Fungal endometritis is an important cause of subfertility in mares because of difficulties in initial detection of infection and failure to attain complete resolution of the uterine infection following treatment. On the basis of culture of endometrial swab specimens, the incidence of fungal endometritis is believed to range from 1% to 5%,1 and infections consist primarily of Candida spp, Aspergillus spp, and Mucor spp.2 A crucial factor in development of an effective therapeutic plan for fungal endometritis is accurate identification of the etiologic agent. Currently, the standard for detection of fungal infections is mycological culture and direct microscopic evaluation of cytologic or biopsy specimens obtained from the endometrium.1 Microscopy often lacks specificity (ie, inability to positively identify an organism), whereas diagnosis by mycological culture often requires a substantial period to allow for the growth phase of mycotic organisms and requires considerable laboratory expertise for accurate identification.3,4
It is common in human clinical laboratories to diagnose mycological infections via in vitro amplification and detection of fungal DNA via molecular techniques.5–8 These assays have the advantages of potentially being more sensitive than other assays and can be performed more rapidly than culture. Prompt identification of a mycological infection allows for rapid institution of antimicrobial treatment, which could potentially improve the clinical outcome. For example, Candida albicans typically is susceptible to fluconazole, whereas Candida krusei and Candida glabrata are inherently resistant to fluconazole.9 Amplification of fungal DNA by PCR assay has been used in equine medicine to provide a rapid diagnosis of mycological infection in horses with fungal keratitis.a Thus, the objective of the study reported here was to develop a broad-range 28S ribosomal DNA qPCR assay for rapid detection of fungal DNA in equine uterine samples.
Coefficient of variation
Belknap E, Barden C, Yin C, et al. Real time PCR as a diagnostic tool for equine fungal keratitis: a preliminary study (abstr), in Proceedings. 36th Annu Conf Am Coll Vet Ophthalmol 2005;7.
Fungal Organism, American Type Culture Collection, Manassas, Va.
Buffer ATL, Qiagen, Valencia, Calif.
DNeasy blood and tissue kit, Qiagen, Valencia, Calif.
QuantiTect SYBR Green PCR kit, Qiagen, Valencia, Calif.
Mastercycler ep realplex, Eppendorf, Hauppauge, NY.
DNA Sequencing Laboratory, College of Biological Sciences, University of California-Davis, Davis, Calif.
Xplorer, version 2.4.2, dnaTools Inc, Fort Collins, Colo.
BLAST, National Center for Biotechnology Information, National Institutes of Health, Bethesda, Md. Available at: blast.ncbi.nlm.nih.gov/. Accessed Jul 27, 2010.
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;2643–2651.
Alexander BDPfaller MA. Contemporary tools for the diagnosis and management of invasive mycoses. Clin Infect Dis 2006; 43(suppl 1):S15–s27.
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: 333–357.
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: 1919–1926.
Tintelnot KDe Hoog GSAntweiler E, et al. Taxonomic and diagnostic markers for identification of Coccidioides immitis and Coccidioides posadasii. Med Mycol 2007; 45: 385–393.
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: 2902–2908.
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: 1216–1223.
Preuner SLion T. Towards molecular diagnostics of invasive fungal infections. Expert Rev Mol Diagn 2009; 9: 397–401.
Espy MJUhl JRSloan LM, et al. Real-time PCR in clinical microbiology: applications for routine laboratory testing. Clin Microbiol Rev 2006; 19: 165–256.
Hoorfar JMalorny BAbdulmawjood A, et al. Practical considerations in design of internal amplification controls for diagnostic PCR assays. J Clin Microbiol 2004; 42: 1863–1868.
Bretagne SCosta J. Towards a molecular diagnosis of invasive aspergillosis and disseminated candidosis. FEMS Immunol Med Microbiol 2005; 45: 361–368.
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: 72–75.