ch40[1].pdf

Advances in Veterinary Dermatology. Edited by Sheila M. F. Torres.

Published 2013 by John Wiley & Sons, Ltd.

8.10

Allergy testing revisited

R.E.W. Halliwell 1 (Chairperson), S. Gilbert 2 (Sec-

retary)

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.

1 University of Edinburgh, Edinburgh, UK

2 Centre V ´ t ´ rinaire Laval, Qu ´ bec, Canada

Margreet Vroom (The Netherlands) asked if biopsies

were taken on positive reactions to ensure these were

not irritant reactions.

Richard Halliwell (UK) acknowledged Veterinary Allergy

Reference Laboratory (VARL) for their sponsorship of this

workshop, which included seven presentations.

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 neg-

ative, so the chance that they were irritant reactions is

unlikely as any irritant reaction would also be seen in the

control dogs.

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 serol-

ogy 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 aller-

gens 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. Reac-

tions 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 reac-

tion against the rawor cooked antigenwas 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 likeli-

hood 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

Reliability of intradermal testing in atopic

dogs (K. Beale)

Karin Beale (USA) presented a study that she had con-

ducted 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 repro-

ducibility (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 investi-

gators in a randomized manner, using a semi-quantitative

scale of ’0’ to ’4’.

Agreement was analysed with Cohen’s weighted

kappa (jw) using a standard linear formula. The investiga-

tors assessed repeatability (comparing scores of 15 dupli-

cates by each investigator) and reproducibility (comparing

the scores for the same 15 allergens amongst investiga-

tors). The Fleiss–Nee–Landis extension of kappa for mul-

tiple raters was used to assess the combined

reproducibility of the three investigators. A statistical soft-

ware program (STATSDIRECT) was used for all calcula-

tions.

Looking at the three individual investigators, the repeat-

ability 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,

ª 2013 The Authors. Journal compilation ª 2013 ESVD and ACVD

305

Workshops

they were injected more ventrally where the skin is thin-

ner.

Reproducibility was done for scores ’0’ to ’4’. Reproduc-

ibility 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 aller-

gens selected to be included in the ASIT based on the IDT

results were almost identical amongst 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 read-

ings was randomized to minimize any effect.

Richard Halliwell asked whether IDT could still be con-

sidered the gold standard.

Jon Plant responded that he did not believe that it could.

Richard Halliwell asked Karen Beale if she does read ’1’

reactions sometimes, or if she tends to go straight from

’0’ to ’2’.

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 anti-

allergic drug withdrawal times before performing intrader-

mal 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 pro-

posed. 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.

Karin Beale conﬁrmed that she does not read ’1’ reac-

tions, 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 elimi-

nating 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.

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 his-

tamine, 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 reac-

tivity. 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 aller-

gen. 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 adminis-

tered 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,

David Robson (Australia) asked with respect to the sta-

tistics 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, irre-

spective 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 rele-

vant 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 investi-

gators.

306

Allergy testing revisited

one used 0.058% hydrocortisone aceponate and one

used 0.015% triamcinolone. The duration of the applica-

tion was from 3 days to 6 weeks. Reactants were hista-

mine 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 adminis-

tration was from 4 to 6 weeks. Reactants were Ascaris

allergen, ﬂea allergen and environmental allergens. OWT

was 0 days.

For tacrolimus, there was one study 20 involving nine

dogs with AD. The study used 0.1% tacrolimus applied

for 4 weeks. Reactants were histamine, lipopolysaccha-

ride, HD and HDM allergens. OWD was 0 days.

For pentoxifylline there was one study 21 involving 10

dogs with AD treated for 4 weeks. Reactants were HDM

allergens. OWD was 0 days.

For ketoconazole, there was one study 22 involving 12

dogs with AD treated for 4 weeks. Reactants were hista-

mine and HDM allergens. OWD was 0 days.

For essential fatty acids there was one study 23 involv-

ing 20 dogs with AD treated for up to 118 weeks. Reac-

tants were histamine and environmental allergens. OWD

was 0 days.

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 reac-

tivity.

Manolis Saridomichelakis commented that he had

thought of that and therefore he cross-tabulated the data

based on the reactant. The outcomewas that if IDT reactiv-

ity to histaminewere excluded, therewould be no changes

in the OWT. There would only be a change in the MWT for

antihistamines. Furthermore, if ﬂea allergen were

excluded, therewould also be no changes in the results.

Richard Halliwell asked if any of these studies used

hydrocortisone aceponate and if there was any difference

between that study and studies using other topical corti-

costeroids.

Manolis Saridomichelakis responded that one study

used hydrocortisone aceponate. IDT results were

affected for 2 weeks and the untreated side of the thorax

was also affected.

Douglas DeBoer (USA) commented that when he lec-

tures to general practitioners (GPs), he always says that

there isn’t really an effect of glucocorticoids on serology

results; however, GPs tell him that laboratories ask them

to discontinue glucocorticoids for a long time before they

can do the test, and he wondered whether anyone from

industry could comment.

Don Wassom (USA) commented that they did a number

of studies on that some years ago at Heska (Fort Col-

lins). The effects of glucocorticoids, whether oral or

injectable, on the outcome of IgE serology over time

was very minimal. However, in one study, levels of aller-

gen-speciﬁc IgE were quantiﬁed in some dogs and when

those dogs were withdrawn from glucocorticoids the

levels of allergen-speciﬁc IgE increased. Whether it

would have inﬂuenced the interpretation of these tests

is questionable. However, the fact that allergen-speciﬁc

IgE tended to increase over time after glucocorticoid

withdrawal suggests that steroid withdrawal might be

useful.

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 study 10

involving eight dogs with FAD treated with methylpred-

nisolone 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 pro-

ceedings; the results apply only to the speciﬁc drugs, dos-

ing regimens and duration of administration; and last, but

not least, reactivity to histamine is not the same as reac-

tivity to allergens injected intradermally.

Manolis Saridomichelakis asked Don Wassom if aller-

gen-speciﬁc IgE was measured for perennial allergens

(HDM) or for seasonal allergens.

Don Wassom did not remember the details of the study

and thought that it might have been done with ﬂea saliva

reactivity.

Manolis Saridomichelakis stated that another factor

might be time. In studies of long duration, the concentra-

tion and exposure to environmental allergens may change

and thus it could make a difference, especially for pollen

allergens. He added that the good thing here is that all

studies assessing the effects of drugs on ASIS were

rather short term (no more than 2 months).

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.

307

Workshops

Ralf Mueller asked Manolis Saridomichelakis to clarify

why this was good.

The conclusion of the study is that some canine sera had

allergen-speciﬁc serum components that seem to bind

peroxidase to give false positive signals. Therewere differ-

ences in signal between different conjugates of the same

mabs and there were differences in signal between mab

5.91 andmab D9when the same conjugatewas used.

Manolis Saridomichelakis explained that it is important

to bear in mind the short half-life of IgE in serum. Indeed,

if environmental allergens are considered, one would not

expect the major changes in the exposure and thus in the

quantity of allergen-speciﬁc IgE in a short-term study that

one might ﬁnd in a long-term study.

Richard Halliwell commented that it appeared that HRP

conjugation should be avoided in all assays.

Richard Halliwell commented that the half-life of IgE in

serum is very short, but it is actually much longer when it

is bound to mast cells, namely 2 to 3 weeks.

Jennifer Bexley agreed, although she was unsure of the

reason.

Ken Lee (USA) said that he had demonstrated some

years ago that the HRP has a carbohydrate molecule that

acts as an epitope that is identical to various carbohy-

drates that are present on other allergens. The IgG from

the serum binds to that epitope on the HRP and also that

on the allergen. The HRP activity is not inhibited when

IgG binds to it. Therefore, it has nothing to do with the

other components in the assay.

Manolis Saridomichelakis emphasized that then differ-

ences in exposure would have less inﬂuence on IDT

results than on results of serology testing.

Enzyme coupling methodology and its

effects on mab speciﬁcity (J. Bexley)

Jennifer Bexley (UK) stated that she initially tried to

assess two different types of monoclonal antibodies

(mab) for canine IgE: mab 5.91 from Bruce Hammerberg

(North Carolina) and mab D9 from Doug DeBoer (Wiscon-

sin). The reactivity of both mabs towards canine IgE and

the level of cross-reactivity towards canine IgG were

checked for validation of the reagents. Both were found

suitable. Next, the levels of IgE towards food allergens

were compared using mab 5.91 conjugated to biotin and

mab D9 conjugated to horseradish peroxidase (HRP).

Mab 5.91 biotin seemed to detect meats (beef, pork,

lamb) while mab D9 HRP detected some of the cereals.

Therefore, it was thought that by combining both mabs,

results would be improved by detecting the full range of

reactivity. Next, by doing an inhibition study, she checked

that the two mabs were not binding the same epitopes of

the IgE. The conclusion was that the mabs bind different

epitopes. Then, the mabs were conjugated to both biotin

and HRP to see which one would be best for the assay.

The author was surprised by the results, as when mab D9

was conjugated to HRP, it picked up the cereals, but did

not do so when conjugated to biotin. Also, mab 5.91

showed a strong signal to beef when conjugated to bio-

tin, but not when conjugated to HRP, although cereal-spe-

ciﬁc IgE was detected. Therefore, the reaction seemed to

be driven by the conjugate rather than by the mab itself.

To answer this question, the assay was run without

including any of the mabs. There was still a signal for the

meat and some of the cereals, in some of the sera. The

possibility that streptavidin was causing this nonspeciﬁc

reaction was discarded and it was concluded that it was

due to the HRP. Next, reactivity of the two different mabs

(D9 and 5.91) was assessed with the same conjugate

(biotin) and with streptavidin alkaline phosphatase (AP).

There was a slight difference between signals, but the

pattern was overall similar. Then, they repeated the same

experiment with AP direct conjugate for both mabs, and

much of the reactivity was lost. However, mab 5.91

picked up more than mab D9 using the same conjugated

label.

Jennifer Bexley asked why it was seen with beef, but

not with rice.

Ken Lee explained that it is because the epitope is not

present in all allergens and rice does not have that carbo-

hydrate. So, yes, HRP should be eliminated from all

assays!

Don Wassom agreed with Ken Lee.

Richard Halliwell commented on the important ﬁnding

that, when exactly the same conjugates are used (even

direct AP), there is some difference in the mab reactivity.

Storage mites and serological reactivity in

canine atopic dermatitis (CAD): have we

underestimated its importance?

(D.L. Wassom)

Don Wassom (USA) noted that his data are based on the

many in vitro IgE tests conducted by Heska Corporation

in Fort Collins, Colorado, USA, over the years. He empha-

sized that improvement in in vitro IgE testing is likely to

come by improving the allergen extracts that are used in

the assays, and that we still do not understand very well

the allergens that dogs, cats and horses recognize in the

extracts as opposed to humans. Major allergens of house

dust mite (HDM) recognized by dogs are der f 15 and der

f 18, so they are different from the major allergens recog-

nized by humans. Optimizing the allergen extracts for

those components that are recognized by animals is

important. Storage mites have some allergens that are

unique and not shared by HDM.

Mites are very important as initiators and perpetua-

tors of allergic skin diseases in dogs. A substantial

number of allergen-speciﬁc immunotherapy (ASIT) pre-

scriptions in the USA and Europe contain mites, and in

northern Europe up to 75% of ASIT prescriptions con-

tain mites only.

308

Allergy testing revisited

This raises the question of whether storage mites are

the primary source of sensitization rather than HDM in

dogs with AD. The rationale for this question is that

although HDM are not common in Colorado, Colorado

dogs suffer from AD and test positive to HDM. Therefore,

if HDM are rare in Colorado, could storage mites account

for the mite reactivity? The storage mite Tyrophagus pu-

trescentiae can be cultured from opened bags of dog

food in Colorado, and Tyrophagus and Dermatophagoides

mites share cross-reacting allergens. Also, there is a sub-

set of dogs that react only to storage mites and not to

HDM. This is the origin of the proposition that there are

unique allergens associated with storage mites that are

not shared by HDM. In fact, in the USA, about 11% of

mite-positive ALLERCEPT tests (Heska) are positive

only for Tyrophagus. However, in Colorado where HDM

are rare, 21% of the animals that test positive for mites

are positive only for Tyrophagus.

Marie Innera (Norway) stated that she sees many dogs

with reactions to storage mites only and a poor response

to ASIT using Tyrophagus. She ﬁnds that many of these

dogs are springer spaniels, and do not respond to an elim-

ination diet trial with hydrolysed diets but are eventually

diagnosed as having food allergy using a home-cooked

diet.

Don Wassom commented that there are a lot of dogs

that are on ASIT with just Tyrophagus immunotherapy

based on the test results. He does not know its efﬁcacy

although Tyrophagus prescriptions that are ordered are

usually reﬁlled, suggesting some favourable responses.

Susanne Ahman (Sweden) reported that she also sees

many dogs being positive only to Tyrophagus or A. siro.

However, she does see a good response to ASIT contain-

ing one of these storage mites as the only allergen.

Richard Halliwell asked Don Wassom if he had done any

cross-inhibition studies.

Allan Bell (New Zealand) commented on the biology of

Tyrophagus, which he said was not as dependent on

humidity as is HDM. He commented on a New Zealand

large animal food company that makes food heavily con-

taminated with A. siro. The company fumigates the food,

which seems to lower the level.

Don Wassom responded that he had and that reactivity

in sera from animals testing positive to both HDM and

storage mites can be inhibited by either antigen. So, there

is no question that mites share cross-reacting allergens.

Most dogs that test positive to mites have antibodies that

cross-inhibit. Usually, dogs that test positive to Tyropha-

gus will test positive to Dermatophagoides but a subset

of dogs test positive to Tyrophagus and negative to Der-

matophagoides.

Don Wassom answered that it seems to be unusual to

ﬁnd HDM in the dry climate of Colorado. However, stor-

age mites are commonly found suggesting that the

requirement of humidity is lower for storage mites than

for HDM.

Richard Halliwell wondered about Acarus siro.

Intradermal testing (IDT) reactivity to dust

mites and storage mites in the south-

eastern USA (P. Hensel)

Patrick Hensel (USA) asked, in view of the fact that up to

75% of clinically normal dogs show positive IDT to HDM,

whether we were dealing with subclinical hypersensitiv-

ity, the use of irritant concentrations for testing, or reac-

tions resulting from cross-reacting allergens.

He reported that he had undertaken two studies 25,26

that were published in Veterinary Dermatology. One

study conducted with his resident attempted to deﬁne

the optimal concentrations for skin testing using mite

allergens. He believed this should be lower than the com-

monly used 250 PNU/mL. The next step was to look for

ideal intradermal test concentrations. Once the new test

concentrations had been selected, they tested dogs with

AD using two different test concentrations, the adjusted

concentrations (that they believed might be better) and

the standard concentrations. Fewer positive reactions

were seen with the adjusted test concentrations and their

conclusion was that the results may be more reliable.

Using six mites – Dermatophagoides farinae (DF),

D. pteronyssinus (DP), Acarus siro (AS), Lepidoglyphus

destructor (LD), Tyrophagus putrescentiae (TP) and Blo-

mia tropicalis (BT) – with different adjusted test concen-

trations, they compared IDT reactivity to these dust mites

(DM) in 24 dogs with atopic dermatitis. The test concen-

trations were: 50 PNU/mL for AS and BT; 75 PNU/mL for

Don Wassom answered that it does usually track with

Tyrophagus and Dermatophagoides. If they are positive

to one, they are positive to all of them. They have not

identiﬁed dogs that test positive for Acarus and negative

to Tyrophagus.

Richard Halliwell asked about cats, as cats recognize dif-

ferent antigens.

Don Wassom said that he does not have enough data to

answer that question.

Douglas DeBoer believes that the piece that has always

been missing in the whole storage mite story is exposure.

Studies showed that open bags of dog food will become

contaminated by storage mites, and unopened bags that

are then sealed properly after being open do not contain

storage mites. These studies looked at the presence of

live and dead mites, but not at the protein. Therefore, a

study should be done on quantifying storage mite protein

to see if unopened bags of dog food contain immunologi-

cally detectable storage mite antigens.

Don Wassom commented that the problem is ﬁrst to

identify the storage mite allergens that are relevant to

dogs and whether they can survive processing and are

able to sensitize via the oral route.

309

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