An Assessment of the Currently Available Molecular Assay for the Diagnosis of Anisakis Sensitization

The diagnosis of allergic reactions to <i>Anisakis</i> remains challenging due to the lack of specific allergens available for routine clinical use. However, the latest version of the multiplex macroarray ALEX-2 now allows the detection of specific IgE against Ani s 1, the major species-specific allergen, as well as Ani s 3 (tropomyosin), a highly cross-reactive molecule with homologs in other allergenic sources. This study aimed to evaluate the potential role of ALEX-2 in diagnosing <i>Anisakis</i> sensitization by comparing it with a previously validated diagnostic algorithm. Serum samples from patients with suspected <i>Anisakis</i> sensitization were consecutively collected at an Italian allergy centre. Diagnosis was based on a history of allergic reactions following seafood consumption, along with negative test results for fish allergy. All patients underwent skin prick testing and specific IgE measurement for <i>Anisakis</i> (p4), <i>Ascaris</i> (p1), shrimp (f24), and <i>Dermatophagoides pteronyssinus</i> (d1), as well as tropomyosins from house dust mites (d205) and shrimp (f351). Additionally, the basophil activation test (BAT) using crude <i>Anisakis</i> extract was performed. Patients were also tested using the ALEX-2 allergy macroarray. Correlation analyses and multiple logistic regression models were applied to assess associations between conventional singleplex tests and the severity of clinical manifestations. A total of 33 eligible subjects were recruited, including 20 females (60.6%). Seven (21.2%) were aged 0-29 years, eleven (33.3%) were 30-60 years old, and fifteen (45.5%) were over 60 years old. ALEX-2 showed positivity for Ani s 1 or Ani s 3 in 39.39% (95% CI: 22.90-57.86%) of subjects with confirmed <i>Anisakis</i> sensitization. A significant correlation was observed between Ani s 3 (r = 0.31 [95% CI: 0.04-0.56], <i>p</i> = 0.01) and <i>Ascaris</i> (r = 0.35 [95% CI: 0.129-0.55], <i>p</i> = 0.004) levels and the severity of clinical reactions. Despite the limitations of this cross-sectional study, including a small sample size, our preliminary findings suggest that the ALEX-2 macroarray may not be sufficiently sensitive for the first-line diagnosis of <i>Anisakis</i> allergy. However, it could provide valuable additional information, as Ani s 1 positivity indicates primary sensitization to the nematode, while Ani s 3 positivity appears to correlate with clinical severity. Larger prospective longitudinal studies are needed to confirm these findings and further assess the predictive value of ALEX-2 in diagnosing <i>Anisakis</i> allergy.