HLA genotyping by NGS in cases with delayed hypersensitivity and
cases with immediate hypersensitivity to penicillins
The clinical characteristics and test results of the 24 patients with
delayed hypersensitivity to penicillins who were subjected to HLA NGS/
NGS-based HLA typing are detailed in Supplemental Tables 1 and
2 . With the exception of patient 17 who had had a bullous exanthema
with edema associated with bacampicillin treatment, none of the other
patients in the cohort had a severe cutaneous adverse reaction (SCAR) as
presentation. Twenty-two of the 24 patients had/displayed both positive
patch test and delayed intradermal test responses, which indicate a
delayed hypersensitivity mechanism Supplemental Table 2 . This
pattern of positivity does not need to be supported by challenges, as
previously reported.15,16 Of the remaining two
patients with negative patch tests and positive delayed-reading
intradermal tests, one (patient 7) underwent challenge with the
amoxicillin experiencing a MPE and the other (patient 22) was not
provoked as he had experienced mild urticaria a few minutes after
benzylpenicillin intradermal tests (Supplemental Tables 1 and
2) . Taking into account the results of skin testing, subjects
classified as group A (i.e., positive only to ampicillin and
amoxicillin, n = 14) were more numerous than those with less selective
responses classified as group B (i.e., positive to benzylpenicillin,
ampicillin and amoxicillin, n = 6) and C (i.e., positive to penicillins
and aminocephalosporins, n = 4). The clinical characteristics and
allergy test results of the 20 subjects with IgE-mediated
hypersensitivity are given in Supplemental Table 3. All
patients were retained after the quality control step of NGS. On the HLA
alleles retrieved in the 24 Italian patients, two HLA alleles,DRB3*02:02:01:02 and DRB3*02:02:01:01 , had a dramatic
enrichment among the seven alleles with an allelic frequency
>20%, compared to the control population of the Allele
Frequency Net Database (Table 1 and Supplemental Tables 4 and
5 ). The alleles DRB3*02:02:01:02 and DRB3*02:02:01:01predicted a very high risk of delayed reaction, which could not be
evaluated precisely because such alleles had not been previously
reported in the general European population. 83% of the patients
(20/24) harbored at least one of the two reported HLA-DRB3alleles, and 71% (11/24) were homozygous for one of the twoHLA-DRB3 alleles or had a compound heterozygous status
(Table 2 and Supplemental Table 4 ). We observed a higher
frequency of DRB3*02:02:01:02 and DRB3*02:02:01:01homozygous genotypes in group A (n=12, 69%) than in group B and C
patients (n=3, 30%). However, no difference of allele frequency was
observed among the 3 groups (Table 4 ).
The allele frequency of the DRB3*02:02 alleles
(DRB3*02:02:01:02 and DRB3*02:02:01:01 ) was significantly
higher in patients with delayed hypersensitivity than in those with
immediate hypersensitivity to penicillins (77% vs. 28%, respectively).
These figures corresponded to an increased risk of delayed reactions in
penicillin hypersensitivity cases harboring DRB3*02:02 alleles,
with an OR of 8.9 (95% CI, 3.4–23.3; P <0.0001) and
an absolute risk difference of 49% (29%–64%; P<0.0001).
The HLA-DRB3 association did not appear to be affected by the severity
of the initial delayed reaction. Indeed, patients with mild reactions
still maintained this association.