Abstract

Background

Over the past two decades, the prevalence of individuals diagnosed with autism spectrum disorder (ASD) has risen dramatically [1], and currently at least 1 in 88 children are diagnosed with ASD in the US [2]. ASD is defined by standardized criteria of qualitative impairments in social interaction, qualitative impairments in communication, and restricted and stereotyped patterns of behavior, interests, and activities [3]. Although these core features define an ASD, recent investigations have described many health, physical, or behavioral co-morbid conditions consistently associated with an ASD such as gastrointestinal disturbances, incontinence, sleep problems, eating disorders, behavioral problems, and sensory processing issues [4].

In further considering the initial clinical presentation of ASD, a significant number of children diagnosed with ASD suffer a loss of previously-acquired skills between 6 and 18 months of age with an estimated incidence of regression among individuals diagnosed with ASDs ranging from 15% to more than 62% of all cases [5]. The importance of postnatal loss of neurological function in those diagnosed with an ASD between 6 and 18 months of age, as observed in affected children who have regressed, is this phenomena is suggestive of neurodegeneration or a type of progressive encephalopathy with an etiological pathogenic basis occurring after birth [5]. To date, the etiology of ASD remains under debate. However, many studies suggest toxicity in individuals diagnosed with an ASD [6]. More specifically, recent evidence suggests that mercury (Hg) may be either a causal factor in or contributory to the brain pathology in ASD, possibly working synergistically with other toxic compounds or pathogens to produce the abnormal brain pathology observed in those diagnosed with an ASD [7].

Hg is a heavy metal that is widespread and persistent in the environment, and infants in the US are exposed to significant levels of environmental Hg through the air, water, and breast milk [8]. In addition to environmental Hg exposure and maternal exposures from the mother’s Hg body burden, dietary intakes, and Hg-containing pharmaceuticals administered to the mother while the child is developing in utero, and injected organic-Hg from Thimerosal-containing childhood vaccines that have been and, in many countries, remain a significant source of Hg exposure for many infants during the first six months of life.

Thimerosal is an organic-Hg compound (49.55% Hg weight) added to vaccines as a preservative, typically at concentrations from 0.005% to 0.01% (12.5 μg Hg or 25 μg Hg per 0.5 mL vaccine dose) [9]. Thimerosal is known to rapidly dissociate into ethyl-Hg chloride, ethyl-Hg hydroxide, and sodium thiosalicylate in saline solutions. As a result of adherence to the recommended routine childhood vaccination schedule in the US during the 1990s infants may have been exposed to bolus doses of Hg nominally ranging from 12.5 μg Hg to 62.5 μg Hg that collectively added up to nominally 200 μg Hg from Thimerosal-containing childhood vaccines during the first six months of life (3 Diphtheria-Tetanus-Pertussis vaccines with 25 μg Hg per dose + 3 Haemophilus influenzae type B vaccines with 25 μg Hg per dose + 3 hepatitis B vaccines with 12.5 μg Hg per dose + 1 influenza vaccine with 12.5 μg Hg per dose, > 50% of all Hg exposure when considering environmental sources of Hg). This dosing pattern continues unabated in many developing nations to the present day. Moreover, despite a call for the removal of Thimerosal from all vaccines in the United States on July 7, 1999 by the American Academy of Pediatrics and United States Public Health Service [10], many children in the US continue to receive significant doses of Hg from the routinely recommended administration of Thimerosal-containing influenza vaccines (where more than 50% of all doses of influenza vaccine continue to contain 0.01% Thimerosal) to pregnant women, infants, and young children [9].

The purpose of the present study was to evaluate concerns about the toxic effects of organic-Hg exposure from Thimerosal in childhood vaccines by conducting a two-phased study with documented exposure to varying levels of Thimerosal from vaccinations. In the first phase, a hypothesis generating study was undertaken to evaluate the relationship between exposure to organic-Hg from a Thimerosal-containing Diphtheria-Tetanus-acellular-Pertussis (DTaP) vaccine in comparison to a Thimerosal-free DTaP vaccine for the risk of ASD in the Vaccine Adverse Event Reporting System (VAERS) database (phase I). In the second phase, a hypothesis testing study was undertaken to evaluate the relationship between organic-Hg exposure from Thimerosal-containing hepatitis B vaccines administered at specific intervals and the risk of ASD in the Vaccine Safety Datalink (VSD) database (phase II).

Methods

Results

Discussion

The present study evaluated the potential relationship between organic-Hg exposure from Thimerosal-containing childhood vaccines and ASD using a two-phase study design. In the first phase, the VAERS database was analyzed for hypothesis generation, and it revealed a significant association between increasing organic-Hg exposure from Thimerosal-containing DTaP vaccine administration in comparison to Thimerosal-free DTaP vaccine for the risk of ASD. In the second phase, the VSD database was analyzed for hypothesis testing, and it revealed that those cases diagnosed with an ASD were at significantly greater risk of exposure to increasing doses of organic-Hg from Thimerosal-containing hepatitis B vaccines administered at three specific intervals within the first six months of life in comparison to controls.

The overall two-phased epidemiological method to evaluate vaccine safety using the VAERS for hypothesis generation (phase I) and the VSD for hypothesis testing (phase II) was previously described by investigators from the CDC and the FDA [16–18]. Specifically, these investigators described that products, such as vaccines, which are intended for healthy people, must be held to a high standard of safety assurance. However, the study of vaccine risks is more complex than for therapeutic products because the exposure is virtually universal for many vaccines, ensuring the chance occurrence of many adverse outcomes in temporal association with vaccines. As a result, these investigators described a two-phased approach by first identifying vaccine safety risks through reports to VAERS (hypothesis generating), and then using the VSD, a consortium of managed care organization, to more rigorously evaluate vaccine-associated risks (hypothesis testing).

It is interesting to consider the specific methods employed to evaluate the potential relationship between organic-Hg exposure from Thimerosal-containing childhood vaccines and the risk of ASD in each of the two phases of the present study. Specifically, in phase I, differences in organic-Hg content were evaluated based upon the differences in Thimerosal content of two different DTaP vaccines manufactured by different companies (i.e., one used Thimerosal as a preservative and the other used 2-phenoxyethanol). Other than the difference in organic-Hg content based upon vaccine manufacturer, these vaccines were similar, being recommended for administration to US infants in the same vaccine schedule at 2, 4, and 6 months of age [19].

Specifically, in phase II, differences in cumulative doses of organic-Hg received at specific intervals during the infant period were evaluated based upon the wide-ranging recommendations for routine hepatitis B vaccine administration. In 1991, the Advisory Committee on Immunization Practices (ACIP) recommended that infants should receive their hepatitis B vaccine doses as follows: first dose between birth and 2 months of age, second dose between 1 and 4 months of age, and third dose between 6 and 18 months of age [20].

Importantly, all told, it is apparent that the differences in organic-Hg exposures observed in both phases of the present study were not the result of a small group of children receiving anomalous exposures to vaccines. Instead, in phase I, these differences were a function of vaccine composition by vaccine manufacturer. Alternatively, phase II assessed varying levels of organic-Hg exposure which resulted from the varying windows recommended for administration of hepatitis B vaccines during the first year of life.

The results observed in the present study also appear to offer important potential biological mechanistic insights into the relationship between the timing and cumulative effects of organic-Hg exposure from Thimerosal-containing vaccines and the risk of receiving a diagnosis of ASD. For example, the effects of later additional organic-Hg exposure from administration of a second or third Thimerosal-containing hepatitis B vaccine did not increase the risk of receiving a diagnosis of ASD in a dose-proportional manner (i.e., odds ratio for second dose of Thimerosal-containing hepatitis B vaccine = 4 and odds ratio for third dose of Thimerosal-containing hepatitis B vaccine = 6). Instead, the odds ratio of receiving an ASD diagnosis after receiving two doses of Thimerosal-containing hepatitis B vaccine (25 μg organic-Hg) by two months of age was similar to the of receiving one dose of Thimerosal-containing hepatitis (12.5 μg organic-Hg) by one month of age. The odds ratio for receiving an ASD diagnosis after three doses of Thimerosal-containing hepatitis B vaccine (37.5 μg organic-Hg) was increased relative to those children receiving one or two doses of Thimerosal-containing hepatitis B vaccine, but the increase observed was significantly below what which would be expected from a cumulative perspective without considering the variable of timing of administration. Our observation of odds ratio being modified by time of exposure is consistent with previous observations regarding other cases of Hg intoxication, in which early exposure is associated with more significant adverse effects (i.e., Hg intoxication susceptibility: fetuses > infants > children > adults) [21].

In addition, the results observed in the present study support the hypothesis that a significant number of children diagnosed with an ASD experience neurodegeneration or a type of progressive encephalopathy with an etiological pathogenic basis occurring after birth. This is the case because the source of exposure examined in the present study was from Thimerosal-containing childhood vaccines administered between birth and 6 months of age, and a significant number of children diagnosed with an ASD have been reported to suffer a loss of previously-acquired skills between 6 and 18 months of age [5]. Because those exposed to Thimerosal-containing vaccines have a statistically significant increased risk of being diagnosed with an ASD, and because administration of Thimerosal-containing vaccines examined in this study was administered between birth and 6 months of age, it is valid to suggest that a significant number of individuals examined in this study who were diagnosed with an ASD suffered neurodegeneration or a type of progressive encephalopathy for which the etiological pathogenic basis was exposure to organic-Hg from Thimerosal administered as a vaccine component.

Importantly, many recent studies support the biologically plausible role of organic-Hg exposure from Thimerosal-containing vaccines in the pathogenesis of ASD [9]. Investigators have examined the distribution of organic-Hg following administration of Thimerosal to animals and infants. For example, administering Thimerosal mimicking the US vaccine schedule of the 1990s to infant monkeys, researchers found that significant levels of Hg were present in the brain (about 40–50 parts-per-billion), and a significant fraction of the Hg was present as inorganic-Hg (about 16 parts-per-billion) that was observed to not significantly decline 120 days following the last dose of Thimerosal [22]. Other investigators undertook further evaluations of the speciation of Hg present in rat tissues following administration of Thimerosal [23]. Interestingly, these researchers observed that administration of Thimerosal resulted in significant brain levels of Hg with 63% present in the form of inorganic-Hg, 13.5% as ethyl-Hg, and, unexpectedly, 23.7% as methyl-Hg.

In addition, investigators observed that Thimerosal-containing vaccine administration to human infants significantly increased the vaccinated infants’ blood Hg levels (with some infants having total blood Hg levels in excess of the safety limits established by the US Environmental Protection Agency) and also significantly increased the vaccinated infants’ hair ethyl-Hg levels (with some infants having total hair Hg levels in excess of the safety limits established by the US Environmental Protection Agency of 1 part-per-million) [9]. Finally, in further research on the distribution of Hg species within the body, investigators recently demonstrated that ethyl-Hg is actively transported across neuronal cellular membranes to the same degree as methyl-Hg, by the L-type neutral amino acid carrier transport (LAT) system [24].

The presence of Hg within brain cells is significant because it can induce cellular damage consistent with the neuronal damage observed in the brains of individuals diagnosed with ASD, and this damage is dependent upon thiol availability, especially glutathione, in that inadequate thiol availability results in significantly greater cellular damage from Hg [7,25]. Many previous studies reveal children diagnosed with ASD have limited thiol availability and decreased glutathione reserve capacity, deficits which make them more susceptible to the toxic effects of Hg exposure from administration of Thimerosal-containing vaccines [9].

Studies have also evaluated the potential for organic-Hg exposure from Thimerosal-containing vaccine administration to induce ASD pathology or clinical symptoms in animal model systems. These studies have yielded significant pathology or clinical symptoms in mice, rats, hamsters, and monkeys that are consistent with those observed in ASD following exposure to Thimerosal-containing vaccines mimicking the 1990s US routine childhood vaccination schedule [9].

The results observed in the present study are also supported by a number of previous epidemiological studies finding an association between organic-Hg exposure from Thimerosal-containing vaccines and ASD, using several epidemiological methods in various databases. For example, Young et al., using an ecological study design, evaluated the relationship between the birth cohort prevalence of specific neurodevelopmental disorder and birth cohort exposures to Hg from Thimerosal-containing childhood vaccines in the VSD [26]. Consistent with the results observed in the present study, it was seen that infants receiving an additional 100 μg organic-Hg from Thimerosal-containing childhood vaccines from birth to 7 months of age, had a significantly increased risk ratio of 2.87 for ASD, and infants receiving an additional 100 μg organic-Hg from Thimerosal-containing childhood vaccines from birth to 13 months of age, had a significantly increased risk ratio of 2.62 for ASD.

As another example, Gallagher and Goodman evaluated the relationship between administration of Thimerosal-containing hepatitis B vaccine administration and the subsequent risk of individual being diagnosed with an ASD, based upon assessment of the National Health Interview Survey (NHIS) 1997–2002 data sets [27]. They reported that boys diagnosed with an ASD in comparison to controls had a 3-fold significantly greater odds ratio for receiving a Thimerosal-containing hepatitis B vaccine during the first month of life.

Previously, Geier and Geier reported on the results of a meta-analysis using statistical modeling to evaluate the relationship between exposure increased Hg doses from Thimerosal-containing vaccines and neurodevelopmental disorder adverse event reports in VAERS [28]. They reported that there was a 1.6-fold significantly increased risk of ASD adverse event reports submitted to VAERS following additional doses of organic-Hg from Thimerosal-containing vaccine administration.

The results of the present study differ from several other cohort studies that failed to find an association between ASD and organic-Hg exposure from Thimerosal-containing childhood vaccines [29–31]. This may have occurred, in part, because other studies examined cohorts with significantly different childhood vaccine schedules and with different diagnostic criteria for outcomes. This difference may have also occurred because these other studies employed different epidemiological methods, especially with respect to the issue of follow-up period for individuals in the cohorts examined. The method used to measure follow-up period for individuals is a critical issue in all studies examining the relationship between exposures and the subsequent risk of an ASD diagnosis. This is the case because the risk of an individual being diagnosed with an ASD is not uniform throughout his/her lifetime. As observed in the present study, the initial mean age for an ASD diagnosis was 4.2 years-old, and the standard deviation of mean age of the initial diagnosis of ASD was 1.54 years-old. Therefore, any follow-up method that fails to consider the lag-time between birth and age of initial ASD diagnosis will likely not be able to observe the true relationship between exposure and the subsequent risk of an ASD diagnosis.

This type of concern is apparent with respect to the study published by Hviid et al. [29]. These investigators reported on a population-based cohort study of all children born in Denmark from January 1, 1990, until December 31, 1996 comparing children vaccinated with a Thimerosal-containing vaccine formulation (early years of the study) with children vaccinated with a Thimerosal-free formulation (latter years of the study) of the same vaccine. In order to evaluate years of follow-up for individuals in the study, these investigators described them in terms of person-years. As a result, these investigators assumed equality of person-years of contribution to the study regardless the age of the individual, despite observing a similar mean age of an ASD diagnosis (4.7 ± 1.7 years-old) as the present study. The consequence in the Hviid et al. [29] study was that an out-of-proportion number of person-years of follow-up was observed for the older children who had received Thimerosal-containing vaccine formulation in comparison to the younger children vaccinated with a Thimerosal-free vaccine formulation, rendering the results of the study uninterpretable. Similar limitations apply to the cohort studies published by Andrew et al. [30] (median age of initial ASD diagnosis = 4.4 years-old) and Verstraeten et al. [31] (median age of initial ASD diagnosis HMO A = 49 months [4.08 years] and HMO B = 44 months [3.67 years]), which used hazard models (i.e., models which presume a constant relative hazard of ASD diagnosis over time) to evaluate follow-up of individuals in their respective examinations of the relationship between Thimerosal-containing vaccine exposures and the subsequent risk of an ASD diagnosis.

In order to further reveal the importance of follow-up period, the following example is informative. In the aforementioned Verstraeten et al. [31] study, the investigators examined a VSD dataset that shared significant overlap with the one examined in the present study. Verstraeten et al. [31] observed a risk ratio = 1.16 (95% confidence interval = 0.78-1.71) for ASD diagnosed among infants receiving 12.5 μg organic-Hg exposure from Thimerosal-containing vaccines administered in the first month of life. This observation is in contrast to the odds ratio = 2.18 (95% confidence interval = 1.74-2.73) observed in the present study when comparing cases to controls for receiving 12.5 μg organic-Hg exposure from Thimerosal-containing hepatitis B vaccine in comparison to receiving 0 organic-Hg exposure from Thimerosal-containing hepatitis vaccine administered in the first month of life. A further analysis of the dataset examined in the present study, using reduced lengths of continuous follow-up among controls presumed to be without an ASD diagnosis, revealed that if the length of continuous follow-up criteria was reduced to the mean age of initial ASD diagnosis + standard deviation of initial ASD diagnosis (5.7 years), the odds ratio = 1.69 (95% confidence interval = 1.35-2.12). Further, if the length of continuous follow-up criteria was reduced further among controls to only the mean age of initial ASD diagnosis (4.2 years), the odds ratio = 1.47 (95% confidence interval = 1.18-1.85). Finally, if the length of continuous follow-up criteria was reduced even further among controls to only the mean age of initial ASD diagnosis – standard deviation of initial ASD diagnosis (2.7 years), the odds ratio = 1.21 (95% confidence interval = 0.97-1.51), a value that, considering the confidence intervals, is consistent with the risk ratio = 1.16 (95% confidence interval = 0.78-1.71) observed in the Verstraeten et al. [31] study. Although the aforementioned odds ratios are still statistically significant (except for the last analysis requiring continuous follow-up criteria among controls to only the mean age of ASD diagnosis – standard deviation), the effect becomes less pronounced in each instance as increasing levels of ambiguity of diagnosis are introduced among controls due to the lack of appropriate follow-up.

Conclusion

The present study provides new epidemiological evidence supporting an association between increasing organic-Hg exposure from Thimerosal-containing childhood vaccines and the subsequent risk of ASD diagnosis. Using a two-phased, hypothesis-generating and hypothesis-testing, epidemiological analytical methodology in two separate databases, organic-Hg exposure from Thimerosal-containing childhood vaccines was determined to be associated with a subsequent diagnosis of an ASD. In addition, in phase II within the VSD, the present study placed special emphasis on requiring an adequate follow-up period in the analysis. Thus, the cases and controls were followed for a sufficient, evidenced-based interval of time, to ensure that they were appropriately classified with respect to their exposures and outcomes, thus helping to ensure the potential for a cause-and-effect relationship between exposure and outcome was not biased or confounded. Future studies should be completed to further evaluate the relationship between other sources of organic-Hg exposure from Thimerosal-containing childhood vaccines and other chronic disorders, and to further explore potential subpopulations and the timing of exposure to organic-Hg from Thimerosal-containing vaccine administration associated with adverse outcomes.

As previously described, routine childhood vaccination is an important public health tool to reduce the morbidity and mortality associated with infectious diseases. However, it is also a public health imperative to end the unnecessary addition of organic-Hg to vaccines in the form of Thimerosal used as a preservative, based on data showing an association between its administration and adverse outcomes.

Abbreviations

(ACIP): Advisory Committee on Immunization Practices; (ASD): Autism spectrum disorder; (BSS): Biologics surveillance summaries; (DTaP): Diphtheria-Tetanus-acellular-Pertussis; (IRB): Institutional Review Board; (KPC): Kaiser Permanente Colorado; (KPNW): Kaiser Permanente North-West; (KPNC): Kaiser Permanente Northern California; (Hg): mercury; (NHIS): National Health Interview Survey; (NIP): National Immunization Program; (CDC): US Centers for Disease Control and Prevention; (FDA): US Food and Drug Administration; (VAERS): Vaccine adverse event reporting system; (VSD): Vaccine safety datalink.

Competing interests

All of the investigators on the present study have been involved in vaccine/biologic litigation.

Authors’ contributions

DG was the main writer and analyzed data, BH was the main computer programmer and reviewed the manuscript structure, ideas and science, JK reviewed the manuscript structure, ideas and science, PG reviewed manuscript structure, ideas, and science, LK edited manuscript structure, and MG evaluated and reviewed manuscript ideas and science. All authors read and approved the final manuscript.

References