Long‐term prognosis of vaccine‐induced contact allergy to aluminium: Third patch‐test with additional test preparations

A high incidence of local itching subcutaneous nodules and aluminium allergy was observed in clinical trials of a new aluminium adsorbed pertussis vaccine in Gothenburg, Sweden, in the 1990s. A total of 495 children with itching nodules were patch tested with aluminium chloride hexahydrate 2% and an empty Finn Chamber®, 377 (76%) with positive reactions. When 241 of them were re‐tested some years later 186 (3 out of 4) had unexpectedly lost their patch test reactivity.


| INTRODUCTION
In clinical trials of a new acellular pertussis toxoid vaccine (aP) in the 1990s in Gothenburg, Sweden, persistent itching subcutaneous nodules (granulomas) at the injection site were reported in 745 of the 76 000 vaccinated children. 1,2e children were aged 3 months to 14 years.The risk for developing itching nodules increased with the number of vaccine doses received.
Additional symptoms were local eczema, hypertrichosis and discolouration of the overlying skin and exacerbation of symptoms during infections.A long delay between vaccination and onset of symptoms (median 3 months) was common.The duration of symptoms may be prolonged (median 6.6 years), but the prognosis is good (86% fully recovered), as reported in a recent long term follow-up study. 3Aluminium-adsorbed vaccines can also induce contact allergy to aluminium, confirmed by an † Died in January 2023.epicutaneous test.[6] Aluminium salts, mostly aluminium hydroxide and phosphate, are used as adjuvants in vaccines to enhance immunogenicity.The exact mechanism is unknown.All vaccines against diphtheria, tetanus, pertussis, hepatitis A and B, human papillomavirus and tick-borne encephalitis are adsorbed to aluminium adjuvants 7 as well as some vaccines against meningococcal and pneumococcal infections.Most antigen extracts used in allergen-specific immunotherapy treatment (ASIT) are also aluminium adsorbed. 8tween 1998 and 2002 all study children with itching granulomas were offered a patch test for aluminium (Patch test I), preceded by an interview and examination of the injection sites. 1 More than 5 years later, 2007-2008, a second patch test (Patch test II) was performed in those who had tested positive in the first study. 2 Patch test I, 377 of the 495 tested children (76%) had positive reactions. 1,2Surprisingly, 3 out of 4 (186/241) had lost their patch test reactions in Patch test II.A negative patch test correlated significantly with ceased clinical symptoms, the time elapsed since the first vaccination, increasing age and the reactivity of the first positive patch test. 2 Such a significant loss of test reactivity had not been reported before, on the contrary, contact allergy so far was considered to persist throughout life.
The principal aim of the present study was to investigate the long-term prognosis of vaccine-induced contact allergy to aluminium in children by performing a third patch test ≥15 years after Patch test I.A second aim was to investigate different aluminium preparations in order to find an optimal compound and test concentration for aluminium.One pregnant woman was excluded.

| Methods
In accordance with the two previous studies, the test material was applied on the upper part of the back using metallic aluminium (an empty Finn Chamber ® ; SmartPractice), and aluminium chloride hexahydrate 2% in petrolatum (ACH 2%) (Chemotechnique Diagnostics ® ).In addition, another three aluminium salts prepared in petrolatum were tested: aluminium chloride hexahydrate 10% (ACH 10%), aluminium lactate 2.4% and aluminium lactate 12.2% (obtained from Sigma Aldrich as aluminium chloride 99% and aluminium L-lactate 95% and prepared by Occupational and Environmental Dermatology Unit, Sahlgrenska University Hospital, University of Gothenburg, Sweden).All preparations were placed in a plastic chamber (IQ-Ultra™, Chemotechnique Diagnostics).All test preparations were applied by two of our staff members with special training in patch test technique and long experience in the Occupational and Environmental Dermatology Unit.
The petrolatum preparations, approximately 25 mg, were applied according to ICDRG guidelines. 9The patches were removed on D2 by the participants themselves.All results were read on D3 and scored according to ICDRG guidelines 9 by two of the authors (Anette Gente Lidholm and Annica Inerot).If there were different outcomes in patch test reactivity to the different test preparations, the strongest positive reaction was to account for the maximum score.Doubtful reactions which did not fulfil the criteria for a one plus reaction were classified as negative.Readings on D7 were not performed due to practical reasons, but all participants were asked to be aware of late reactions and, in that case, to contact us for a second reading.
The participants were interviewed about ongoing symptoms and additional aluminium-containing vaccinations.The injection site from the vaccine trials was inspected and examined for remaining nodules by two of the authors (Birger Trollforsand Elisabet Bergfors) as in the previous studies. 1,2

| Long-term prognosis of contact allergy to aluminium
An overview of the three testings and the results is shown in Figure 2.
Of the 20 persons who had positive reactions to ACH 2% and/or an empty Finn Chamber ® in Patch test II (group A), five were still positive to these compounds in Patch test III.Seven were negative to ACH 2% and the Finn Chamber ® but positive to ACH 10% and/or the lactate preparations.Eight had negative reactions to all five tested compounds (Table 2).
A total 2 out of 11 persons who lacked reactivity in Patch test II but had ongoing symptoms from their itching nodule, or had a new nodule (group B), had turned positive to ACH 2% and Finn Chamber ® in Patch test III.Another two had positive reactions only to ACH 10% and the lactate preparations.For detailed information of the reactivity scores, see Tables 1 and 2.
None of the 31 participants contacted us with a late reaction.

| The outcome of five different aluminium preparations
Of the five tested aluminium preparations, the highest number of positive reactions, 14, was observed in aluminium lactate 12.2%, followed by 12 positive reactions to ACH 10%.There was no significant difference between the higher and lower concentrations for either ACH (2% and 10%) (P = 0.13) or aluminium lactate (2.4% and 12.2%) (P = 0.63).Three of 5 individuals with doubtful reactions to ACH 2% had positive reactions to ACH 10% or aluminium lactate 12.2%.The long-term prognosis of patch-test-verified hypersensitisation to aluminium is poorly studied, even if a loss of patch test reactivity has been described by some authors. 10ide from a short report on four children in Denmark 1992, 5 the prognosis was first described in our previous study on a repeated patch test in children from the vaccine trials in Gothenburg (2013). 2 Including the present study the same cohort we can now report a long-time prognosis for aluminium allergy for more than 20 years.The cause, or causes, for the persistence or the loss of positive patch test reactivity is poorly known but widely discussed.

| Clinical course
Avoidance of the sensitiser, weak positive reactions misinterpreted as negative and persistence rates between specific allergens and allergen groups are theories favouring the loss of sensitivity. 11 the total exposure to systemically available aluminium ions decreases the exposure to the allergen at patch testing will decrease as well and-potentially-cause a weaker, or a loss of reactivity.This has been shown for gold 12,13 and may be an explanation to loss of reactivity with increased age.In our cohort, the systemic load of aluminium ions may have decreased with time since the vaccinations in the trials.On the other hand, most of the children received a booster dose of pertussis vaccine at 10 years of age 3  In summary, the young persons in our study might certainly have got a reload of aluminium ions by new aluminium adsorbed vaccines which may have affected the patch test results.But, after all, the loss of reactivity is so apparent that a reload seems to be of minor importance in this cohort.
In the present study, two persons tested positive in Patch test I, negative in Patch test II but then positive again in Patch test III.
Individual variation in test reactivity has been seen after repeated patch tests for several antigens including aluminium, 14 and may be due to immunological factors and differences in patch test technique. 15,16

| Optimal test preparations
In the 1990s, when the children with itching nodules were first tested within the vaccine trials, the only commercial aluminium preparations available were ACH 2% and metallic aluminium (an empty Finn Chamber ® ).Recent studies suggest that the 2% concentration of ACH may lead to false-negative results and that a 10% preparation may be preferable. 17On the other hand, ACH 2% was demonstrated to be sufficient to trace aluminium hypersensitivity in small children in three Swedish studies with altogether 601 patch tested children.65% of those aged 1-2 years scored +++ to this concentration. 18Similar findings have been observed in both a Danish and a French study on children, where 93% and 100% respectively exhibited a positive reaction to ACH 2%. 19,20trospectively, taking into consideration today's knowledge that ACH 10% is preferable in patch testing for aluminium allergy in older children and adults, we should have considered applying an additional patch of this preparation in Patch test II.
In Patch test III, ACH 10% and aluminium lactate 2.4% and 12.2% were added to the weaker compounds.However, no significant difference was found in the number of positive reactions to any of the aluminium formulas in this small material.
None of the participants in Patch test III reacted to the empty Finn Chamber ® exclusively.The role of metallic aluminium in patch testing for aluminium allergy is, in our opinion, and as earlier reported, of minimal benefit. 21 has been proposed that aluminium should be included in the baseline series for children.However, test results must always be interpreted in relation to clinical symptoms.In our experience many parents are prone to refrain from vaccinating their children in fear for adverse events. 22We are seriously concerned that this could be a consequence if an aluminium allergy is accidentally discovered in a baseline series including aluminium.At the time for Patch test I and II, there were no recommendations on the amount of a petrolatum preparation to be applied.Since then, the dose of the sensitisers in each type of test chamber has been standardized. 9These recommendations were followed in Patch test III.
When interpreting and comparing the results of three patch tests in a cohort followed for about 20 years, one has to be aware of the continuous development and changes in test procedures and recommendations.
T A B L E 2 Thirty-one individuals with vaccine-induced itching nodules: Outcome (scores) in Patch test III for patch testing with aluminium chloride hexahydrate 2% and 10%, aluminium lactate 2.4% and 12.2% and an empty Finn chamber.Our young participants were not always aware of their vaccination history.Data concerning additional vaccinations may be somewhat uncertain and thereby another source of error.
The greatest, and most regrettable, limitation of the study is the small number of participants remaining when they were halved by the COVID-19 pandemic.This loss has probably influenced the results.
Reading the patch tests only on D3 may be another limitation since late reactions may be underestimated.No late reactions were reported.

| Clinical relevance based on all three studies
In our different roles as clinical physicians, the great benefit of the long-term studies which now have come to an end is that we can inform parents to children with recently detected itching subcutaneous nodules and aluminium allergy that the symptoms, however troublesome they are, will come to an end sooner or later and that the contact allergy, in contrast to earlier belief, also has a very good prognosis.This information is of great importance for parents and of course for the growing children themselves and, in the long term, to the Child Health vaccination programmes in general.recommend that further vaccination with aluminium adsorbed vaccines can go on despite earlier proven aluminium contact allergy. 19,22e risk for new itching vaccination granulomas is low once the original one has vanished and the itching has resolved or nearly resolved and the recommended paediatric vaccination programmes can be fulfilled when the children grow older.Infants with onset of symptoms already after the first or second dose should be judged individually concerning continued vaccination.

2. 1 |
Patients All subjects in the present study had persistent itching nodules after vaccination in the Gothenburg Pertussis Vaccine Trials and a positive patch test for aluminium in 1998-2002 (Patch test I). 1 They had also participated in the second patch test study in 2007-2008 (Patch test II). 2 The third patch test was performed in January-March 2020.The participants were divided into two groups: (A) persons with positive patch tests for aluminium both in 1998-2002 and 2007-2008; (B) persons with a positive test in 1998-2002 and a negative in 2007-2008 but either still having symptoms from their itching nodules at that time or were free from symptoms in 2007-2008 but had a new itching nodule after an aluminium containing vaccine given later on in life.
Three individuals still had intermittent itching subcutaneous nodules on the former injection site 17, 18 and 20 years after the onset, respectively.One of them had a mild (+) positive reaction in Patch test III while the two others showed no reactivity.None of them reported any new itching granulomas after later vaccination with aluminium adsorbed vaccines or any symptoms after topical aluminium exposure.One of the participants reported local eczema after the use of aluminium-containing antiperspirants and sunscreens.Approximately half of the remaining used aluminium-containing antiperspirants daily without complaining of local eczema.The others still used aluminium-free products after being diagnosed with aluminium allergy in childhood.Twenty-four of the 31 participants had received one or some aluminium adsorbed vaccines after Patch test II (hepatitis A and B, tickborne encephalitis).Three of them reported a new itching nodule at the injection site after these vaccinations.None of the participants in Patch test III had been exposed to ASIT.6 | DISCUSSION 6.1 | Prognosis of contact allergy to aluminium All three tests were performed with ACH 2% and an empty Finn Chamber ® in accordance with the standard procedure in 1998 when the first children were tested.The maintenance of the same test procedure was a prerequisite for a long-term prognosis.The main finding in the present study is that the loss of reactivity that was seen in about 77% of the children during the 5-10 years between Patch test I and II has continued.Another 75% of the now young adults lost their reactivity during the next 10-12 year until Patch test III.Even if the number of tested this time is small the tendency is clear and not unexpected.The fact that seven individuals still had remaining positive reactions to ACH 2% and Finn Chamber ® as late as 18-20 years after the first test is perhaps more interesting.
and thereby a systemic reload of aluminium ions.Only a few of them experienced new itching nodules after the booster doses and typically with a much shorter symptom duration after re-exposure compared to the initial T A B L E 1 A total of 31 individuals with vaccine-induced itching nodules: Outcome (scores) for patch testing with aluminium chloride hexahydrate 2% in petrolatum and an empty Finn Chamber ® in Patch test I, II and III.reload of aluminium ions could also be gained by vaccination with other aluminium adsorbed vaccines later on in life.Most girls in our cohort received two doses of vaccine against human papillomavirus (HPV) in school at 10-12 years and as many as 24 of the 31 participants reported that they had received one or some doses of vaccines against hepatitis A and B and tick-borne encephalitis between Patch test II and III.

6 . 3 |
Positive reactions from + to +++ were regarded as delayed hypersensitivity.Doubtful reactions '?+' were regarded as negative results.Group A: Positive reactions in both Patch test I and II.Group B: Positive reactions in Patch test I but no reactivity in Patch test II.Sources of error: Limitations Test applications and the readings were performed by experienced staff and dermatologists (Annica Inerot and Anette Gente Lidholm) in the study in order to minimize errors due to the testing procedure.The dose per area of a sensitiser is the most decisive factor for a patch test.The outcome in our three studies was probably affected by the different size of the plastic chambers used in Patch test I and II (the original plastic IQ Chamber with an inside area of 81 mm 2 ) and in Patch test III (the IQ-ultra™ Chamber with an inside area of 64 mm 2 ).
Contact allergy to aluminium caused by vaccination with aluminiumadsorbed vaccines in childhood declines with time in a majority of the affected individuals as demonstrated by repeated patch tests during more than 20 years.In those few still positive in patch test the clinical relevance is low.By following the cohort from the Gothenburg Pertussis VaccineTrials for so many years, and in addition to earlier findings we F I G U R E 1 A total of 31 individuals with vaccine-induced itching nodules who were patch tested three times with aluminium chloride hexahydrate 2% in petrolatum and an empty Finn chamber ® : Age at first vaccination with aluminium adsorbed vaccine and at Patch test I, II and III.Outcome (overall score) of the three tests.
Positive reactions from + to +++ were regarded as delayed hypersensitivity.Doubtful reactions '?+' were regarded as negative results.Group A: Positive reactions in both Patch test I and II.Group B: Positive reactions in Patch test I but no reactivity in Patch test II.