As previously published by investigators from the CDC and the US Food and Drug Administration (FDA), products, such as vaccines, which are intended for healthy people, must be held to a high standard of safety assurance [14]. However, these previous investigators described that the study of vaccine risks is more complex than for therapeutic products because the exposure is nearly universal for many vaccines, ensuring the chance occurrence of many subsequent temporally associated adverse outcomes after the administration of vaccines. As a result, these investigators described using the VSD, a consortium of HMOs, to more rigorously evaluate post-vaccination associated risks (hypothesis testing).

The present study revealed cases diagnosed with PDD were overall, among both male and female cases analyzed separately, and on a dose-dependent basis, significantly more likely than controls to receive increased organic Hg exposure from Thimerosal in Hib-containing vaccines administered at specific intervals within the first 15 months of life. The results of the present study are consistent with and extend the previous ecological birth cohort study [7] and case-control studies [8, 9] of the potential relationship between organic Hg exposure from Thimerosal-containing childhood vaccines and PDD in the VSD database. The size and magnitude of the observed relationship on a per microgram organic Hg basis are similar to the previously discussed VSD studies and suggest that the phenomena observed in the VSD database is more than mere chance. In addition, the significant association observed between organic Hg exposure and subsequently diagnosed PDD is consistent with several other studies examining other databases such as the National Health Interview Survey (NHIS) [15] and the Vaccine Adverse Event Reporting System (VAERS) [8, 16].

The results of the present study stand in contrast to six previous epidemiological studies that failed to find a significant relationship between Hg exposure from Thimerosal-containing childhood vaccines and diagnosed PDD [17–22]. As recently reviewed, the aforementioned studies were found to have significant methodological concerns, and, as a consequence, their potential ability to contribute to understanding the potential relationship between organic Hg exposure from Thimerosal-containing childhood vaccines and diagnosed PDD was found to be not interpretable [23].

In further support of the biological plausibility of the phenomena observed in the present study, investigators have examined a number of different lines of scientific inquiry. For example, investigators recently published a critical review on the mechanisms of how limited thiol availability, abnormal sulfation chemistry, and decreased glutathione reserve capacity in children diagnosed with PDD could make them more susceptible to the toxic effects of Thimerosal routinely administered as part of childhood immunization schedules [24]. Another study demonstrated that human neuronal systems exposed to low concentrations of Thimerosal were able to induce cellular damage similar to that observed in the neuropathological studies of subjects diagnosed with a PDD [25]. Finally, several recent studies of Thimerosal exposure in animal model systems resulted in neuropathological and behavioral symptoms similar to those associated with PDD [26–30].

Strengths/Limitations

In considering the strength of the results observed, the VSD observations made were based upon retrospective assessment of prospectively collected medical records of patients enrolled in various HMOs. As such, the VSD data examined were collected independently of the study design and were, in fact, collected as part of the routine healthcare individuals received as part of the participation with their respective HMOs. Thus, at the times of the collection of these records, the healthcare providers probably were not thinking about the potential association between vaccine exposures and potential subsequent adverse health outcomes.

The study design used to evaluate the potential relationship between exposure and outcome was another significant strength of the present study. Cases examined in the present study had to be enrolled from birth and were required to be continuously enrolled until a medical diagnosis of PDD was made. Moreover, 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 a PDD. As a result, 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. In addition, cases diagnosed with a PDD were specifically evaluated to ensure that only those cases diagnosed with an PDD following vaccine administration were considered in the present analyses.

An additional strength of the present study is that it was decided, a priori, to ensure adequate amounts of data for our analyses and that the included controls would be HMO enrolled continuously from birth until they were at least the mean age of initial diagnosis of PDD plus twice the standard deviation of the mean age of initial diagnosis of PDD. For cases diagnosed with a PDD, it was possible to mathematically evaluate the mean and standard deviation of age for initial PDD diagnoses within the VSD. From this information, it was possible to estimate how many additional potential diagnoses of PDD were missed. It was decided, a priori, to ensure adequate amounts of data for our analyses and that controls had to be continuously enrolled in the VSD from birth until they were at least 7.22 years old (mean age of initial diagnosis of PDD +2× standard deviation of the mean age of initial diagnosis of PDD). Based on the data for age of initial diagnosis for PDD, this was a sufficient period to ensure that, with further follow-up, those controls without PDD would probably not receive a PDD diagnoses in the VSD (mathematically there is a <2.5 % chance of these individuals being diagnosed with a PDD with additional follow-up time beyond 7.22 years). As shown in Table 7, when the length of follow-up of controls examined was reduced to only the mean age of initial diagnosis of PDD (i.e., cases/controls were followed for same length of time and mathematically there is an about 50 % error), the overall adverse effects observed on a per microgram basis from increasing exposure to organic Hg exposure from Thimerosal in Hib-containing vaccines completely disappear. Clearly, these analyses establish the importance of following controls in any study of PDDs for a sufficient period to ensure that the risk of a control being subsequently diagnosed with a “case” condition is as small as possible, subject to the limitations of the records that are accessible for study.

Table 7 A summary of organic Hg exposure from Thimerosal in Hib-containing vaccines among cases diagnosed with a PDD in comparison to controls with a reduced length of follow-up1 Full size table

A further strength of the present study was the specific methods employed to evaluate differences in cumulative doses of organic Hg received at specific intervals during the infant period were not the result of small group of children receiving anomalous exposure to vaccines. Instead, the subjects examined were exposed to different levels of organic Hg based upon differences in the Thimerosal content of licensed Hib vaccines [13] and the recommendations on the ages for the administration of Hib vaccines to children made by the 1991 Advisory Committee on Immunization Practices recommendations [31].

However, the results of the present study may have a number of potential limitations. For example, the results observed may have occurred from unknown biases or cofounders present in the datasets examined. This seems unlikely because the control outcome of febrile seizure (i.e., an outcome that is not biologically plausibly linked to postnatal organic Hg exposure from Thimerosal-containing vaccines) was examined, using the same methodology employed for PDD. As shown in Tables 4 and 5, no similar patterns of significant associations were observed as those that were found for organic Hg exposure from Thimerosal in Hib-containing vaccines and the subsequent risk of PDD diagnosis.

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 addition, it was observed that there were similar order-of-magnitude odds ratios for increasing organic Hg exposure among cases diagnosed with a PDD in comparison to controls for discrete point estimates and logistic regression estimates.

Still, other potential limitations of the present study include the possibilities that some of the individuals in VSD examined may have had more subtle neurological dysfunction, which 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. In addition, it is possible that some of the organic Hg exposures from the Hib-containing vaccines were different than the estimated amount. For example, ActHIB vaccine manufactured by Aventis Pasteur was always Thimerosal free, but it was usually reconstituted with Thimerosal-containing Tripedia, but it is possible that some doses were reconstituted with just plain saline. Similarly, some Hib vaccines by Merck and Company, Inc. may have had 12.5-μg organic Hg per dose, and not the assigned 0-μg organic Hg per dose. In addition, it is possible that some very small number of doses of HibTITTER vaccine manufactured by Lederle may have come in single-dose vials with 0-μg organic Hg per dose, and not the assigned 25-μg organic Hg per dose. 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. At this time, data analysis restrictions imposed by the CDC prevent us from studying more than one type of vaccine subjects received, despite the fact that it is very likely that the subjects examined incurred other organic Hg exposures from other Thimerosal-containing childhood vaccines. It is also likely that subjects examined received Hg exposures from dental amalgams, fish, or other environmental sources. While all these other sources of Hg may play a significant involvement in the pathogenesis PDD, these unaccounted for Hg exposures would actually tend to bias the results observed toward 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 reported may be the result of other components of the vaccines studied, which in isolation or synergistically interacted with the organic Hg exposures examined in the present study. These possibilities seem remote, since multiple different brands of Hib-containing vaccines were examined (with many different amounts/types of adjuvants and trace constituents), but a significant overall and dose-dependent relationship was observed among cases diagnosed with a PDD in comparison to controls for increasing organic Hg exposure. Moreover, any effects of other components of vaccines working in isolation or synergistically would tend to bias the results observed toward the null hypothesis because they were not considered in the statistical analyses undertaken.

Additionally, a potential limitation of the present study is there may be differences in the general health status among the subjects examined independent of exposure to organic Hg from Hib-containing vaccine administration. As described previously by investigators from the CDC [32], confounding of this sort is a general problem for studies of adverse reactions to prophylactic interventions, as they may be withheld from some individuals precisely because they are already at high risk of the adverse event, and, as a consequence, studies that fail to adequately control for such confounding factors are likely to underestimate the risk of adverse events attributable to vaccination. In order to help minimize that potential effect in our analyses, we utilized a reference group that received 25 μg organic Hg exposure from Hib-containing vaccines in all of our statistical analyses. Despite our attempt to minimize these phenomena in our analyses, we did observe that there was an apparent protective association for increased organic Hg exposure from Thimerosal in Hib-containing vaccines for our control outcome of febrile seizure compared to controls. As a consequence, there was a “healthy vaccine” effect in our data. Since this phenomenon is apparent in the data examined, the significantly increased positive dose-dependent associations observed for the PDD probably underestimates the true extent of the relationship between organic Hg exposure from Thimerosal in Hib-containing vaccines and the subsequent PDD outcome studied.

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.