Mammalian Meat Allergy and IgE to Alpha-gal in in Central Virginia: findings from a COVID-19 vaccine and patient cohort

Abstract

Background:

IgE to galactose-alpha-1,3-galactose (alpha-gal) is linked with tick bites and an important cause of anaphylaxis and urticarial reactions to mammalian meat. The “alpha-gal syndrome” (AGS) is recognized as being common in the southeastern USA, however prevalence studies are lacking and open questions remain about risk factors and clinical presentation of alpha-gal sensitization.

Objective:

Here we characterized the prevalence, as well as presentation and risk factors, of AGS and alpha-gal IgE sensitization in adults in central Virginia recruited without regards to history of allergic disease.

Methods:

Adults in central Virginia, primarily University of Virginia Health employees, were recruited as part of a COVID-19 vaccine study. Subjects provided at least one blood sample and answered questionnaires about medical and dietary history. ImmunoCAP was used for IgE assays and ABO blood group was assessed by reverse typing using stored serum. Biobanked serum from COVID-19 patients was also investigated.

Results:

Of 267 enrollees, median age was 42, 76% were female and 43 (16%) were sensitized to alpha-gal (cut-off 0.1 IU/mL), of which mammalian meat allergy was reported by 7 (2.6%). Sensitized subjects were i) older, ii) had higher total IgE levels but similar frequency of IgE to common respiratory allergens, and iii) were more likely to report tick bites than non-sensitized subjects. Among those who were sensitized, alpha-gal IgE levels were higher among meat allergic than non-allergic subjects (GM 9.0 vs 0.5 IU/mL, P<0.001). Mammalian meat and dairy consumption was common in individuals with low-level sensitization.

Conclusion:

In central Virginia AGS is a dominant cause of adult food allergy with a prevalence approaching or exceeding 2%.

INTRODUCTION

The alpha-gal syndrome (AGS) is mediated by IgE to galactose-alpha-1,3-galactose (alpha-gal) and a cause of allergic reactions to “red” meat and other mammalian products that contain the alpha-gal glycan.^1–3 Tick bites are strongly associated with AGS and bites from Amblyomma americanum (the lone star tick) are understood as the primary driver of alpha-gal IgE sensitization in the USA.^4–8 Accordingly, AGS cases have a regional distribution and are most frequently recognized in southeastern states where A. americanum ticks are established.^{7, 9} Reactions in AGS are typically delayed between 2 and 6 hours after the oral consumption of mammalian meat or dairy, with symptoms ranging from mild gastrointestinal (GI) distress to severe anaphylaxis.^{10, 11}

Recent reports have raised the possibility that in areas where alpha-gal sensitization is prevalent, AGS could be an unrecognized cause of some cases labeled as “irritable bowel syndrome”.^12–14 Two studies have also suggested that alpha-gal sensitization may be an independent risk factor for coronary artery disease (CAD).^{15, 16} Mechanistic studies are lacking, but a working model to explain a CAD association posits that subclinical inflammation in coronary arteries occurs in sensitized individuals who have ongoing exposure to alpha-gal via dietary intake of mammalian meat and/or dairy.¹⁷ Accordingly, we have hypothesized that there are many individuals in our community who are sensitized to alpha-gal but lack overt allergic symptoms and therefore do not specifically avoid mammalian meat or dairy. To date there has been little investigation of alpha-gal sensitization, symptomatic AGS prevalence, and information about mammalian meat and dairy consumption in unselected adult populations. Open questions also remain about the influence of factors such as age, race, ABO blood group and traditional atopy on the risk of developing AGS. Here we sought to investigate these questions in a COVID-19 vaccine employee cohort recruited at the University of Virginia without regard to allergic disease. As a validation cohort for local sensitization prevalence, IgE to alpha-gal was assessed in patients seen in a COVID-19 follow-up clinic.

METHODS

COVID-19 Vaccine Cohort

This cohort study was approved by the University of Virginia (UVA) institutional review board (IRB), and all participants provided verbal and written consent. Adults employed or affiliated with UVA, largely healthcare workers, were recruited between 2020 and 2022 by flyer and email to participate in a study because of interest in learning about their anti-SARS-CoV-2 IgG antibodies following COVID-19 vaccines, as previously described.^{18, 19} In the Fall of 2021, the study was expanded to include adults from the greater Charlottesville community. Inclusion criteria for the current analysis includes all subjects who provided a blood sample, received at least one COVID-19 vaccine, and completed a questionnaire with demographic, medical, and dietary history. Many of the subjects participated in more than one study visit. After the initial wave of enrollment between December 2020 and March 2021, the questionnaire was modified to include more detailed history about diet, post-prandial GI symptoms, and tick bite history (see online supplement). There were no inclusion or exclusion criteria related to preexisting comorbidities, including allergic conditions. Blood samples were processed and serum was isolated and banked at −30 °F before being assayed. For most subjects, alpha-gal IgE was assayed in samples collected at the day 21 post-booster timepoint. For subjects recruited >21 days after their primary vaccine series, the first serum sample available was assayed.

COVID-19 Patient Cohort

Adult COVID-19 patients were recruited within the UVA hospital and from a COVID-19 outpatient follow-up clinic as part of a UVA-IRB approved study, as previously described.²⁰ At least one serum sample was obtained from each subject after COVID-19 infection and banked in the UVA serum biorepository at −30°C before being assayed. The first serum sample received from each patient was screened for alpha-gal IgE antibodies. Information regarding age, sex, race, and COVID-19 severity was obtained for all subjects.

IgE and ABO Blood Type Assays

Total and specific IgE (sIgE) antibodies were measured by ImmunoCAP 250 (Thermo Fisher/Phadia, Waltham, Massachusetts) using commercially available reagents and expressed in IU/mL Common inhalant (dust mite [Dermatophagoides pteronyssinus], oak tree, Timothy grass, ragweed), food (egg, wheat, peanut, shrimp) and venom (honey bee and yellow jacket) allergens were assayed to characterize atopic status, with atopy defined as sIgE ≥0.35 IU/mL to one or more of these allergens. ABO blood group status was assessed by reverse-typing using commercially available reference cells (Bio-Rad, Dreieich, Germany) according to the manufacturer’s instructions.

Statistical Analysis

Alpha-gal sensitization was defined as alpha-gal sIgE ≥0.1 IU/mL and AGS cases were defined as self-report of mammalian meat allergy in conjunction with an alpha-gal sIgE ≥0.1 IU/mL. Positive antibody levels were expressed as geometric means (GM) with 95% confidence interval. Mann-Whitney U test was used to compare continuous data between two groups. Categorical variables were compared with Pearson’s χ² test. Factors associated with alpha-gal sensitization were assessed using logistic regression. Optimal alpha-gal sIgE and alpha-gal sIgE/total IgE ratio cutoffs were determined by receiver operator characteristic (ROC) analysis using the Youden Index. Proportional confidence intervals were calculated with the Wald method. Odds ratios were determined with univariable (OR) and multivariable (aOR) logistic regression. Statistical analysis was performed with GraphPad Prism 8 (GraphPad Software, San Diego California).

RESULTS

COVID-19 Vaccine and Patient Cohorts

Of the 267 participants in the COVID-19 vaccine study, 202 (76%) were female, the median age was 42 (IQR 31–55), and 216 (81%) were White (Table 1). Most subjects (n=241, 90.3%) were recruited before the inclusion criteria was expanded to include community members who may not have been affiliated with UVA. Overall, the cohort was not enriched for atopic individuals on the basis of total IgE levels (GM 22.5 IU/mL [95%CI 18.6–27.2]), self-report of allergic disease or IgE testing to a panel of inhalants, foods and stinging insects (Figure 1). Allergic rhinitis was reported by 29%, atopic dermatitis by 7.1%, asthma by 7.5% and recurrent hives by 4.1%. Interestingly, 5 subjects, equating to 2% of the cohort, reported eosinophilic esophagitis. IgE to alpha-gal (cut-off ≥ 0.1 IU/mL) was detected in 43 (16% [95% CI 12.2–21]) subjects in their screening sample. Mammalian meat allergy was reported by 8 (3%) subjects, 7 of whom were sensitized to alpha-gal. Accordingly, 16% (95%CI 7.8–30) of the sensitized subjects reported symptoms consistent with AGS, equating to a frequency of symptomatic AGS of 2.6% (95%CI 1.2–5.4) among this population.

Table 1.

Demographics and baseline characteristics of the COVID-19 vaccine cohort.

Characteristics of Vaccine Cohort	Total Cohort (n=267)	Alpha-gal IgE ≥0.1 IU/mL (n=43)	Alpha-gal IgE <0.1 IU/mL (n=224)	P
Age, median (IQR)	42 (31–55)	60 (44–68)	40 (31–52)	<0.001
Sex, Females, n (%)	202 (75.7%)	31 (72.1%)	171 (76.3%)	0.55
Race and ethnicity				0.05
White, n (%)	216 (80.9%)	42 (97.7%)	174 (77.7%)
Black, n (%)	18 (6.7%)	0 (0.0%)	18 (8.0%)
Hispanic, n (%)	6 (2.3%)	0 (0.0%)	6 (2.7%)
Asian, n (%)	26 (9.7%)	1 (2.3%)	25 (11.2%)
Other, n (%)	1 (0.4%)	0 (0.0%)	1 (0.5%)
Total IgE (IU/mL), GM [95% CI]	22.5 [18.6–27.2]	48.8 [30.7–77.5]	18.7 [15.1–23.2]	<0.001
Mammalian Meat Allergy, n (%)	8 (3.0%)	7 (16.3%)	1 (0.5%)	<0.001
Allergic Rhinitis, n (%)	76 (28.5%)	14 (32.6%)	62 (27.7%)	0.52
Atopic Dermatitis, n (%)	19 (7.1%)	2 (4.7%)	17 (7.6%)	0.49
Recurrent Hives, n (%)	11 (4.1%)	1 (2.3%)	10 (4.5%)	0.52
Asthma, n (%)	20 (7.5%)	4 (9.3%)	16 (7.1%)	0.62
EoE, n (%)	5 (1.9%)	1 (2.3%)	4 (1.8%)	0.81
COVID infection, n (%)	43 (16.1%)	8 (18.6%)	35 (15.6%)	0.63
Eats Red Meat Regularly, n (%)	209 (78.3%)	31 (72.1%)	178 (79.5%)	0.28
Eats Dairy Regularly, n (%)	246 (92.1%)	41 (95.3%)	205 (91.5%)	0.39

Figure 1.

Levels and prevalence of IgE to inhalant, food and stinging insect allergens. Geometric Mean with 95% Confidence Interval for all values ≥0.1 IU/mL, compared using Mann-Whitney U Test. Prevalence of specific IgE ≥0.35 IU/mL, expressed as percentage and compared using Pearson’s χ² test.

Of the 125 COVID-19 patients, 46% were female and the median age was 55 (IQR 45–67) (Table 2). This cohort was more diverse than the vaccine cohort, with 52% of the patients White, 19% Black and 25% Hispanic. Nearly all (85%) of the patients were hospitalized for COVID-19 before recruitment, with 65% having been hospitalized in the intensive care unit. Questionnaire information on medical and dietary history was not available for many of these patients, however, total IgE levels (GM 27.6 [95% CI 20.2–37.6]) suggest this cohort was not highly atopic. Alpha-gal sIgE was detected in 18 of these patients (14%). Combining the 392 individuals studied in the two cohorts equated to a sensitization prevalence of 15.6% (95% CI 12.3–20%).

Table 2.

Demographics and characteristics of the COVID-19 patient cohort

Characteristics	Total Cohort (n=125)	Sensitized (n=18)	Non-sensitized (n=107)	P
Age, median (IQR)	55 (45–67)	62 (44.75–67.25)	54 (45–66)	0.38
Sex, Females, n (%)	57 (45.6%)	2 (11.1%)	55 (51.4%)	0.002
Race and ethnicity
White, n (%)	64 (51.6%)	9 (50.0%)	55 (51.4%)	0.49
Black, n (%)	24 (19.2%)	2 (11.1%)	22 (20.6%)
Hispanic, n (%)	31 (24.8%)	7 (38.9%)	24 (22.4%)
Asian, n (%)	2 (1.6%)	0 (0.0%)	2 (1.9%)
Other, n (%)	4 (3.2%)	0 (0.0%)	4 (3.7%)
Total IgE (IU/mL), GM [95% CI]	27.6 [20.2–37.6]	90.7 [40.5–202.8]	22.6 [16.3–31.3]	0.002
Hospitalized, n (%)	106 (84.8%)	15 (83.3%)	91 (85.1%)	0.85
Admitted to ICU, n (%)	81 (64.8%)	12 (66.7%)	69 (64.5%)	0.86
On Mechanical Ventilation, n (%)	61 (48.8%)	9 (50.0%)	52 (48.6%)	0.91

Investigating Risk Factors for Alpha-Gal IgE Sensitization Vaccine cohort

In the vaccine cohort (Table 1), sensitized subjects were older (median 60 [IQR 44–68] years) than non-sensitized subjects (median 40 [IQR 31–52] years), P <0.001. Alpha-gal sensitization rates were similar among men and women, but varied by race and ethnicity (P=0.05). White participants represented 98% of the sensitized group versus 78% of the non-sensitized group, whereas there were no Black participants who were sensitized compared to 8% in the non-sensitized group. Total IgE levels were higher in alpha-gal sensitized subjects (GM 48.8 [95% CI 30.7–77.5]) compared to non-sensitized subjects (GM 18.7 [95% CI 15.3–23.2]), P <0.001. Additional risk factors for alpha-gal sensitization were investigated in a subset of the cohort who filled out a detailed questionnaire on exposures and co-morbidities (n=207) (Table 3). Tick and/or chigger bites were significantly associated with alpha-gal sensitization using three separate questions: i) any history of tick or chigger bites, ii) bites in the past 3 years and iii) history of itchy bites with a bite in the past 3 years. We included the word chigger in the questionnaire because our experience suggests that many patients in this area mistake bites from larval stage seed ticks with bites from trombiculid “chigger” mites and because it remains plausible that chiggers could also drive alpha-gal sensitization²¹. Expression of the B antigen blood group (i.e. – individuals with either B or AB blood type) was less frequent among sensitized (12%) versus non-sensitized subjects (22%), but this did not achieve statistical significance (P =0.15). As defined by IgE ≥0.35 IU/mL to one or more of the ten allergens tested, atopy trended but was not significantly more common in alpha-gal sensitized (56%) versus non-sensitized (41%) subjects, P =0.08. Sensitization to insect venom (32% versus 8%, P <0.001), and to a lesser degree food (17% versus 8%, P =0.07), but not inhalants (37% versus 36%, P =0.96), was associated with alpha-gal sensitization. Analysis of individual allergen responses revealed that IgE to peanut, shrimp and yellow jacket were significantly over-represented among alpha-gal sensitized subjects (Figure 1; Supplemental Table E1). Logistic regression modeling accounting for age, sex, race and ethnicity, tick/chigger bites, ABO blood group and atopy revealed that age, White race, and a history of itchy bites in the past 3 years were factors significantly associated with alpha-gal sensitization (Table 4).

Table 3.

Risk factors for alpha-gal sensitization.

Risk Factor	Total group (n = 207)	Alpha-gal IgE ≥0.1 IU/mL (n=41)	Alpha-gal IgE <0.1 IU/mL (n=166)	P
Tick Bites
Any history of tick or chigger bites, n (%)	134 (64.7%)	37 (90.2%)	97 (58.4%)	<0.001
Tick or chigger bites in past 3 years, n (%)	86 (41.6%)	31 (75.6%)	55 (33.1%)	<0.001
Itchy tick or chigger bites and in past 3 years, n (%)	56 (27.1%)	28 (68.3%)	28 (16.9%)	<0.001
Blood Group
B or AB Blood Group, n (%)	42 (20.3%)	5 (12.2%)	37 (22.3%)	0.15
Atopy
IgE to any allergen*, n (%)	91 (44.0%)	23 (56.1%)	68 (41.0%)	0.08
IgE to 1 or more inhalant allergens, n (%)	75 (36.2%)	15 (36.6%)	60 (36.1%)	0.96
IgE to 1 or more food allergens, n (%)	20 (9.7%)	7 (17.1%)	13 (8.0%)	0.07
IgE to 1 or more venom allergens, n (%)	27 (13.0%)	13 (31.7%)	14 (8.4%)	<0.001

^*cut-off of ≥0.35 IU/mL for IgE to panel of inhalant (dust mite [D. pteronyssinus], oak tree, Timothy grass, ragweed), food (egg, wheat, peanut, shrimp) and venom allergens (honey bee and yellow jacket)

Table 4.

Univariate and multiple variable regression analysis of risk factors for alpha-gal sensitization.

Risk Factors for alpha-gal sensitization	Univariate Model OR [95% CI]	Multivariable Model aOR* [95% CI]
Age	1.08 [1.05–1.11]	1.06 [1.03–1.09]
Sex [M]	1.30 [0.59–2.74]	0.76 [0.25–2.11]
Race [W]	12.3 [2.54–221]	5.43 [1.02–101]
Atopy	1.84 [0.93–3.71]	2.30 [0.97–5.63]
Itchy Tick or Chigger Bitesa	10.6 [5.00–23.7]	6.58 [2.84–15.81]
B or AB Blood Group	0.48 [0.16–1.23]	0.46 [0.12–1.47]

^*included age, sex, race, atopy, tick/chigger bite and ABO blood group in the model

Patient Cohort

In the patient cohort (Table 2), age was not significantly different between sensitized and non-sensitized subjects, but sensitization was disproportionally greater in men (23.5%) than women (3.5%), P=0.002. Evaluation by race and ethnicity revealed alpha-gal sensitization rates trended higher in Hispanic subjects (23%) than in White (14%) or Black (8%) subjects, but this was not statistically significant. As in the vaccine cohort, total IgE was higher in the sensitized than the non-sensitized group (91 IU/mL vs 23 IU/mL, P=0.002)

Diet, Gastrointestinal Symptoms and Cardiovascular Disease in Relation to Alpha-Gal IgE

We next sought to investigate diet and also GI and CVD co-morbidities in relation to alpha-gal status in the vaccine cohort (Supplemental Table E2). Over 70% of the vaccine cohort routinely consumed both mammalian meat and dairy, and the frequency was similar between sensitized and non-sensitized participants. Among the 207 subjects who completed questionnaires asking detailed information regarding GI symptoms following meals (including abdominal pain, heartburn, bloating, nausea, vomiting, and diarrhea), there was no association between GI symptoms (or diagnoses) and alpha-gal sensitization, though antacid use ≥ once per week was significantly associated with alpha-gal sensitization (OR 6.9 [95%CI 1.9–28.4]) (Supplemental Table E3). No association was seen for alpha-gal IgE and the CVD comorbidities of coronary artery disease, diabetes or hypertension, and no CVD medications had significant association with alpha-gal IgE with the exception of statin use (OR 4.0 [95%CI 1.5–10.4]). In multiple variable regression modeling accounting for age, sex, and race, the use of statins was no longer significantly associated with alpha-gal sensitization (aOR 0.9 [95%CI 0.3–2.9]), however the association with frequent antacid use remained significant (aOR 7.1 [95%CI 1.5–38.4]).

Alpha-gal IgE levels in sensitized individuals with and without symptoms

Reported AGS symptoms in the vaccine cohort included anaphylaxis, hives, GI symptoms, and one subject with isolated GI symptoms. There were no significant demographic differences between the seven subjects with AGS and those who were sensitized but asymptomatic (Supplemental Table E4). Geometric mean alpha-gal sIgE levels in symptomatic AGS cases were 9.0 IU/mL [95% CI 2.8–29.4], compared to 0.5 IU/mL [95% CI 0.3–0.7] among the asymptomatic group, P <0.001 (Figure 2A). Total IgE trended, but was not significantly higher among AGS cases (Figure 2B). Accordingly, alpha-gal/total IgE ratios were also significantly higher among AGS cases, P <0.001 (Figure 2C). ROC analysis revealed area under the curve of 0.94 [95%CI 0.86–1.00] for alpha-gal sIgE levels and 0.92 [95% CI 0.85–1.00] for alpha-gal sIgE/total IgE ratios (not shown). In this cohort, optimal performance characteristics for symptomatic AGS was a cut-off of ≥1.5 IU/mL for alpha-gal sIgE (sensitivity 100% and specificity of 86%) and a cutoff of ≥3.4% for alpha-gal sIgE/total IgE (sensitivity of 100%, specificity of 78%) (Figure 2D and E).

Figure 2:

Alpha-gal sIgE levels (A), total IgE levels (B), and alpha-gal sIgE/Total IgE ratios (C) compared between symptomatic and asymptomatic but sensitized patients. Optimal cutoffs of alpha-gal sIgE (D) and alpha-gal sIgE/total IgE (E) ratios determined by sensitivity and specificity analysis.

DISCUSSION

By investigating adults recruited for reasons related to the COVID-19 pandemic and without any inclusion criteria related to allergic disease or tick bite history, this report addresses important knowledge gaps in our understanding of AGS in an area of the US where the syndrome is known to be common.¹⁰ To date there have been very few studies that have described AGS prevalence. Thompson et al. used national testing results to confirm the strong geographic distribution of AGS cases in the southeast, and, while acknowledging the likelihood of underestimation, they also provided county-level prevalence estimates.²² Their observation of a peak AGS rate of 1.2% in a county in south-central VA (Charlotte County) generally supports our finding that AGS prevalence was 2.6% (95%CI 1.2–5.4) in the greater Charlottesville area.

The association of tick bites with alpha-gal sensitization was not unexpected based on current understanding of AGS, including data from other regional and international cohorts, and studies in animal models.²³ The fact that the strongest association was for pruritic tick bite history (aOR 6.6 [95%CI 2.8–15.8]) likely reflects the relevance of a local immune response at the bite site in inducing or enhancing IgE production. In the vaccine cohort we found that increased age was associated with alpha-gal sensitization and a similar trend was observed in the patient cohort. This aligns with findings from population-based studies in Europe as well as the report by Thompson et al. in the USA,^{22, 24–26} though this was not seen in a high-risk cohort in Germany.²⁷ It could well be that an age association is explained by a higher cumulative number of tick bites that can occur over time, an effect that would be less apparent in populations selected for frequent tick exposures. Regardless of the explanation, this is distinct from most respiratory and food allergies, where sIgE levels tend to wane with age.²⁸

Evaluating the relationship between sex and alpha-gal sensitization we found discrepant results between the two cohorts. Male sex was associated with sensitization in the patient but not the employee cohort. The explanation for this discrepancy is not clear, but we would highlight that there are inconsistent findings relating to sex and alpha-gal sensitization in the literature. For example, a cross-sectional study of adults in Denmark (n=2297) and Spain (n=444) found a higher frequency of alpha-gal sensitization in males than females in Denmark, but not Spain²⁴. Investigation of a large military cohort revealed an association between male sex and new-onset alpha-gal sensitization, but when accounting for military occupation the finding lost significance.²⁹ Thus, differences in alpha-gal sensitization by sex observed in many cross-sectional cohorts may well be explained by differences in occupational or recreational exposures to ticks.

We also found that alpha-gal sensitization varied by race and ethnicity. Of note, Black subjects had relatively low rates of alpha-gal sensitization in both the employee (0%) and patient (8.3%) cohorts. The explanation behind this is not clear, but is consistent with a recent investigation of alpha-gal sensitization in military personnel³⁰ and is also supported by a recent case-control study of AGS in North Carolina⁵. Given the homology between alpha-gal and B blood group antigen and the higher incidence of B blood group among Black individuals, ABO blood group is an important consideration^31–34. However, associations of alpha-gal sensitization with race and ethnicity persisted in models accounting for ABO blood group, suggesting that differences in urban/rural residence, occupation and recreational activities could be more relevant factors. To our knowledge, there has been little investigation of AGS in Hispanic populations. There were very few Hispanic individuals in the vaccine cohort but in the COVID-19 patient cohort, which was 25% Hispanic, we observed a trend toward increased alpha-gal sensitization in Hispanic as compared to non-Hispanic White or Black subjects. We speculate that this trend could reflect occupational risk of tick exposure, particularly for Hispanic men, in which construction and grounds maintenance are among the top occupations^{35, 36}. Interestingly, all Hispanic patients who were sensitized to alpha-gal were men.

Here we observed that B blood group was less frequent among alpha-gal sensitized subjects, but this did not reach statistical significance. These findings were similar to results from a case-control investigation of AGS in Virginia we previously reported and also a study in Germany^{10, 37}, but differ from findings reported by other groups where B blood group was inversely associated with AGS.^{32, 34} The explanation for the discrepant findings is not clear, but suggests that systematic investigation in larger cohorts is warranted.

Investigators have reached varying conclusions about the relevance of traditional atopy as a risk factor for developing AGS.^{26, 38, 39} Here we found that total IgE was significantly higher among sensitized subjects, though this did not extend to an increased level or frequency of IgE to common inhalant allergens, nor self-report of asthma or allergic rhinitis. Among the 10 allergens tested, only IgE to peanut, shrimp and yellow jacket had individual associations with alpha-gal sensitization. The relationship with yellow jacket could be explained by a shared environmental risk for exposure to stinging insects and ticks, though there is also evidence for homology between some tick and insect antigens.⁴⁰ The connection with peanut is interesting in view of the fact that, like alpha-gal, peanut sensitization is often thought to occur through the skin.⁴¹ Overall, our interpretation of this data is that the higher levels of total IgE seen here and in many other AGS cohorts is not explained by a high burden of traditional atopy (i.e. – a tendency to make IgE to common respiratory or food allergens) among those sensitized to alpha-gal, but rather that higher total IgE levels likely reflect a history of bites and stings in the skin which robustly promote IgE.⁴²

Most of the alpha-gal sensitized subjects did not report a history of meat allergy despite frequently consuming mammalian meat and dairy. This observation is consistent with results from a high-risk cohort in Germany and a colorectal screening cohort in North Carolina^{27, 43}. It is also consistent with other forms of food sensitization where population-based investigation has revealed that IgE sensitized individuals very often consume foods to which they make IgE⁴⁴. While these data depart from findings in mice where food-specific IgE promote aversion and food avoidance⁴⁵, they could have implications related to two recent reports suggesting that alpha-gal IgE sensitization is a risk factor for CAD^{15, 16}. We have hypothesized that repeated exposure to dietary alpha-gal, while often insufficient to cause recognizable allergy symptoms in individuals with low-level alpha-gal sIgE, could nonetheless lead to CAD by promoting chronic sub-clinical inflammation in sensitized individuals.¹⁷ We did not observe any association between alpha-gal sIgE and CVD or CVD-related diseases in this cohort, but this study was not powered to capture rare events in a relatively young and healthy adult population. Accordingly, we anticipate that large studies assessing the interaction between alpha-gal sIgE and diet with prospective monitoring would be needed to address this question.

Multiple investigators have recently reported on AGS cases presenting with isolated GI symptoms^{12, 13, 46, 47}. Although many of these cases presented with discrete episodes of severe pain after ingestion of red meat, it has been proposed that, in areas where lone star tick bites are common, alpha-gal sIgE could be a potential cause of chronic GI symptoms that otherwise masquerade as “Irritable Bowel Syndrome” (IBS)^{14, 46}. In the current cohort we did not find associations between alpha-gal sIgE sensitization and any GI co-morbidities (including heartburn), though frequent antacid use was more common among those who were sensitized. These findings are similar to those observed in a high-risk group of 46 forestry workers in Kentucky where antacid use more than one time per week (and also heartburn more than one time per week) were associated with alpha-gal sensitization.⁴⁸ Our interpretation of this data is that AGS is unlikely to be a major cause of IBS-like symptoms at a community level (even in areas where sensitization prevalence is high), but can nonetheless be a cause of isolated GI distress in select patients. We think it is less likely, but it is also plausible that antacid use could lead to alpha-gal sensitization via perturbations in the GI tract.

A strength of this study is that we obtained alpha-gal symptom questionnaire data with no a priori knowledge of subjects’ alpha-gal sensitization status, which allowed us to investigate diagnostic thresholds of alpha-gal sIgE and sIgE/total IgE ratios to distinguish symptomatic and asymptomatic sensitized subjects. In line with previous reports, higher alpha-gal IgE levels and sIgE/total IgE ratios were strongly associated with symptomatic AGS. Optimal Youden’s cut-offs for predicting symptomatic AGS (sIgE level of ≥1.5 IU/mL; sIgE/total IgE ratio of ≥3.4%) aligned reasonably well with previously reported optimal alpha-gal IgE cut-offs of ≥0.54 IU/mL and ≥2.0 IU/mL.^{5, 49, 50} While our sample size is relatively small for this ROC analysis, our serologic and questionnaire findings confirm that low-level alpha-gal sIgE often does not lead to clinically appreciable symptoms.

There are several limitations to consider. We did not have detailed information on medical, diet and allergy history in the COVID-19 patient cohort, but this group was still relevant as a replication cohort for our observation about alpha-gal sIgE prevalence in this region of Virginia. The employee vaccine cohort was mostly female and White and was working age. These factors and also socio-economic considerations that were not captured in our questionnaire may limit the generalizability to the community as a whole. Accordingly, we acknowledge our prevalence estimates to be approximations. We also did not carry out challenges to confirm symptomatic AGS, though such challenges are not commonly done given the delay in symptom onset. For some subjects detailed questions about diet and symptoms occurred only at a follow-up visit, so any changes that occurred in these variables between enrollment and follow-up may not have been captured. This study was not designed to prove or disprove our hypothesis about connections between alpha-gal sensitization and CVD, but did provide convincing evidence that many individuals in our community routinely consume mammalian meat and dairy despite being sensitized to alpha-gal.

In conclusion, investigating healthcare workers and community members in central Virginia recruited as part of COVID-19 studies we found a prevalence of alpha-gal sensitization of ~15% and the prevalence of symptomatic AGS of ~2.6%. Alpha-gal sensitization was associated with tick bite history, higher total IgE and older age, but not traditional atopic conditions or inhalant sensitization. The majority of alpha-gal sensitized subjects had low-level sIgE, consumed mammalian meat and dairy without discernable symptoms, and did not disproportionally carry diagnoses of IBS. Alpha-gal IgE testing is critical to confirm an AGS diagnosis in patients with relevant clinical history. On the other hand, providers should be aware that many members of the community can be sensitized to alpha-gal but nonetheless tolerate mammalian meat and dairy without obvious allergic symptoms.

HIGHLIGHTS.

What is already known about this topic?

IgE to alpha-gal is an important cause of a tick-acquired mammalian meat allergy, often referred to as the alpha-gal syndrome (AGS), but questions about AGS prevalence, risk factors and presentation remain incompletely understood.

What does this article add to our knowledge?

In this study of adults recruited without regard to allergic disease in a region of Virginia where lone star ticks are established alpha-gal sensitization prevalence was 16% and symptomatic AGS prevalence was 2.6% (95%CI 1.2–5.4).

How does this study impact current management guidelines?

In central Virginia, and likely other parts of the country where lone star ticks are established, AGS should be appreciated as a major cause of adult food allergy.

Abbreviations:

Alpha-gal

galactose-alpha-1,3-galactose

AGS

alpha-gal syndrome

CAD

coronary artery disease

COVID-19

coronavirus disease 2019

CVD

cardiovascular disease

EoE

eosinophilic esophagitis

GI

gastrointestinal

GM

geometric mean

IBS

irritable bowel syndrome

IgE

Immunoglobulin E

IRB

institutional review board

OR

odds ratio

sIgE

specific IgE

UVA

University of Virginia