• 1

    Edgar RC: Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26: 2460, 2010.

  • 2

    Edgar RC: UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nat Methods 10: 996, 2013.

  • 3

    Edgar RC: MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32: 1792, 2004.

  • 4

    Edgar RC: MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics 5: 113, 2004.

  • 5

    Price MN, Dehal PS, Arkin AP: FastTree: computing large minimum evolution trees with profiles instead of a distance matrix. Mol Biol Evol 26: 1641, 2009.

  • 6

    Price MN, Dehal PS, Arkin AP: FastTree 2: approximately maximum-likelihood trees for large alignments. PLoS One 5: e9490, 2010.

  • 7

    Elewski BE: Onychomycosis: treatment, quality of life, and economic issues. Am J Clin Dermatol 1: 19, 2000.

  • 8

    Ghannoum MA, Hajjeh RA, Scher R, et al: A large-scale North American study of fungal isolates from nails: the frequency of onychomycosis, fungal distribution, and antifungal susceptibility patterns. J Am Acad Dermatol 43: 641, 2000.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 9

    Litz CE, Cavagnolo RZ: Polymerase chain reaction in the diagnosis of onychomycosis: a large, single-institute study. Br J Dermatol 163: 511, 2010.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10

    da Cunha KC, Sutton DA, Gené J, et al. Pithomyces species (Montagnulaceae) from clinical specimens: identification and antifungal susceptibility profiles. Med Mycol 52: 748, 2014.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 11

    Farwa U, Abbasi SA, Mirza IA, et al: Non-dermatophyte moulds as pathogens of onychomycosis. J Coll Physicians Surg Pak 21: 597, 2011.

  • 12

    Green BJ, Mitakakis TZ, Tovey ER: Allergen detection from 11 fungal species before and after germination. J Allergy Clin Immunol 111: 285, 2003.

  • 13

    Hini? V, Lang C, Weisser M, et al: Corynebacterium tuberculostearicum: a potentially misidentified and multiresistant Corynebacterium species isolated from clinical specimens. J Clin Microbiol 50: 2561, 2012.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14

    Zehnder M, Guggenheim B: The mysterious appearance of enterococci in filled root canals. Int Endod J 42: 277, 2009.

  • 15

    Shemer A, Gupta AK, Amichai B, et al: Increased risk of tinea pedis and onychomycosis among swimming pool employees in Netanya Area, Israel. Mycopathologia 181: 851, 2016.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 16

    Ioannidou D, Maraki S, Krasagakis S, et al: The epidemiology of onychomycoses in Crete, Greece, between 1992 and 2001. J Eur Acad Dermatol Venereol 20: 170, 2006.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 17

    García-Martos P, Domínguez I, Marín P, et al: Onychomycoses caused by non-dermatophytic filamentous fungi in Cádiz [in Spanish]. Enferm Infecc Microbiol Clin 18: 319, 2000.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 18

    Ebihara M, Makimura K, Sato K, et al: Molecular detection of dermatophytes and nondermatophytes in onychomycosis by nested polymerase chain reaction based on 28S ribosomal RNA gene sequences. Br J Dermatol 161: 1038, 2009.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 19

    Gupta AK, Carviel JL, Abramovits W: Efficacy of tofacitinib in treatment of alopecia universalis in two patients. J Eur Acad Dermatol Venereol 30: 1373, 2016.

  • 20

    Gupta AK, Drummond-Main C, Cooper EA, et al: Systematic review of nondermatophyte mold onychomycosis: diagnosis, clinical types, epidemiology, and treatment. J Am Acad Dermatol 66: 494, 2012.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 21

    Mikailov A, Cohen J, Joyce C, et al: Cost-effectiveness of confirmatory testing before treatment of onychomycosis. JAMA Dermatol 152: 276, 2016.

  • 22

    Feng X, Ling B, Yang X, et al: Molecular Identification of Candida Species Isolated from Onychomycosis in Shanghai, China. Mycopathol 180: 365, 2015.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 23

    Otaševi? S, Barac A, Pekmezovic M, et al: The prevalence of Candida onychomycosis in Southeastern Serbia from 2011 to 2015. Mycoses 59: 167, 2016.

  • 24

    Arrua JMM, Rodrigues LAS, Pereira FO, et al: Prevalence of Candida tropicalis and Candida krusei in onychomycosis in João Pessoa, Paraiba, Brazil from 1999 to 2010. An Acad Bras Cienc 87: 1819, 2015.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25

    Guclu E, Soypacaci Z, Yildirim M, et al: First case of continuous ambulatory peritoneal dialysis peritonitis due to Candida sake. Mycoses 52: 280, 2009.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 26

    Presterl E, Daxböck F, Graninger W, et al: Changing pattern of candidaemia 2001-2006 and use of antifungal therapy at the University Hospital of Vienna, Austria. Clin Microbiol Infect Off Publ Eur Soc Clin Microbiol Infect Dis 13: 1072, 2007.

    • Search Google Scholar
    • Export Citation
  • 27

    Palmisano A, Benecchi M, De Filippo M, et al: Candida sake as the causative agent of spondylodiscitis in a hemodialysis patient. Spine J Off J North Am Spine Soc 11: e12, 2011.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 28

    Dorko E, Jautová J, Tkáciková L, et al: The frequency of Candida species in onychomycosis. Folia Microbiol (Praha) 47: 727, 2002.

  • 29

    Crozier WJ: Two cases of onychomycosis due to Candida zeylanoides. Australas J Dermatol 34: 23, 1993.

  • 30

    Nguyen NH, Suh S-O, Marshall CJ, et al: Morphological and ecological similarities: wood-boring beetles associated with novel xylose-fermenting yeasts, Spathaspora passalidarum gen. sp. nov. and Candida jeffriesii sp. nov. Mycol Res 110: 1232, 2006.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 31

    Veraldi S, Chiaratti A, Harak H: Onychomycosis caused by Aspergillus versicolor. Mycoses 53: 363, 2010.

  • 32

    Nenoff P, Ginter-Hanselmayer G, Tietz H-J: [Fungal nail infections - an update. Part 2 - From the causative agent to diagnosis - conventional and molecular procedures]. Hautarzt Z Dermatol Venerol Verwandte Geb 63: 130, 2012.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 33

    Moreno G, Arenas R: Other fungi causing onychomycosis. Clin Dermatol 28: 160, 2010.

  • 34

    Torres-Rodríguez JM, Madrenys-Brunet N, Siddat M, et al: Aspergillus versicolor as cause of onychomycosis: report of 12 cases and susceptibility testing to antifungal drugs. J Eur Acad Dermatol Venereol JEADV 11: 25, 1998.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 35

    Tsang C-C, Hui TWS, Lee K-C, et al: Genetic diversity of Aspergillus species isolated from onychomycosis and Aspergillus hongkongensis sp. nov., with implications to antifungal susceptibility testing. Diagn Microbiol Infect Dis 84: 125, 2016.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 36

    Nouripour-Sisakht S, Mirhendi H, Shidfar MR, et al: Aspergillus species as emerging causative agents of onychomycosis. J Mycol Medicale 25: 101, 2015.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 37

    Fernández MS, Rojas FD, Cattana ME, Sosa M de LÁ, Mangiaterra ML, Giusiano GE: Aspergillus terreus complex: an emergent opportunistic agent of Onychomycosis. Mycoses 56: 477, 2013.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 38

    Nazareth SW, Gonsalves V: Halophilic Aspergillus penicillioides from athalassohaline, thalassohaline, and polyhaline environments. Front Microbiol 5: 412, 2014.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 39

    Gibbons JG, Salichos L, Slot JC, et al: The evolutionary imprint of domestication on genome variation and function of the filamentous fungus Aspergillus oryzae. Curr Biol CB 22: 1403, 2012.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 40

    Itabashi T, Hosoe T, Toyasaki N, et al: [Allergen activity of xerophilic fungus, Aspergillus restrictus]. Arerugi Allergy 56: 101, 2007.

Fungal Diversity and Onychomycosis

An Analysis of 8,816 Toenail Samples Using Quantitative PCR and Next-Generation Sequencing

Annette Joyce DPM1, Aditya K. Gupta MD, PhD, FRCP(C)2, Lars Koenig PhD3, Randall Wolcott MD4, and Jessie Carviel PhD2
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  • 1 Joyce Podiatry, Westminster, MD. Dr. Joyce is now with Dermfoot Educational Ventures, LLC, Pawley's Island, SC.
  • | 2 Mediprobe Research Inc, London, Ontario, Canada.
  • | 3 Research and Testing Laboratory, Lubbock, TX.
  • | 4 Southwest Regional Wound Care Clinic, Lubbock, TX.
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Background:

Onychomycosis is a fungal infection of the nail that is often recalcitrant to treatment and prone to relapse. Traditional potassium hydroxide and culture diagnosis is costly and time-consuming. Therefore, molecular methods were investigated to demonstrate effectiveness in diagnosis and to quantify the microbial flora present that may be contributing to disease.

Methods:

A total of 8,816 clinically suspicious toenail samples were collected by podiatric physicians across the United States from patients aged 0 to 103 years and compared with a control population (N = 20). Next-generation sequencing and quantitative polymerase chain reaction were used to identify and quantify dermatophytes, nondermatophyte molds, and bacteria.

Results:

Approximately 50% of suspicious toenails contained both fungi and bacteria, with the dermatophyte Trichophyton rubrum contributing the highest relative abundance and presence in 40% of these samples. Of the remaining 50% of samples, 34% had bacterial species present and 16% had neither. Fungi only were present in less than 1% of samples. Nondermatophyte molds contributed to 11.0% of occurrences in fungus-positive samples. All of the control samples were negative for fungi, with commensal bacterial species composing most of the flora population.

Conclusions:

Molecular methods were successful in efficiently quantifying microbial and mycologic presence in the nail. Contributions from dermatophytes were lower than expected, whereas the opposite was true for nondermatophyte molds. The clinical significance of these results is currently unknown.

Corresponding author: Annette Joyce, DPM, 423 Lumbee Circle, Pawley's Island, SC 29585. (E-mail: drjoycepodiatry@gmail.com)