• 1.

    Dascanio JJSchweizer CLey WB. Equine fungal endometritis. Equine Vet Educ 2001; 13: 324329.

  • 2.

    Coutinho da Silva MAAlvarenga MA. Fungal endometritis. In: McKinnon ASquires EVaala W, et al, eds. Equine reproduction. 2nd ed. Danvers, Mass: Wiley-Blackwell Publishing, 2011;26432651.

    • Search Google Scholar
    • Export Citation
  • 3.

    Alexander BDPfaller MA. Contemporary tools for the diagnosis and management of invasive mycoses. Clin Infect Dis 2006; 43(suppl 1):S15s27.

    • Search Google Scholar
    • Export Citation
  • 4.

    Chen SCAHalliday CLMeyer W. A review of nucleic acid-based diagnostic tests for systemic mycoses with an emphasis on polymerase chain reaction-based assays. Med Mycol 2002; 40: 333357.

    • Search Google Scholar
    • Export Citation
  • 5.

    Vollmer TStormer MKleesiek K, et al. Evaluation of novel broad-range real-time PCR assay for rapid detection of human pathogenic fungi in various clinical specimens. J Clin Microbiol 2008; 46: 19191926.

    • Search Google Scholar
    • Export Citation
  • 6.

    Tintelnot KDe Hoog GSAntweiler E, et al. Taxonomic and diagnostic markers for identification of Coccidioides immitis and Coccidioides posadasii. Med Mycol 2007; 45: 385393.

    • Search Google Scholar
    • Export Citation
  • 7.

    Jaeger EEMCarroll NMChoudhury S, et al. Rapid detection and identification of Candida, Aspergillus, and Fusarium species in ocular samples using nested PCR. J Clin Microbiol 2000; 38: 29022908.

    • Search Google Scholar
    • Export Citation
  • 8.

    Kurtzman CPRobnett CJ. Identification of clinically important ascomycetous yeasts based on nucleotide divergence in the 5′ end of the large-subunit (26S) ribosomal DNA gene. J Clin Microbiol 1997; 35: 12161223.

    • Search Google Scholar
    • Export Citation
  • 9.

    Preuner SLion T. Towards molecular diagnostics of invasive fungal infections. Expert Rev Mol Diagn 2009; 9: 397401.

  • 10.

    Espy MJUhl JRSloan LM, et al. Real-time PCR in clinical microbiology: applications for routine laboratory testing. Clin Microbiol Rev 2006; 19: 165256.

    • Search Google Scholar
    • Export Citation
  • 11.

    Hoorfar JMalorny BAbdulmawjood A, et al. Practical considerations in design of internal amplification controls for diagnostic PCR assays. J Clin Microbiol 2004; 42: 18631868.

    • Search Google Scholar
    • Export Citation
  • 12.

    Bretagne SCosta J. Towards a molecular diagnosis of invasive aspergillosis and disseminated candidosis. FEMS Immunol Med Microbiol 2005; 45: 361368.

    • Search Google Scholar
    • Export Citation
  • 13.

    Hinrichs KCummings MRSertich PL, et al. Clinical significance of aerobic bacterial flora of the uterus, vagina, vestibule, and clitoral fossa of clinically normal mares. J Am Vet Med Assoc 1988; 193: 7275.

    • Search Google Scholar
    • Export Citation

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Development of a broad-range quantitative polymerase chain reaction assay to detect and identify fungal DNA in equine endometrial samples

Ryan A. Ferris DVM, MS1, Katy Dern DVM2, Julia K. Veir DVM, PhD3, Jennifer R. Hawley BS4, Michael R. Lappin DVM, PhD5, and Patrick M. McCue DVM, PhD6
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  • 1 Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80521.
  • | 2 Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80521.
  • | 3 Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80521.
  • | 4 Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80521.
  • | 5 Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80521.
  • | 6 Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80521.

Abstract

Objective—To develop a broad-range 28S ribosomal DNA quantitative PCR (qPCR) assay for detection of fungal DNA in equine endometrial samples.

Sample—12 fungal samples from a clinical diagnostic laboratory and 29 samples obtained from 17 mares.

Procedures—The qPCR assay was optimized with commercially acquired fungal organisms and validated with samples obtained from the clinical diagnostic laboratory. Subsequently, 29 samples from 17 mares suspected of having fungal endometritis were evaluated via the qPCR assay and via traditional fungal culture and endometrial cytology. Amplicons from the qPCR assay were subjected to genetic sequencing to identify the organisms.

Results—The qPCR assay theoretically had a detection threshold of 2 organisms of Candida albicans. Fungal DNA was amplified from all 12 fungal samples from the commercial diagnostic laboratory. Fungal identification by use of genetic sequencing was successful for 34 of 36 amplicons from the 12 samples assayed. A fungal agent was identified via qPCR assay and genetic sequencing in all 12 samples; in contrast, a fungal agent was identified in only 8 of 12 samples via standard fungal culture and biochemical analysis. The qPCR assay detected fungal DNA in samples from 12 of 17 mares suspected of having fungal endometritis.

Conclusions and Clinical Relevance—A rapid, sensitive, and repeatable qPCR assay was developed for detection of fungal DNA from equine endometrial samples. The qPCR may prove to be clinically useful as an adjunct to microbial culture and cytologic examination to provide identification of fungal organisms in a timely manner.

Abstract

Objective—To develop a broad-range 28S ribosomal DNA quantitative PCR (qPCR) assay for detection of fungal DNA in equine endometrial samples.

Sample—12 fungal samples from a clinical diagnostic laboratory and 29 samples obtained from 17 mares.

Procedures—The qPCR assay was optimized with commercially acquired fungal organisms and validated with samples obtained from the clinical diagnostic laboratory. Subsequently, 29 samples from 17 mares suspected of having fungal endometritis were evaluated via the qPCR assay and via traditional fungal culture and endometrial cytology. Amplicons from the qPCR assay were subjected to genetic sequencing to identify the organisms.

Results—The qPCR assay theoretically had a detection threshold of 2 organisms of Candida albicans. Fungal DNA was amplified from all 12 fungal samples from the commercial diagnostic laboratory. Fungal identification by use of genetic sequencing was successful for 34 of 36 amplicons from the 12 samples assayed. A fungal agent was identified via qPCR assay and genetic sequencing in all 12 samples; in contrast, a fungal agent was identified in only 8 of 12 samples via standard fungal culture and biochemical analysis. The qPCR assay detected fungal DNA in samples from 12 of 17 mares suspected of having fungal endometritis.

Conclusions and Clinical Relevance—A rapid, sensitive, and repeatable qPCR assay was developed for detection of fungal DNA from equine endometrial samples. The qPCR may prove to be clinically useful as an adjunct to microbial culture and cytologic examination to provide identification of fungal organisms in a timely manner.

Contributor Notes

Address correspondence to Dr. Ferris (rferris@colostate.edu).