Fungal polymerase chain reaction testing in equine ulcerative keratitis.
Abstract: To assess the diagnostic utility of fungal polymerase chain reaction (PCR) in forty-three horses with naturally acquired corneal ulcers presenting to a private practice. Methods: Routine evaluation of cytologic, histologic, and microbiologic samples was performed. Two PCR approaches were compared - generic and specific fungal nested PCR followed by sequencing and quantitative PCR (qPCR). PCRs were applied to pure control fungal cultures, corneal tissue from ulcerated eyes and in a subset of 9 horses, to swabs from contralateral normal eyes. Results: The expected fungus was identified by nested PCR and qPCR in all control fungal cultures. In all fungal culture-positive affected eyes (10/43), one or more fungi were identified by nested PCR and 4/10 were positive by qPCR. In 6/10 animals, the same fungus was identified by nested PCR and culture. Of these 6, only three were positive by qPCR. Fungal agents were identified by morphology in 8/10 horses. Diagnosis of fungal keratitis was reserved for only those cases in which the same fungus could be identified by PCR, culture, and morphology (5 horses). In 33/43 culture-negative affected eyes and in 6/9 unaffected eyes, one or more fungi were identified by nested PCR in 26 samples and by qPCR in 2 samples. Apart from Aspergillus spp, similar fungi were identified in affected and control eyes. Most eyes harbored mixed bacterial and fungal agents. Conclusions: Nested PCR results confirmed all cytologically positive cases of fungal keratitis. Nested PCR identified a greater spectrum of agents than either culture or qPCR.
© 2012 American College of Veterinary Ophthalmologists.
Publication Date: 2012-12-10 PubMed ID: 23227970DOI: 10.1111/vop.12004Google Scholar: Lookup
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- Journal Article
Summary
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The research evaluated the efficacy of fungal polymerase chain reaction (PCR) tests in diagnosing horses with naturally occurring corneal ulcers. It revealed that the nested PCR technique proved more beneficial than culture testing or quantitative PCR in identifying a broader range of causal agents.
Objective of the Study
- The study aimed to evaluate the diagnostic efficacy of fungal polymerase chain reaction (PCR) tests in identifying a range of pathogens that cause corneal ulcers in horses.
Methodology
- The research studied forty-three horses with naturally acquired corneal ulcers that were presented to private practices. These horses were subjected to standard cytologic, histologic, and microbiologic evaluations.
- Two different PCR methodologies were compared, which included both a generic and a specific fungal nested PCR. After the initial PCR, a sequencing step and quantitative PCR (qPCR) were performed.
- The test was also performed on pure fungal cultures derived from the corneal tissues of the ulcerated eyes. Additionally, a subset of nine horses had swabs taken from their unaffected eyes for comparison.
Results
- All the control fungal cultures showed the expected fungus when subjected to both the nested PCR and qPCR.
- Out of 43 affected eyes, ten were found to be culture-positive for fungus. Nested PCR could identify one or more fungi in all ten samples, whereas qPCR was positive only for four samples.
- In six of these ten culture-positive cases, the same fungus was identified by both the nested PCR and culture methods. Of these six, qPCR identified the fungus in only three cases.
- Morphological identification was possible for eight of the ten culture-positive cases.
- However, the study preserved the diagnosis of fungal keratitis for those cases where the same fungus was identified by all three tests, i.e., PCR, culture, and morphological examination. This was true for five horses.
- In 33 of the 43 culture-negative cases and six unaffected eyes, nested PCR identified one or more fungi in 26 samples. However, qPCR could only identify fungal presence in two instances.
- Most of the affected eyes hosted mixed bacterial and fungal microorganisms, with Aspergillus species being the most common fungi in both affected and unaffected eyes.
Conclusions
- The study concluded that nested PCR tests confirmed all cytologically positive cases of fungal keratitis.
- It also highlighted nested PCR as a more efficient method than either culture or qPCR for identifying a broader range of pathogens in both affected and unaffected eyes.
Cite This Article
APA
Zeiss C, Neaderland M, Yang FC, Terwilliger G, Compton S.
(2012).
Fungal polymerase chain reaction testing in equine ulcerative keratitis.
Vet Ophthalmol, 16(5), 341-351.
https://doi.org/10.1111/vop.12004 Publication
Researcher Affiliations
- Section of Comparative Medicine, Yale University School of Medicine, New Haven, CT 06520, USA. caroline.zeiss@yale.edu
MeSH Terms
- Animals
- Bacterial Infections / diagnosis
- Bacterial Infections / microbiology
- Bacterial Infections / veterinary
- Corneal Ulcer / microbiology
- Corneal Ulcer / veterinary
- Horse Diseases / diagnosis
- Horse Diseases / microbiology
- Horses
- Mycoses / diagnosis
- Mycoses / veterinary
- Polymerase Chain Reaction / methods
- Polymerase Chain Reaction / veterinary
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