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

Ryan A. Ferris Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80521.

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Katy Dern Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80521.

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Julia K. Veir Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80521.

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Jennifer R. Hawley Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80521.

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Michael R. Lappin Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80521.

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Patrick M. McCue Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80521.

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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.

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.

Materials and Methods

SamplesAspergillus fumigatus (ATCC 1022) and C albicans (ATCC 10231) were acquired from a commercial companyb and used to develop the qPCR assay. Twelve samples obtained from a clinical diagnostic laboratory were used to evaluate the qPCR assay. Finally, 29 samples obtained from 17 mares suspected of having fungal endometritis were evaluated with the qPCR assay.

Development of a 28S ribosomal qPCR assay to detect fungal organisms—Serial logarithmic dilutions of A fumigatus and C albicans in sterile (PCR-grade) PBS solution were used as control samples. Calcium alginate–tipped swabs were dipped in the logarithmic aliquots to mimic clinical samples collected from the uterus. These swab specimens were placed in 1.0 mL of sterile PBS solution for 1 hour and then vortexed for 1 minute. Each sample was poured into a 1.5-mL microcentrifuge tube and centrifuged at 10,000 × g for 3 minutes. Supernatant was removed, and the pellet was resuspended in 200 μL of sterile PBS solution. Fungi were lysed by incubation at 95°C for 10 minutes. Following cell lysis, 180 μL of tissue lysis bufferc and 20 μL of proteinase K were added, and the mixture was briefly centrifuged and then incubated at 54°C for 1 hour. Extraction of DNA was performed with a commercial tissue extraction kit.d Quantitation of fungal DNA was performed by use of a real-time PCR reaction that was adapted from an established protocol.5 Briefly, a volume of 20 μL that contained 10 μL of commercial fluorescent PCR dye,e 0.4 μL of NL1 primer (5′-GCATAT-CAATAAGCGGAGGAAA AG-3′),8 0.4 μL of 260R (5′-TTAGCTTTAGA TGRARTTTACCACC-3′),5 5 μL of extracted template DNA, and 4.2 μL of sterile water was used in the real-time PCR assay. All reactions were performed in a thermocyclerf; conditions were 5 minutes at 95°C, which was followed by 40 cycles (each cycle consisted of a denaturation step of 10 seconds at 95°C followed by an annealing step of 35 seconds at 60°C). The DNA from amplicons with positive results was submitted to a commercial laboratoryg for sequence analysis. Chromatograms were analyzed,h and results were compared with published sequences by use of a sequence-similarity tooli at default settings.

Assay sensitivity for common fungal organisms was estimated from serial logarithmic dilutions of A fumigatus and C albicans in sterile PCR-grade PBS solution. The sensitivity experiment was repeated with sterile saline (0.9% NaCl) solution recovered after uterine lavage of mares with negative results for bacterial and fungal cultures to evaluate for the presence of PCR inhibitors in the uterus of mares. The DNA extracted from cultured samples of Klebsiella pneumoniae, Pseudomonas aeruginosa, and Streptococcus equi subsp zooepidemicus obtained from the Veterinary Diagnostic Laboratory at Colorado State University was used to confirm a lack of cross-reactivity of primers with common bacterial uterine pathogens. Interassay CV was determined by repeating the qPCR assay (15 replicates) for 1 extraction of both C albicans and A fumigatus.

Evaluation of extraction techniques to isolate fungal DNA—The efficiency of DNA extraction was evaluated after exposure of swab specimens to various concentrations (10−1 to 10−6 g of DNA) of A fumigatus and C albicans DNA. Ten swab specimens from each concentration were soaked in sterile PBS solution for 2 hours, vortexed, and centrifuged; DNA was then extracted as described previously. A second set of 10 swab specimens was placed in sterile PBS solution and immediately vortexed and centrifuged; DNA was then extracted as described previously. The Ct at which the minimum threshold was exceeded was compared between extraction groups to semiquantitatively determine whether the total amount of DNA extracted differed between the 2 extraction techniques.

Comparison of traditional microbial culture and the qPCR assay to detect fungal organisms—The qPCR assay was subsequently evaluated for its use in detecting fungal pathogens from agar plates obtained from the Veterinary Diagnostic Laboratory at Colorado State University. Twelve samples were submitted for traditional microbial culture and analysis via qPCR assay. The interassay CV for the qPCR fungal assay was determined by performance of the assay on all 12 samples 5 times. The purposes of this experiment were to determine whether the relative detectable amount of DNA in the sample was similar between qPCR assays, whether DNA sequencing could be used to identify the same organism in each assay, and whether the same organism would be identified via traditional fungal culture and the qPCR assay.

Use of the qPCR assay to detect fungal organisms in mares suspected of having fungal endometritis—A total of 29 samples (endometrial swab specimens, biopsy specimens, or low-volume lavage samples) obtained from 17 mares suspected of having fungal endometritis were submitted by veterinarians in private practice or at referral microbiological laboratories. A diagnosis of fungal endometritis was based on results of microbial culture or cytologic evaluation (or both) of endometrial samples. Additional samples were collected from 46 mares considered to be reproductively normal on the basis of a lack of ultrasonographically detectable intrauterine fluid during a 4-week period, no growth of pathogens on microbial culture of endometrial samples, and no inflammatory cells or microbial pathogens observed during cytologic examination of endometrial samples.

Statistical analysis—The Ct in which the minimum threshold was exceeded was compared between groups by use of a Student t test. Comparisons in frequency of identification of fungal organisms between microbial culture and the qPCR assay were performed with the Fisher exact test. Values were considered significantly different at P < 0.05.

Results

Development of 28S ribosomal qPCR assay to detect fungal organisms—The broad-spectrum primers were able to amplify fungal DNA from A fumigatus and C albicans as determined by a positive signal detected by the thermocycler (data not shown). Logarithmic dilution of fungal organisms in uterine lavage fluid revealed that the assay was able to detect a minimum of 2 × 10−14 g of fungal DNA. The typical C albicans organism contains approximately 37 fg of DNA. Theoretically, the assay has a detection limit of 2 organisms. There was no difference in the minimum detectable limits of fungal DNA in PBS solution or uterine lavage fluid (data not shown). The interassay CV of the logarithmic dilutions of A fumigatus DNA from 15 replicates of a single extraction was 6%. The primers used in the qPCR assay did not amplify DNA of bacterial organisms commonly isolated from horses with endometritis.

Evaluation of fungal DNA extraction techniques—The Ct for the 2 fungal DNA extraction techniques did not differ significantly (data not shown).

Comparison of traditional microbial culture and the qPCR assay to detect fungal organisms—The qPCR assay was able to amplify fungal DNA from all 12 samples (Table 1). The interassay CV, as determined by performing the qPCR assay multiple times on all 12 samples (5 replicates/sample), was < 4%. The qPCR assay and subsequent genetic sequencing could be used to identify the genus and species of fungi in all 12 samples, compared with identification of only 8 of 12 samples via fungal culture and biochemical methods. Identification via biochemical methods and DNA sequencing was similar for 6 of 8 samples. Results of the qPCR assay followed by genetic sequencing were obtained within 3 days after sample acquisition. In contrast, microbial culture required a mean of 3 days (range, 2 to 5 days) to enable detection of a fungal organism and an additional 18 days (range, 11 to 42 days) for determination of genus and species.

Table 1—

Identification of fungal organisms via qPCR assay with subsequent DNA sequence analysis or via traditional fungal culture with biochemical analysis.

Sample No.Interassay CV (%)*qPCR assay and DNA sequence analysisFungal culture and biochemical analysis
12Microsporum canisM canis
21Aspergillus fumigatusTrithelcium roseae (previously A fumigatus)
32A fumigatusFungus (not identified)
41Geomyces pannorum (previously Trichophyton sp)Trichophyton sp
52Penicillium spPenicillium sp
65Candida orthopsilosisYeast (not identified)
73C orthopsilosisYeast (not identified)
82Trichosporon spTrichosporon sp
92C orthopsilosisC orthopsilosis (previously Torulopsis glabrata)
102Pichia guilliermondii (previously Candida guilliermondii)P guilliermondii
111A fumigatusFungus (not identified)
124Candida parapsilosisC orthopsilosis (previously T glabrata)

Determined on the basis of Ct values.

Performed at the Colorado State University Diagnostic Laboratory.

Use of the qPCR assay to detect fungal organisms in mares suspected of having fungal endometritis—The qPCR assay detected fungal DNA in samples from 12 of the 17 mares. Sequence analysis of the amplicon provided identification of the genus and species for all 12 samples (Table 2). Yeast-type fungal pathogens were identified in 10 of the 12 mares, whereas hyphate-type fungal organisms were identified in the other 2 mares. Fungal culture and qPCR assay for fungal DNA both yielded negative results for all uterine samples collected from the 46 reproductively normal mares.

Table 2—

Identification of fungal organisms via qPCR assay with subsequent DNA sequence analysis of samples collected from 17 mares suspected of having fungal endometritis.

Mare No.SourceCt valueqPCR assay and DNA sequence analysisSuspicion of fungal endometritis
1Uterine swab specimen18Candida tropicalisPositive results for fungal culture
2Uterine swab specimenNANo fungal organism detectedPositive results for fungal culture
3Uterine swab specimen21C orthopsilosisPositive results for fungal culture
4Uterine swab specimen21P guilliermondiiPositive results for fungal culture
5Uterine swab specimen15Clavispora lusitaniaePositive results for fungal culture
6Uterine swab specimen19Candida glabrataT glabrata on fungal culture
7Uterine swab specimen14Candida parapolymorphaPositive results for fungal culture
8AFungal isolate from a uterine swab specimen cultured on a blood agar plate15Dipodascus capitatusPositive results for fungal culture
8BUterine swab specimen22Ascomycota spPositive results for fungal culture*
8CLow-volume uterine lavage15Ascomycota spPositive results for fungal culture*
9Uterine swab specimenNDNo fungal organism detectedPositive results for fungal culture
10Uterine swab specimen22C tropicalisYeast identified on cytologic examination of endometrial swab specimen
11Uterine swab specimenNDNo fungal organism detectedPositive results for fungal culture
12Low-volume uterine lavageNDNo fungal organism detectedHistory of previous fungal endometritis
13Uterine swab specimenNDNo fungal organism detectedHistory of previous fungal endometritis
14Uterine swab specimen18A fumigatusFungal hyphae identified on cytologic examination of endometrial swab specimen
15Uterine biopsy specimen19Candida terebraYeast identified on cytologic examination of endometrial swab specimen
16AClitoral fossa swab specimen31Candida albicansYeast identified on cytologic examination of endometrial swab specimen
16BClitoral sinus swab specimen26C albicansYeast identified on cytologic examination of endometrial swab specimen
16CVestibule swab specimen22C albicansYeast identified on cytologic examination of endometrial swab specimen
16DVaginal swab specimen21C albicansYeast identified on cytologic examination of endometrial swab specimen
16EUterine swab specimen30C albicansYeast identified on cytologic examination of endometrial swab specimen
17AClitoral fossa swab specimen32P guillermondiiYeast identified on cytologic examination of endometrial swab specimen
17BClitoral sinus swab specimen29P guillermondiiYeast identified on cytologic examination of endometrial swab specimen
17CVestibule swab specimen25P guillermondiiYeast identified on cytologic examination of endometrial swab specimen
17DVaginal swab specimenNDNo fungal organism detectedYeast identified on cytologic examination of endometrial swab specimen
17ECervix swab specimenNDNo fungal organism detectedYeast identified on cytologic examination of endometrial swab specimen
17FLow-volume uterine lavage14P guillermondiiYeast identified on cytologic examination of endometrial swab specimen
17GUterine swab specimen21P guillermondiiYeast identified on cytologic examination of endometrial swab specimen

Specimen was obtained after 8A fungal growth was identified.

NA = Not available. ND = Not detected.

Discussion

The use of molecular techniques to diagnose bacterial, fungal, and viral infections in clinical laboratories has exponentially increased during the past decade.10 Real-time PCR amplification combined with sequencing of PCR products is a more rapid technique than is traditional microbial culture. In the present study, 8 hours typically was required for the detection of fungal DNA with the qPCR assay, and approximately 72 hours was required to determine identification of the organisms via DNA sequencing. The interval from sample acquisition to pathogen identification was largely a result of the time required for overnight shipment of the amplicon to a diagnostic laboratory for DNA sequencing.

It has been suggested that amplification inhibitors may adversely affect PCR diagnostic testing, which can cause false-negative results.11 The qPCR assay detected similar concentrations of fungal DNA diluted in sterile PBS solution or uterine lavage fluid, which suggested that amplification inhibitors are not important in the uterus. The sensitivity of the assay was similar for both diluents, which suggested that important PCR inhibitors were not present in uterine lavage fluid.

The qPCR assay detected fungal DNA in samples obtained from 12 of 17 clinically affected mares in which fungal endometritis was diagnosed via cytologic examination or microbial culture of endometrial samples. The inability of the assay to detect fungal DNA in samples from the other 5 mares could have been attributable to a fungal DNA load that was below the sensitivity of the assay or the presence of amplification inhibitors, or it is possible that the fungal cultures yielded false-positive results.11

It should be mentioned that fungal endometritis was originally diagnosed in 5 of the 17 mares via cytologic examination of endometrial samples, whereas microbial culture of uterine samples obtained from these 5 mares yielded negative results for fungal growth. These 5 mares had positive results for fungal DNA via the qPCR assay, and an organism was identified.

The qPCR assay appeared to be more sensitive than was routine microbial culture. One reason suggested for this is the ability of qPCR assays to amplify DNA of nonviable organisms and free fungal DNA.12 The uterus is considered to be a privileged immunologic site from which no fungal organisms should ever be recovered. Consequently, detection of any fungal DNA from a uterine sample would be clinically relevant.13 Another issue is possible contamination of the sample with fungal DNA from equine skin or fecal material during sample collection. Care must be taken during sample collection to minimize contamination of samples submitted for microbial culture or qPCR assay.

Unfortunately, the qPCR assay cannot be used to determine antimicrobial susceptibility for fungal organisms. Culture of fungal organisms currently remains the standard for determination of antimicrobial susceptibility. However, fungal organisms detected in equine uterine samples generally have clearly defined susceptibilities to antifungal drugs, and identification of a fungal organism via qPCR assay will provide valuable information for development of a treatment plan. Thus, the rapid return of qPCR assay results can allow for empirical administration of drugs likely to be effective on the basis of genus and species identification of the organisms. In the future, a genetics-based approach to antifungal susceptibility may be possible.

In the study reported here, a rapid, sensitive, and repeatable qPCR assay was developed for detection of fungal DNA in equine uterine samples. The qPCR assay may prove to be clinically useful as an adjunct to microbial culture and cytologic examination of uterine samples for identification of fungal organisms in a timely manner and allow for early development and implementation of a treatment plan.

ABBREVIATIONS

Ct

Threshold cycle

CV

Coefficient of variation

qPCR

Quantitative PCR

a.

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.

b.

Fungal Organism, American Type Culture Collection, Manassas, Va.

c.

Buffer ATL, Qiagen, Valencia, Calif.

d.

DNeasy blood and tissue kit, Qiagen, Valencia, Calif.

e.

QuantiTect SYBR Green PCR kit, Qiagen, Valencia, Calif.

f.

Mastercycler ep realplex, Eppendorf, Hauppauge, NY.

g.

DNA Sequencing Laboratory, College of Biological Sciences, University of California-Davis, Davis, Calif.

h.

Xplorer, version 2.4.2, dnaTools Inc, Fort Collins, Colo.

i.

BLAST, National Center for Biotechnology Information, National Institutes of Health, Bethesda, Md. Available at: blast.ncbi.nlm.nih.gov/. Accessed Jul 27, 2010.

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