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.

Strengths/limitations

A strength of the present study involved examination of two separate cohorts of children from two separate databases (i.e., VAERS and VSD). The observations made in VAERS were based upon retrospective assessment of passively reported adverse events. In contrast, the VSD observations were made based upon retrospective assessment of prospectively collected medical records of patients enrolled in various HMOs. Despite these intrinsic differences in data-collection methods between the two databases, in both databases organic-Hg exposure from Thimerosal-containing vaccine administration was consistently associated with a subsequent diagnosis of an ASD.

The study design used to evaluate the relationship between exposure and outcome was another significant strength of the present study. In phase I of the study, adverse event reports in VAERS were analyzed. The method employed to examine VAERS ensured that the exposures to the various types of vaccines studied occurred prior to the outcomes described in the adverse event reports, since those reporting the subsequent adverse outcomes in association with the vaccines listed on the adverse event reports. In phase II, cases had to be enrolled from birth and were required to be continuously enrolled until a medical diagnosis of an ASD was made and controls had to be enrolled from birth for a sufficient time period to ensure that there was a very small chance that, during additional follow-up, any of the controls would be medically diagnosed with an ASD. As a result, any factors associated with enrollment (i.e., adjustment for potential independent variables between cases and controls were not necessary because enrollment was from birth) or healthcare-seeking behavior (i.e., adjustment for potential access/availability of healthcare was continuous among cases and controls) were minimized.

For cases diagnosed with an ASD, it was possible to mathematically evaluate the mean and standard deviation of age for initial ASD diagnoses within the VSD. From this information, it was possible to estimate how many additional potential diagnoses of ASD were missed. It was decided, a priori, to ensure adequate amounts of data for our analyses, that controls had to be continuously enrolled in the VSD from birth until they were at least 7.29 years-old (mean age of initial diagnosis of ASD + 2× standard deviation of mean age of initial diagnosis of ASD). Based on the data for age of initial diagnosis for ASD, this was a sufficient period to ensure that, with further follow-up, those controls without an ASD would probably not receive an ASD diagnoses in the VSD (mathematically there is a < 2.5% chance of these individuals being diagnosed with an ASD with additional follow-up time beyond 7.29 years). In addition, cases diagnosed with an ASD were specifically evaluated to ensure that only those cases diagnosed with an ASD following vaccine administration were considered in the present analyses.

Another strength of the study was that the VSD and VAERS data were collected independently of the study design used in the present study. The VSD data records analyzed, in particular, were collected as part of the routine healthcare individuals received as part of the participation with their respective HMOs, and as such, the healthcare providers in no way were thinking about the potential association between vaccine exposures and potential health outcomes.

However, the results of the present study may have a number of potential limitations. It is possible the results observed may have occurred from unknown biases or cofounders present in the datasets examined. This seems unlikely because other control outcomes (i.e. outcomes that are not biologically plausibly linked to postnatal organic-Hg exposure from Thimerosal-containing vaccines) were examined, such as a diagnosis of congenital anomalies (ICD-9 code: 759.9) in the VSD database, using the same methodology employed for ASD. No similar patterns of significant associations were observed as that found for organic-Hg exposure from Thimerosal-containing hepatitis B vaccine administration and the subsequent risk of ASD diagnosis. For example, exposure to 12.5 μg organic-Hg from Thimerosal-containing hepatitis B vaccines administered in the first month of life in comparison to 0 μg organic-Hg from Thimerosal-containing hepatitis B vaccines administered in the first month of life among cases and controls revealed similar risk of exposure (odds ratio = 1.03, p > 0.50). Similarly, adverse event reports with the control outcome symptom of febrile convulsion (VAERS code: 10016284) were searched for in VAERS using the same methodology employed for ASD. The results of that analysis failed to reveal a similar pattern of a significant association observed for organic-Hg exposure from Thimerosal-containing DTaP vaccine in comparison to Thimerosal-free DTaP vaccine administration and the risk of reported ASD. There were 48 adverse event reports with febrile convulsions reported to VAERS following administration of Thimerosal-containing DTaP vaccines in comparison to 41 adverse event reports with febrile conclusions reported to VAERS following administration of Thimerosal-free DTaP vaccines (risk ratio = 1.06, p > 0.50).

It is also possible that the VAERS and VSD databases examined in the present study may have limitations. As described previously, VAERS may have shortcomings, such as underreporting, difficulty in determining causal relationship, lack of precise denominators. Nevertheless, as previously described by investigators from the CDC, almost all of these types of shortcomings would apply equally to VAERS reports after vaccines administered to similar populations [32], such as the Thimerosal-containing and Thimerosal-free DTaP vaccines examined in the present study. As a result, the comparison of vaccines administered to similar populations should provide accurate relative qualitative and quantitative relationships between vaccine exposures and adverse outcomes [32]. The VSD may also have limitations because despite having vaccination and medical records linked by computer, the cohort in VSD is restricted to a limited population derived from several different participating HMOs. The validity of the databases examined in the present study is significantly supported by the consistency of the results observed in VAERS and VSD.

Another potential limitation of the present study is that the results observed may be the result of statistical chance. However, such a possibility would be unlikely given the limited number of statistical tests performed, the highly significant results observed (most p-values observed were < 0.01), and the consistency in the direction and magnitude of the results observed in two separate epidemiological databases.

Still, other potential limitations of the present study include the possibilities that some of the individuals in the cohorts in VAERS and VSD examined may have had more subtle neurological dysfunction that was not brought to the attention of their healthcare providers, healthcare providers may have misdiagnosed some individuals, or some vaccine exposures may not have been appropriately classified. These limitations, while possibly present in the data examined in the current study, should not have significantly impacted the results observed because it is unclear how differential application would have occurred to the study cohorts examined based upon the Thimerosal doses that the individuals received. Moreover, misclassification occurring in the data examined would tend to bias any results observed toward the null hypothesis, since such effects would result in individuals being placed in the wrong exposure and/or outcome categories examined, and result in decreased statistical power to determine true potential exposure-outcome relationships.

In addition, another potential limitation of the present study is that exposures to other sources of Hg were not evaluated. It is very likely that the individuals examined in the present study incurred other organic-Hg exposures from other Thimerosal-containing childhood vaccines, dental amalgams, fish, or other environmental sources. While these other sources of Hg may play a significant involvement in the pathogenesis ASD, these unaccounted for Hg exposures would actually tend to bias the results observed towards the null hypothesis because they potentially would confound the specific exposure classifications of Hg examined. For example, individuals classified as having lower organic-Hg exposure from Thimerosal-containing vaccines may have actually received high doses of Hg from other sources, and individuals having higher organic-Hg exposure from Thimerosal-containing vaccines may have actually received low doses of Hg from other sources, with the net result tending to minimize the magnitude of the associations observed.

It is also possible that the findings may be the result of the vaccines themselves or other components of the vaccines studied, which in isolation or synergistically, interacted with the organic-Hg exposures examined in the present study. The former seems remote, since in our study of DTaP vaccines in VAERS for example, as summarized in Table 3, the composition of the Thimerosal-free DTaP vaccine in comparison to the Thimerosal-containing DTaP vaccine indicates that vaccine components such as antigens, aluminum, formaldehyde, and other trace constituents were more present overall in the Thimerosal-free DTaP vaccine in comparison to the Thimerosal-containing DTaP vaccine. We suggest that in future studies additional examination should be undertaken further evaluate the potential adverse consequences of the vaccines themselves or other vaccine components both as a function of exposure and timing of exposure.

Table 3 The composition of the DTaP vaccines under study in the VAERS Full size table

Another limitation of the present study is that individuals diagnosed with an ASD were based upon the information contained in the automated adverse event reports reported to VAERS or in the ICD-9 codes recorded within the automated medical records in VSD. As such, no additional medical information was provided about the individuals examined, such as whether the individuals diagnosed with an ASD experienced a regression. As discussed previously, it would be important to examine such phenomena in future studies, since regressive ASD would be expected to be associated with a neurological insult occurring in the early postnatal period, whereas non-regressive ASD would be expected to be more associated with a neurological insult occurring in the prenatal period. It would be worthwhile in future studies to attempt to further examine the potential relationship between timing of Hg exposure and the initial onset of ASD symptoms.

Finally, the current study suffers from the potential limitation that analyses were not conducted to further explore the precise timing and cumulative doses of organic-Hg from all Thimerosal-containing childhood vaccines associated with maximum adverse consequences. In future studies it would be worthwhile to further explore these precise-timing and cumulative-doses phenomena. In addition, it would be valuable to evaluate other neurodevelopmental outcomes, as well as other covariates such as gender, race, birth weight, etc., that may further affect the magnitude of the adverse effects found.