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This study assessed the value of polymerase chain reaction (PCR) for making a diagnosis of feline herpesvirus (FHV-1) infection, and for differentiating this from Chlamydophila felis and Mycoplasma spp. infection in a clinical setting in Canada. We compared the frequency of positive FHV-1 PCR test results from 48 clinical cases of ocular disease in cats suspected to be due to FHV-1 between 1 research and 2 commercial laboratories in Canada. We also compared PCR results for Chlamydophila felis and Mycoplasma spp. between the 2 commercial laboratories. The prevalence of FHV-1 infection in the cats ranged from 4% to 21%. The prevalence of Chlamydophila felis was 2% and 17% and the prevalence of Mycoplasma spp. was 11% and 27%. Agreement between FHV-1 culture and PCR results at the research laboratory was substantial (κ = 0.76). There was slight agreement (κ < 0.20) between the 3 laboratories for FHV-1 PCR and between the 2 commercial laboratories for both Chlamydophila felis (κ = 0.2) and Mycoplasma spp. (κ = 0.07) PCR.
Comparaison des épreuves de la réaction en chaîne par la polymérase pour le diagnostic de l’herpèsvirus félin, Chlamydophila felis, et de l’infection à Mycoplasma spp. chez les chats atteints d’une maladie oculaire au Canada. Cette étude a évalué la valeur de la réaction en chaîne par la polymérase (RCP) pour poser un diagnostic d’une infection par l’herpèsvirus félin (FHV-1) et pour la différenciation de cette infection de celle par Chlamydophila felis et par Mycoplasma spp. dans un contexte clinique au Canada. Nous avons comparé la fréquence des résultats positifs des épreuves de RCP pour le FHV-1 parmi 48 cas cliniques de maladie oculaire chez les chats dont la cause suspectée était le FHV-1 entre 1 laboratoire de recherche et 2 laboratoires commerciaux au Canada. Nous avons aussi comparé les résultats de RCP pour Chlamydophila felis et Mycoplasma spp. entre les 2 laboratoires commerciaux. La prévalence de l’infection à FHV-1 chez les chats variait de 4 % à 21 %. La prévalence de Chlamydophila felis était de 2 % et de 17 % et la prévalence de Mycoplasma spp. était de 11 % et de 27 %. La concordance entre la culture de FHV-1 et les résultats de RCP au laboratoire de recherche était presque parfaite (κ = 0,76). Il y avait peu de concordance (κ < 0,20) entre les 3 laboratoires pour la RCP du FHV-1 et entre les 2 laboratoires commerciaux pour Chlamydophila felis (κ = 0,2) et pour la RCP de Mycoplasma spp. (κ = 0,07).
(Traduit par Isabelle Vallières)
Feline herpesvirus (FHV-1) is a double-stranded, enveloped DNA virus (1). Infection with FHV-1 is widespread and approximately 90% of cats are seropositive for the virus (2–4). Primary FHV-1 infection most often occurs in neonatal or young cats and usually results in upper respiratory tract disease (2,5,6). Ocular manifestations during primary infection are usually bilateral and consist mainly of conjunctivitis, although corneal ulceration may also occur (6,7). Latency of FHV-1 is established primarily in the trigeminal ganglion following primary infection (8–10) in about 80% of cats exposed to FHV-1, and 45% of latently infected cats will shed virus periodically throughout life (11).
Ocular manifestations of FHV-1 infection in adult cats are primarily the result of reactivation of latent virus (2,6). Reactivation often occurs following natural stresses such as change in housing, parturition, and lactation, but may also be iatrogenically induced following immunosuppressive therapy (7,11–13). During reactivated infection, signs of upper respiratory tract disease are often absent and ocular manifestations are usually unilateral (2,6). Clinical signs range from mild conjunctivitis to variable degrees of keratitis (5,6). Superficial, linear, branching ulcers, known as “dendritic ulcers” are considered pathognomonic for FHV-1 infection. Small ulcers may coalesce to form larger geographic lesions (7).
The primary differential consideration for feline conjunctivitis is infection with Chlamydophila felis. This agent is primarily a conjunctival pathogen and, in contrast to FHV-1, does not affect the cornea. Clinical manifestations in acute cases include ocular discharge, conjunctival hyperemia, and often dramatic chemosis. The condition may present unilaterally but usually becomes bilateral within 1–3 wk (14,15). Nasal discharge and sneezing may occur in some cats but this is more likely to occur in cats < 9 mo of age and is extremely rare without concurrent conjunctivitis (16,17). Mycoplasma spp. (M. felis and M. gatae) have also been implicated as causes of conjunctivitis in cats (18). Clinical manifestations include conjunctival hyperemia, thickening, discharge, and the presence of friable pseudomembranes (15). The pathogenicity of Mycoplasma spp. is debated as this organism can be isolated from the conjunctiva of normal cats, and experimental infection cannot be achieved without immuno-suppression (19,20).
A presumptive diagnosis of FHV-1 infection is often made on the basis of clinical signs. A diagnosis of FHV-1 can be presumed when pathognomonic dendritic ulcers are present. Confirmation of FHV-1 infection requires laboratory detection of the virus. Laboratory confirmation of the virus helps to eliminate other potential infectious agents and to guide therapy. Polymerase chain reaction (PCR) is the most sensitive method of detecting FHV-1 in ocular tissues (21–23). Anecdotally, however, PCR test results seem inconsistent, leading many veterinary ophthalmologists to abandon PCR testing as a means of diagnosis, instead relying on clinical history and presentation alone.
In a comparison of 6 PCR tests for FHV-1, all assays were equally likely to detect vaccine virus, and concordant results were obtained for 9 of 15 conjunctival biopsy samples; however, test sensitivity varied greatly (24). The PCR assays were performed in 1 research laboratory and method variability was minimized by using similar reaction volumes and the same Taq polymerase and thermocycler (24). In the clinical setting, however, veterinarians rely on commercial laboratories for PCR testing and more variation would be expected in testing methods. In Canada there is a limited number of laboratories that offer FHV-1 PCR tests. These tests are often conveniently offered as ocular disease PCR panels, which include tests for FHV-1 as well as the primary agents considered in differential diagnoses, Chlamydophila felis and Mycoplasma spp. The objective of this study was to investigate the value of PCR testing for making a diagnosis of FHV-1 infection, and for differentiating this from Chlamydophila felis and Mycoplasma spp. in a clinical setting. We compared the frequency of positive FHV-1 PCR test results from clinical cases of ocular disease suspected to be due to FHV-1 between 3 different laboratories, and the agreement between results performed in different laboratories. Agreement between PCR test results and FHV-1 culture performed in a research laboratory was also evaluated as was the relative sensitivity compared to FHV-1 culture. Because the commercial laboratories concurrently run PCR for Chlamydophila felis and Mycoplasma spp., we also evaluated detection of these pathogens in our population and agreement between those PCR tests.
The 48 cats used in this study were clinical patients of the ophthalmology service at the Western College of Veterinary Medicine teaching hospital in Saskatoon, Saskatchewan. To be included in the study the cats had to have clinical signs that were most consistent with a diagnosis of feline herpesvirus ocular disease. Patients included cats or kittens with historical or current upper respiratory tract disease and bilateral conjunctivitis with or without corneal ulceration, and cats with unilateral conjunctivitis with or without dendritic or geographic corneal ulceration. Animals received a complete ophthalmic examination including neurophthalmic examination, Schirmer tear test (Schirmer Tear Test Strips; Alcon Canada, Mississauga, Ontario), fluorescein staining (Fluorets, Bausch & Lomb, Markham, Ontario), intra-ocular pressure measurement by rebound tonometry (Tonvet, Tiolat, Helsinki, Finland), slit-lamp biomicroscopy (Osram 64222, Carl Zeiss Canada, Don Mills, Ontario), and indirect ophthalmoscopy (Heine Omega 200, Heine Instruments, Kitchener, Ontario). Cats were excluded from the study if they had received any antiviral therapy in the 2 wk prior to examination or if examination revealed concurrent intraocular disease.
Samples were collected by a veterinary ophthalmologist or ophthalmology resident at the University of Saskatchewan between June 2005 and June 2008. Four corneal (n = 7) or conjunctival (n = 41) swabs were collected consecutively from each cat by rolling dry cotton-tipped applicators along the ventral conjunctiva or region of corneal ulceration following the application of 0.5% proparacaine hydrochloride (Alcaine, Alcon, Mississauga, Ontario). Each sample was placed into a sterile tube containing 0.5 mL of sterile saline. Samples were then stored at 4°C for a maximum of 72 h until they were transported to the laboratories by courier. Swabs were randomly assigned for shipment to laboratories A (research), B (commercial), and C (commercial). Laboratory A received 2 swabs, 1 for culture and 1 for PCR, while laboratories B and C each received 1 swab. All laboratories were located in Ontario. The travel distances by courier were approximately 2866 km to laboratory A, 2976 km to laboratory B, and 2962 km to laboratory C.
For PCR completed in the research laboratory (A), DNA was extracted from swabs using a QIAamp DNA mini kit (Qiagen, Mississauga, Ontario). The swab was placed in a 1.5 mL microcentrifuge tube containing 300 μL of saline and incubated at 37°C for 10 min on a shaker. Approximately 200 μL of saline was then removed and placed in a fresh 1.5 mL tube, and DNA was extracted according to the manufacturer’s protocol. Polymerase chain reaction was performed with 5 μL of template DNA, polymerase (GoTaq; Promega, Madison, Wisconsin, USA), primers FHV1f 5′-CGG GAA AAT CCA GTA CGA GT-3′ and FHV-1r 5′-AGG AAG AGT TCG GCG GTA TT-3′, which amplified a region in the FHV-1 terminase gene, and appropriate buffer and nucleotides (Promega). Every sample was also subjected to PCR-amplification with primers for the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH)-f 5′-AGC CTT CTC CAT GGT GGT GAA GAC-3′ and GAPDH-r 5′-CGG AGT CAA CGG ATT TGG TCG-3′ to verify presence of suitable DNA. Amplifications consisted of initial incubation at 95°C for 2 min, followed by 40 cycles of 95°C for 30 s; 58°C for 30 s, and 72°C for 30 s; with a final extension at 72°C for 5 min. Amplicons were detected as 200 (FHV) or 320 (GAPDH) base pair bands after gel electrophoresis and nucleic acid staining (SybrGreen, Promega). Only samples with positive GAPDH PCR results were included in the study. During each amplification, controls consisting of samples lacking DNA (negative control) and samples with DNA derived from in vitro infection of Crandell Reese Feline Kidney (CRFK) cells with FHV (positive control) were included. The identity of selected PCR products was verified through direct sequencing (Laboratory Services Division, Guelph, Ontario). Details regarding the PCR methods of the commercial laboratories were not available.
The CRFK cells (American Tissue Culture Collection, Manassas, Virginia, USA) were cultured in Dulbecco’s Modified Eagle Medium with 10% horse serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine (all from Sigma, Burlington, Ontario) at 37°C. Near confluent cells were inoculated with 200 μL of saline derived from 1 ocular swab. Cells were examined daily for 7 d for cytopathic effects, as described previously (25).
Descriptive statistics, McNemar’s chi-squared (χ2) test, and kappa (κ) statistics were calculated using a commercial software program (Stata/SE v9.2; StataCorp, College Station, Texas, USA). The McNemar’s χ2 was used to determine if cats were significantly more likely to test positive on 1 test or the other (26). Kappa was used to measure the agreement beyond chance and was judged according to the following guidelines: < 0.2 = slight agreement; 0.2–0.4 = fair; 0.4–0.6 = moderate; 0.6–0.8 = substantial; and > 0.8 = almost perfect (27). These statistics were calculated independently for the results of each PCR as compared to culture results and then for each comparison of the various PCR results.
We chose to compare PCR and culture results with culture considered the gold standard for FHV-1 testing. The relative sensitivity and specificity of PCR as compared to culture was calculated as follows:
where: TP, FP, TN, FN = true positive, false positive, true negative, and false negative test results, respectively.
Logistic regression was used to determine whether the odds of a positive culture or PCR result for FHV at laboratory A differed depending on the location of the swab (conjunctiva versus cornea), type of lesion (conjunctivitis versus keratitis), or age of the cat.
All swabs received at laboratory A had amplifiable DNA, thus, none were eliminated from PCR testing. The results of FHV-1 culture and PCR testing completed at the research laboratory, as well as FHV-1, Chlamydophila felis, and Mycoplasma spp. testing completed at 2 commercial laboratories, are shown in Table 1. Test results for all pathogens were highly variable between the 3 laboratories. The prevalence of FHV-1 infection in cats suspected to have FHV-1-associated ocular disease was 13% by culture, and 21%, 4%, and 21% by PCR at 1 research and 2 commercial laboratories, respectively. The prevalence of Chlamydophila felis in cats suspected to have FHV-1 associated ocular disease was 2% and 17% by PCR in commercial laboratories B and C, respectively. The prevalence of Mycoplasma spp. in cats suspected to have FHV-1 associated ocular disease was 11% and 27% by PCR at commercial laboratories B and C, respectively.
Compared with culture, there was substantial agreement for PCR completed at the research laboratory, but only slight agreement for PCR results from the commercial laboratories (Table 2). There was slight agreement between the 3 laboratories for FHV-1 PCR and slight agreement between the 2 commercial laboratories for both Chlamydophila felis and Mycoplasma spp. PCR.
The relative sensitivity and specificity of PCR results when compared to culture was variable (Table 3). Polymerase chain reaction completed in laboratory A had the highest sensitivity (100%), while the sensitivities of PCR at the other laboratories were low (16.7% and 0% for laboratories B and C, respectively). The specificity was high for laboratory A (93.2%) and B (97.5%), but was lower for laboratory C (75.7%).
There was no difference in the odds of a positive culture or PCR result for FHV from laboratory A based on location of the swab, conjunctiva versus cornea (P = 0.65 for culture, and 0.65 for PCR), type of lesion, conjunctivitis versus keratitis (P = 0.65 for culture, and 0.65 for PCR), or age of the cat (P = 0.82 for culture, and 0.56 for PCR).
Overall, the detection rate of FHV-1 in cats with clinical signs considered consistent with infection by this organism was low, ranging from 4% to 21%, depending on the laboratory. Agreement between PCR assays by different laboratories for FHV-1, Chlamydophila felis, and Mycoplasma spp. was also very low. Compared to culture results, the sensitivity and specificity of the different PCR tests was highly variable.
The detection rate of FHV-1 by PCR in this study is consistent with those previously published. Rates of FHV-1 DNA detection by PCR in cats with suspected FHV-1-associated ocular disease were reported to vary from 9% to 89% (21,27–34). Similarly, rates of FHV-1 DNA detection in clinically normal cats ranged from 3% to 49% (21,30,32,33,35). Reasons for such different detection rates may include variation in sample collection, sample storage, shipping time, DNA extraction, amplification, and amplicon detection (24,36). It is possible that the sample collection method may have affected FHV-1 detection rates in this study. Methods for sample collection include the use of cotton or dacron swabs, cytology brushes, or conjunctival biopsy. In this study, samples were collected by conjunctival swabbing using a cotton swab, as this is the method most likely to be used in clinical practice. Recently it was shown that DNA yields from dry swabs or brushes were higher than from fluid-suspended swabs, and that dry swabs yielded greater amounts of DNA than brushes (36). Samples in this study were suspended in saline after collection, which may have reduced DNA yields. To the authors’ knowledge, controlled clinical studies to compare quantity of DNA obtained with conjunctival biopsy, brush cytology, cotton swabs, or dacron swabs are not available. Performing the conjunctival swab is easy and minimally invasive; however, it may not maximize yield of infectious agent DNA. Further investigation may be warranted to compare sample collection techniques.
Temperature was not controlled during transit time, nor was transit time to the various laboratories recorded. However, variability in storage and transit as well as transit times previously had no apparent effect on PCR testing outcome (37). In fact, transit times of up to 14 d did not affect FHV-1 PCR results with 1 assay (37). In this study, all samples from an individual cat travelled similar distances from Saskatchewan to Ontario on the same day, thus transit time and temperatures encountered were likely similar for swabs taken from the same cat. Temperatures during transit may have been variable between swabs from different cats as they were collected at different times of the year, and thus may have affected detection rates. Although DNA was not quantified in swabs, since all samples were obtained in a similar fashion by 2 individuals, and randomized before being sent to laboratories A, B, or C, variability in DNA content was likely random and minimal. Since all PCR swabs tested at laboratory A had amplifiable DNA, it is likely that samples sent to the other laboratories also had amplifiable DNA. Differences in PCR protocols for detection of FHV-1, Chlamydophila felis, and Mycoplasma spp. by laboratories may have affected the results in this study. However, in a clinical setting, veterinarians rely on commercial laboratories for PCR testing and variation in protocols should minimally affect results. It was not our objective to compare different PCR protocols, but rather, we were primarily interested in the utility of the available commercial PCR tests in confirming a clinical diagnosis of FHV-1 infection.
The reasons for low FHV-1 detection rates found in this study and other studies are unknown. It is possible that there is a low overall incidence of FHV-1 infection or, more likely, that the methods employed did not optimize recovery of viral DNA. Patient selection could have influenced the rate of FHV-1 detection, since more virus is present and hence detectable during the acute stages of the disease (7,21). Stage of disease should have less effect on PCR compared to other testing methods, since PCR is considered the most sensitive diagnostic method (21–23).
The use of PCR as a diagnostic test for FHV-1-associated disease is complicated by the fact that clinically normal cats may shed virus and that viral DNA can be found in the cornea of cats following recovery from experimental infection, as well as in conjunctival and corneal tissue of clinically normal cats (22,38–41). Three potential scenarios to be considered when virus is detected in a clinical case are that the presence of virus may be unrelated to the primary disease, may be a result of reactivation secondary to a primary disease, or may be the cause of the primary disease. In light of these considerations, as well as the apparent low detection rate and variability in DNA detection, the diagnosis of FHV-1 infection should not be based solely on the laboratory test results but should also incorporate presence of clinical features consistent with the disease. CVJ
Financial support was received from the University of Guelph Pet Trust Foundation and the Winn Feline Foundation.
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