Our results emphasize the validity of a diagnostic test that has not been properly evaluated to date. In this assessment, a cut-off value for sIgE to alpha-gal ≥ 0.1 kUA/L was able to diagnose 97% of patients with symptoms after exposure to potential alpha-gal sources, with good internal and external validity. In addition, the characteristics of the patients in this group, suggest a relationship between sensitization to this oligosaccharide and a history of tick bites, as previously described [2].

The most common method to confirm the diagnosis of AGS is the use of serologic testing for alpha-gal sIgE levels ≥ 0.1 kUA/L [11]. Moreover, the classic features of AGS include the presence of positive sIgE levels to alpha-gal [7]. Other diagnostic methods are available, such as skin testing with commercial extracts of mammalian meat, but they have lower sensitivity, or prick-to-prick testing with cooked meats, or skin prick and/or intradermal testing with cetuximab or gelatin, which present a strong correlation with alpha-gal sIgE levels [11]. In addition, it should be taken into account that patients with anaphylaxis due to AGS and indolent systemic mastocytosis can present with lower values of sIgE to alpha-gal [12]. However, none of the patients in our study presented mastocytosis and the determination of sIgE levels to alpha-gal allowed us to diagnose AGS with a good validity of the results.

Other researchers have proposed an integrative diagnostic methodology that combines medical history with anti-alpha-gal IgE titers in a machine learning algorithm because it is possible that the cutoff value of sIgE that reveals clinical reactivity might not be well determined since AGS can be highly influenced by the presence of cofactors and the delayed nature of the reaction [13]. Although the presence of cofactors was not taken into account in the analysis, 11 individuals from Group 1 (9 men and 2 women) had presented them in the initial reaction, highlighting their role in the diagnosis.

Current strategies for diagnosing AGS explained that the relevance of positive testing to alpha-gal sIgE levels with a cut-off value ≥ 0.35 kU/L remains unclear because it is not able to predict the severity of the reactions [14]. Other authors have shown that the cut-off for sIgE to alpha-gal associated with a positive predictive value of > 95% probability of presenting meat allergy was 2.00 kU/L [15]. In our study, the results revealed that a cut-off for sIgE to alpha-gal ≥ 0.1 kUA/L was able to diagnose AGS in all patients with a positive predictive value of 82%, although the performance of a challenge-proven meat allergy test could modify the diagnosis of some patients. However, if the cut-off point applied was ≥ 2 kUA/L as in the study of Mabelante et al. [15], the positive predictive value of specific IgE to alpha-gal would be 93%.

The predictive test cut-offs in food allergens offers guidance in determining successful oral tolerance [16]. The validation of sIgE for the diagnosis of egg allergy in children has also been reported, with a sensitivity of 0.91 and positive predictive value of 0.94 to sIgE levels for egg white ≥ 0.35 kUA/L [17]. In our study, a cut-off value of ≥ 0.1 kUA/L also showed high sensitivity and specificity (0.97 and 0.89 respectively), and a relatively high positive predictive value (0.82), with sensitivity being higher in men and specificity being higher in women. In addition, a previous study on sIgE to alpha-gal to diagnose red meat allergy showed a sensitivity of 1.00 and specificity of 0.92, which were higher than in our population, but had a lower positive predictive value (≤ 50%). Futhermore, as in our study, the proportion of men with AGS symptoms was higher than women, since 55.6% of the subjects were male, although the authors did not perform an analysis adjusted by sex as we did [8].

The analysis of internal validity parameters showed a high positive probability ratio and a low false negative rate in all populations, with a higher positive probability ratio in women than in men. In our population, women were more likely to test positive than men given that most patients in Group 1 were male and most of patients in Group 2 were women. Unlike other studies describing the limited utility of sIgE levels to alpha-gal ≥ 0.35 kUA/L to confirm the diagnosis of mammalian meat allergy in 118 patients showing a high sensitivity (85%) but poor specificity (32%), and including 30 false positives [18], our results illustrate the analysis of internal validity parameters in detail. The differences in sensitivity and specificity compared to other studies [8] are probably due to the use of a lower cut-off point and the comparison with populations from other geographic regions. On the other hand, the analysis of external validity parameters revealed a high negative predictive value (0.98) and global value (0.92), which represented proof of the validity of sIgE levels to alpha-gal for AGS diagnosis in our geographical area.

Some patients in the control group showed a subclinical sensitization to alpha-gal because they tolerated mammalian meat without developing allergic symptoms. The determination of sIgE antibodies can provide essential information on the etiology of the disease because they are produced following exposure of a susceptible individual to an allergen [19], but sensitization is not the same as clinical allergy, and the presence of IgE does not confirm a diagnosis of AGS [20]. The only patient with alpha-gal allergy and sIgE to alpha-gal < 0.1 kUA/L was a woman diagnosed years ago, when she presented with sIgE levels ≥ 0.1 kUA/L, thus being an example of a false negative result. A possible explanation is that sIgE levels alpha-gal tend to decrease over time, except in patients with a history of anaphylaxis, whose levels are maintained [21].

The main limitation of this study was the small sample size, since it made it difficult to carry out comparative statistical analyses between the two groups of patients. Other limiting factors were the heterogeneity of the sample, the lack of analysis of cofactors according to sex, the severity of the reaction, and the time between the performance of the study and the presentation of the reaction since they could influence the final diagnosis. Nevertheless, to our knowledge, this is the first detailed analysis on sIgE to alpha-gal adjusted by sex as a diagnostic tool in AGS.

In conclusion, we described the diagnostic validity of sIgE levels to alpha-gal in AGS in our population. In accordance with these results, sIgE to alpha-gal is a useful diagnostic tool, although it requires further evaluation to explore its correlation with other factors, other available diagnostic techniques or other potential diagnostic tools.