Allergy testing revisited
Richard Halliwell (UK) acknowledged Veterinary Allergy Reference Laboratory (VARL) for their sponsorship of this workshop, which included seven presentations.
Patch testing and IgE/IgG testing for canine adverse food reactions (R.S. Mueller)
Ralf Mueller (Germany) presented a summary of a recently published study that evaluated patch and serology testing for food-specific IgE and IgG in normal dogs and atopic dogs, many of which also had adverse food reactions, as proven via elimination diet trial.1
The study included 10 healthy dogs and 23 dogs with atopic dermatitis (AD). Patch and serology testing for allergen-specific IgE and IgG were undertaken for allergens commonly found in dog food including beef, chicken, pork, lamb, turkey, fish (both raw and cooked), wheat, corn and potato. For the patch test, allergens were applied on the lateral thorax under occlusion. Reactions were evaluated at 24, 48 and 72 hours.
Overall, there were 46 positive patch test reactions and 137 negative reactions, with a mean of three reactions for each affected dog. The allergens implicated in each dog’s condition were identified by sequential oral single-antigen challenges. There were 29 of 46 true positives and 136 of 137 true negatives. Most reactions were against beef with the majority appearing after 48 hours. The pattern of reaction against the raw or cooked antigen was inconsistent.
For the IgE serology testing, there were 13 positive reactions and 145 negative reactions. Two of 13 were true positives and 117 of 145 were true negatives. For IgG, there were 23 positive reactions and 135 negative reactions. Eight of 23 were true positives and 113/135 were true negatives. Beef was again the most common reaction and there were no reactions to fish, chicken or turkey. Therefore, there were fewer true positive and fewer true negative reactions than in the patch testing.
The negative predictive value (NPV) for serology and patch testing, respectively, ranged from 80.75% to 99.3%, while the positive predictive value (PPV) ranged from 15.4% to 63%. Although the NPV is fairly high, it is important also to look at the positive and negative likelihood ratios (PLR and NLR), as the values depend on the prevalence of the disease in the population. For patch testing, the PLR was 8.8, which increases the probability of a positive result being correct by 40%, while the NLR was 0.04, which halves the probability that a negative result is truly negative. Therefore, the results may not be as good as the NPV and PPV might suggest.
Margreet Vroom (The Netherlands) asked if biopsies were taken on positive reactions to ensure these were not irritant reactions.
Ralf Mueller responded that biopsies were not taken, as owners would not have allowed that. However, there were 137 negative reactions of which 136 were true negative, so the chance that they were irritant reactions is unlikely as any irritant reaction would also be seen in the control dogs.
Reliability of intradermal testing in atopic dogs (K. Beale)
Karin Beale (USA) presented a study that she had conducted 4 years ago with Gabriel Ferrer-Canals, Jon Plant and Valerie Fadok.2 Intradermal testing (IDT) is typically considered the gold standard for selection of allergens for allergen-specific immunotherapy (ASIT). However, four other studies have shown no difference in outcome whether ASIT is based on IDT or serum allergy testing. Therefore, the question addressed by their study was ‘How reliable is the IDT?’
They assessed this using two parameters: repeatability (consistency of the reading of the duplicates) and reproducibility (between the three investigators). IDTs were performed on 12 atopic dogs using 51 different allergens, and 15 allergens were selected for blinded duplicates by the technician and were injected at the end of the test. The readings were undertaken by three individual investigators in a randomized manner, using a semi-quantitative scale of ‘0’ to ‘4’.
Agreement was analysed with Cohen’s weighted kappa (κW) using a standard linear formula. The investigators assessed repeatability (comparing scores of 15 duplicates by each investigator) and reproducibility (comparing the scores for the same 15 allergens amongst investigators). The Fleiss–Nee–Landis extension of kappa for multiple raters was used to assess the combined reproducibility of the three investigators. A statistical software program (STATSDIRECT) was used for all calculations.
Looking at the three individual investigators, the repeatability was fair with investigators #1 and #2, while it was moderate with investigator #3. Factors that influenced the repeatability were that duplicates were injected at the end of the test, so they were temporally separated. Also, they were injected more ventrally where the skin is thinner.
Reproducibility was done for scores ‘0’ to ‘4’. Reproducibility was good for ‘4’, fair for ‘0’, ‘2’ and ‘3’, and poor for ‘1’. One reason for the poor reproducibility for ‘1’ was that one of the investigators did not score reactions of ‘1’. The juggling of the three investigators to read the tests, while also seeing appointments, led to assessments being done at different times after injections. Therefore, the reliability of IDT was assessed as being fair. Nevertheless, the allergens selected to be included in the ASIT based on the IDT results were almost identical amongst investigators.
Richard Halliwell asked Karen Beale if she does read ‘1’ reactions sometimes, or if she tends to go straight from ‘0’ to ‘2’.
Karin Beale confirmed that she does not read ‘1’ reactions, which definitely had an effect on the statistics for both the ‘0’ and ‘1’ scores.
Jon Plant (USA) commented that because the ‘1’ score was not used by one investigator, the kappa value for the three investigators for the score of ‘1’ was negative, implying that the agreement on ‘1’ was worse than by chance alone. He also mentioned that he was unaware of treatment recommendations when the statistics were done, and thus could not comment on any effect of eliminating all ‘1’ reactions.
Karin Beale confirmed that the results of the IDT were correlated with the history and seasonality of the clinical signs. As all the investigators tended to select allergens for ASIT based upon reactions of ‘2’ or greater, concerns regarding ‘1’ scores was eliminated.
David Robson (Australia) asked with respect to the statistics whether different groups were evaluated, for example ‘0’, ‘1’ and ‘2’. The rationale for this would be that in most cases ‘0’ and ‘1’ reactions would not be included in ASIT vaccines, whereas ‘2’ or more are, irrespective of whether they are ‘2’, ‘3’ or ‘4’. Using the results in this way might have led to better correlations between the investigators as being more clinically relevant with respect to immunotherapy formulation.
Karin Beale agreed, but said that this was not assessed, although it could have led to a different outcome.
Jon Plant also agreed, but suggested further that it could have been better to just grade reactions as positive or negative.
Ralf Mueller commented that he had done a similar study when working in Melbourne. Although the results differed between investigators, almost identical allergens were included in the ASIT vaccines.
Patrick Hensel (USA) asked what the timeframe was between the readings of the tests by the different investigators.
Karin Beale replied that the first investigator read the IDT 8 minutes after completing the test. The goal was to have the second read right after the first, and the third right after the second, but there was sometimes some delay. However, there was never more than 15 minutes between the different readings, and the order of the readings was randomized to minimize any effect.
Richard Halliwell asked whether IDT could still be considered the gold standard.
Jon Plant responded that he did not believe that it could.
Drug interference in intradermal testing and serology: an evidence-based review (M. Saridomichelakis)
Manolis Saridomichelakis (Greece) and Thierry Olivry conducted this study for the International Committee on Atopic Diseases of Animals (ICADA) and the results are currently under consideration for publication. The study looked at evidenced-based recommendations for antiallergic drug withdrawal times before performing intradermal testing (IDT) and allergen-specific IgE serology (ASIS). The authors looked at three Internet databases. In addition, a search was done in the proceedings of the World, American and European Congresses of Veterinary Dermatology. Two different withdrawal times were proposed. The optimal withdrawal times (OWTs), which had no drug interference on the test results, and the minimal withdrawal times (MWTs), which may be associated with a small inhibitory effect that may not affect interpretation of the results.
Results for IDT (based on immediate reactions only)
For antihistamines, there were three studies.3–5 One comprised 18 dogs with flea allergy dermatitis (FAD), one comprised six normal dogs and one comprised 10 dogs with house dust mite (HDM) reactivity. Two used hydroxyzine and one used cetirizine. Reactants were histamine, flea allergen and HDM allergen. OWT was 1 week and MWT was 2 days.
For oral glucocorticoids, there were five studies.5–9 One comprised 10 dogs with FAD, one comprised 10 dogs with FAD and 11 dogs with atopic dermatitis (AD), one comprised eight dogs with FAD, one comprised five normal dogs and one comprised 10 dogs with HDM reactivity. Two used prednisone and three used prednisolone. The duration of the glucocorticoid administration was from 3 days to 6 weeks. Reactants were histamine, anti-canine IgE, flea allergen, pollen allergens and HDM allergen. OWT was 21 days and MWT was 7 days.
For injectable glucocorticoids there was only one study,10 composed of eight dogs with FAD. The study used methylprednisolone acetate, which was administered on two occasions, 1 month apart. The reactant was flea allergen. OWT was unknown, but MWT was 28 days.
For topical glucocorticoids, there were four studies.11–14 One included 16 normal dogs and seven pruritic dogs, and the other three studies comprised 10 dogs with AD. Three used 0.1% hydrocortisone +/<minus> pramoxine, one used 0.058% hydrocortisone aceponate and one used 0.015% triamcinolone. The duration of the application was from 3 days to 6 weeks. Reactants were histamine and anti-canine IgE. OWT was 2 weeks and MWT was 0 days, especially if the test was not done on the treated side.
For otic glucocorticoids, there were two studies.15,16 One included eight normal dogs and the second enrolled 20 dogs with atopic dermatitis (AD). The drugs used were 0.088% betamethasone and 0.1% mometasone, which were applied for 2 weeks. Reactants were histamine, anti-canine IgE and environmental allergens. OWT was 14 days and MWT was 0 days.
For ciclosporin there were four studies.8,17–19 One included six dogs with AD, the second eight dogs with FAD, the third 16 dogs with AD and the fourth involved four dogs sensitized to Ascaris. The duration of administration was from 4 to 6 weeks. Reactants were Ascaris allergen, flea allergen and environmental allergens. OWT was 0 days.
For tacrolimus, there was one study20 involving nine dogs with AD. The study used 0.1% tacrolimus applied for 4 weeks. Reactants were histamine, lipopolysaccharide, HD and HDM allergens. OWD was 0 days.
For pentoxifylline there was one study21 involving 10 dogs with AD treated for 4 weeks. Reactants were HDM allergens. OWD was 0 days.
For ketoconazole, there was one study22 involving 12 dogs with AD treated for 4 weeks. Reactants were histamine and HDM allergens. OWD was 0 days.
For essential fatty acids there was one study23 involving 20 dogs with AD treated for up to 118 weeks. Reactants were histamine and environmental allergens. OWD was 0 days.
Results for ASIS
For oral glucocorticoids, there were two studies.8,24 One included 15 dogs with AD and the other eight dogs with FAD treated with prednisone or prednisolone for 3 to 7 weeks. OWT was 0 days.
For injectable glucocorticoids there was one study10 involving eight dogs with FAD treated with methylprednisolone acetate two times 1 month apart. OWT was less than 28 days.
For ciclosporin, there were three studies.8,18,19 One included eight dogs with FAD, another included 16 dogs with AD and the third included four normal dogs. The duration of treatment was 4 to 7 weeks. There was no major influence and OWT was 0 days.
Manolis Saridomichelakis pointed out that this study had several limitations. The main ones were: the small number of studies, which included many abstracts of proceedings; the results apply only to the specific drugs, dosing regimens and duration of administration; and last, but not least, reactivity to histamine is not the same as reactivity to allergens injected intradermally.
Richard Halliwell felt that the histamine wheal will return well before the allergen-induced wheal after withdrawal from corticosteroids, and asked if anybody else shared his feeling.
Ralf Mueller agreed and he also believed that there are differences between allergen groups and that flea and HDM reactivity return quicker than pollen allergen’s reactivity.
Manolis Saridomichelakis commented that he had thought of that and therefore he cross-tabulated the data based on the reactant. The outcome was that if IDT reactivity to histamine were excluded, there would be no changes in the OWT. There would only be a change in the MWT for antihistamines. Furthermore, if flea allergen were excluded, there would also be no changes in the results.