother 2015;16:213–26CrossRefGoogle ScholarPubMed
Ruangritchankul, K, Chindamporn, A, Worasilchai, N, Poumsuk, U, Keelawat, S, Bychkov, A. Invasive fungal disease in university hospital: a PCR-based study of autopsy cases. Int J Clin Exp Pathol 2015;8:14840–52Google ScholarPubMed
Babouee Flury, B, Weisser, M, Prince, SS, Bubendorf, L, Battegay, M, Frei, R et al. Performances of two different panfungal PCRs to detect mould DNA in formalin-fixed paraffin-embedded tissue: what are the limiting factors? BMC Infect Dis 2014;14:692–700CrossRefGoogle ScholarPubMed
Chamilos, G, Luna, M, Lewis, RE, Bodey, GP, Chemaly, R, Tarrand, JJ et al. Invasive fungal infections in patients with hematologic malignancies in a tertiary care cancer center: an autopsy study over a 15-year period (1989–2003). Haematologica 2006;91:986–9Google Scholar
Rickerts, V, Mousset, S, Lambrecht, E, Tintelnot, K, Schwerdtfeger, R, Presterl, E et al. Comparison of histopathological analysis, culture, and polymerase chain reaction assays to detect invasive mold infections from biopsy specimens. Clin Infect Dis 2007;44:1078–83CrossRefGoogle ScholarPubMed
Alexander, BD. Diagnosis of fungal infection: new technologies for the mycology laboratory. Transpl Infect Dis 2002;4(suppl 3):32–7CrossRefGoogle ScholarPubMed
Avni, T, Leibovici, L, Paul, M. PCR diagnosis of invasive candidiasis: systematic review and meta-analysis. J Clin Microbiol 2011;49:665–70CrossRefGoogle ScholarPubMed
Baskova, L, Buchta, V. Laboratory diagnostics of invasive fungal infections: an overview with emphasis on molecular approach. Folia Microbiol (Praha) 2012;57:421–30CrossRefGoogle ScholarPubMed
Bialek, R, Konrad, F, Kern, J, Aepinus, C, Cecenas, L, Gonzalez, GM et al. PCR based identification and discrimination of agents of mucormycosis and aspergillosis in paraffin wax embedded tissue. J Clin Pathol 2005;58:1180–4CrossRefGoogle ScholarPubMed
Borman, AM, Linton, CJ, Miles, SJ, Johnson, EM. Molecular identification of pathogenic fungi. J Antimicrob Chemother 2008;61(suppl 1):i7–12CrossRefGoogle ScholarPubMed
Buitrago, MJ, Bernal-Martinez, L, Castelli, MV, Rodriguez-Tudela, JL, Cuenca-Estrella, M. Performance of panfungal - and specific - PCR-based procedures for etiological diagnosis of invasive fungal diseases on tissue biopsy specimens with proven infection: a 7-year retrospective analysis from a reference laboratory. J Clin Microbiol 2014;52:1737–40CrossRefGoogle ScholarPubMed
Gharizadeh, B, Norberg, E, Loffler, J, Jalal, S, Tollemar, J, Einsele, H et al. Identification of medically important fungi by the pyrosequencing technology. Mycoses 2004;47:29–33CrossRefGoogle ScholarPubMed
Gholinejad-Ghadi, N, Shokohi, T, Seifi, Z, Aghili, SR, Roilides, E, Nikkhah, M et al. Identification of mucorales in patients with proven invasive mucormycosis by polymerase chain reaction in tissue samples. Mycoses 2018;61:909–15CrossRefGoogle ScholarPubMed
Khot, PD, Fredricks, DN. PCR-based diagnosis of human fungal infections. Expert Rev Anti Infect Ther 2009;7:1201–21CrossRefGoogle ScholarPubMed
Kourkoumpetis, TK, Fuchs, BB, Coleman, JJ, Desalermos, A, Mylonakis, E. Polymerase chain reaction-based assays for the diagnosis of invasive fungal infections. Clin Infect Dis 2012;54:1322–31CrossRefGoogle Scholar
Munoz-Cadavid, C, Rudd, S, Zaki, SR, Patel, M, Moser, SA, Brandt, ME et al. Improving molecular detection of fungal DNA in formalin-fixed paraffin-embedded tissues: comparison of five tissue DNA extraction methods using panfungal PCR. J Clin Microbiol 2010;48:2147–53CrossRefGoogle ScholarPubMed
Salehi, E, Hedayati, MT, Zoll, J, Rafati, H, Ghasemi, M, Doroudinia, A et al. Discrimination of aspergillosis, mucormycosis, fusariosis, and scedosporiosis in formalin-fixed paraffin-embedded tissue specimens by use of multiple real-time quantitative PCR assays. J Clin Microbiol 2016;54:2798–803CrossRefGoogle ScholarPubMed
Sambrook, J, Russell, DW. Purification of nucleic acids by extraction with phenol:chloroform. CSH Protoc 2006;2006:pdb.prot4455Google ScholarPubMed
Praweswararat, P. Invasive fungal sinusitis: seven years experiences in Rajavithi Hospital. Asian Rhinology Journal 2015;2:33–9Google Scholar
Roongrotwattanasiri, K, Fooanant, S, Naksane, L. Invasive fungal rhinosinusitis in Maharaj Nakorn Chiang Mai Hospital. J Med Assoc Thai 2007;90:2524–8Google ScholarPubMed
Soontrapa, P, Larbcharoensub, N, Luxameechanporn, T, Cheewaruangroj, W, Prakunhungsit, S, Sathapatayavong, B et al. Fungal rhinosinusitis: a retrospective analysis of clinicopathologic features and treatment outcomes at Ramathibodi Hospital. Southeast Asian J Trop Med Public Health 2010;41:442–9Google ScholarPubMed
Michael, RC, Michael, JS, Ashbee, RH, Mathews, MS. Mycological profile of fungal sinusitis: an audit of specimens over a 7-year period in a tertiary care hospital in Tamil Nadu. Indian J Pathol Microbiol 2008;51:493–6CrossRefGoogle Scholar
Bhatt, VR, Viola, GM, Ferrajoli, A. Invasive fungal infections in acute leukemia. Ther Adv Hematol 2011;2:231–47CrossRefGoogle ScholarPubMed
Pastor, FJ, Guarro, J. Alternaria infections: laboratory diagnosis and relevant clinical features. Clin Microbiol Infect 2008;14:734–46CrossRefGoogle ScholarPubMed
Hamilos, DL. Allergic fungal rhinitis and rhinosinusitis. Proc Am Thorac Soc 2010;7:245–52CrossRefGoogle ScholarPubMed
Lee, B, Baker, G. Environment and the distribution of microfungi in a Hawaiian mangrove swamp. Pacific Science 1972;26:11–19Google Scholar
Lau, A, Chen, S, Sorrell, T, Carter, D, Malik, R, Martin, P et al. Development and clinical application of a panfungal PCR assay to detect and identify fungal DNA in tissue specimens. J Clin Microbiol 2007;45:380–5CrossRefGoogle ScholarPubMed
Figure 0

Table 1. Primers used in PCR for ZP3 housekeeping gene, panfungal detection and differentiating specific fungi