Abstract
Background/Aim: The etiology of autism spectrum disorders (ASD) is currently unknown. Few studies have explored the role of Cytomegalovirus (CMV) and Epstein Barr Virus (EBV) as potential etiological factors of ASD. The aim of the present study was to evaluate the seropositivity rate and antibody titre to CMV and EBV in children with ASD compared to same-aged healthy controls. Patients and Methods: We compared the seropositivity rate and titre of antibodies to CMV and EBV in 54 children with ASD (19 with autistic disorder and 35 with non-autistic disorder ASD) and in 46 controls. Results: Seropositivity rate and titre of the two antibodies were not dissimilar between cases and controls. However, considering only patients with ASD, those seropositive for CMV tended to test worse to the major severity scales than the seronegative ones. Conclusion: Titre and seropositivity rate of antibodies to CMV and EBV are similar between children with ASD and healthy controls.
The Autism Spectrum Disorders (ASD) are multiple cognitive and developmental disorders and include Autistic Disorder (AD), Asperger Syndrome, Rett Syndrome, Childhood Disintegrative Disorder and PDD-NOS (Pervasive Developmental Disorder Not Otherwise Specified). According to the definition of the American Psychiatric Association, significantly abnormal or deficient social interaction, impaired communication and language abilities and considerable narrow pattern of activities and interests are the mainstays of ASD diagnosis. The prevalence of ASD is dramatically increasing. In fact, in the United States, it was estimated that ASD affected 1:2,000 children before the 80s and now is diagnosed in 1:88 newborns (1, 2), with a specific male dominance (5:1 ratio between males and females).
Nevertheless the prevalence trend and the enormous interest aroused by such complex and fascinating disorders, most cases of ASD lack a definitive etiological agent (1, 3). In fact, genetic predisposition and punctual gene abnormalities are thought to be able to explain only a small percentage of the cases of ASD, approximately less than 20% (4). In the other cases, it is generically stated that ASD develop from an interaction between genetic and environmental factors (5). Several authors have proposed different etiological hypotheses of ASD that include infections (6-11), vaccine employment (12, 13), vitamin deficiency (14-16), pesticide exposure (17), paracetamol use (18-20).
In this sense, a novel unifying hypothesis of the etio-pathogenesis of ASD has been proposed by our group (3). We suggested that ASD are disorders of the immune system whose onset, in individuals with a background of genetic and environmental susceptibility (the latter likely due to vitamin D deficiency), can ultimately be triggered by an infection (notably a viral infection). The effects of this deranged immune response could, in turn, result in focal damages to specific central nervous system areas.
Therefore, viral infections have repeatedly been invoked as risk factors in the development of ASD (11, 13, 21-25), mainly the ones caused by herpesviridae (26-34). Several case reports have associated cytomegalovirus (CMV) infections with the onset of ASD (35-43). A study conducted in Egypt assessed the prevalence of anti-CMV IgG in patients with autism and age-matched healthy controls and a significant higher seropositivity rate was registered in the autistic group (43.3% vs. 7%) (44).
As regards to Epstein Barr Virus (EBV), it represents an actual obscure and challenging matter of study, since this virus has, above all others and more recurrently, been implicated as both an environmental trigger factor and as a direct causative agent of central nervous system immunopathology of disorders such as multiple sclerosis (45-48) or autoimmune encephalomyelitis (49).
In this sense, according to the above-quoted unifying hypothesis, a study on possible virus-induced autoimmune phenomena that could affect the developing brain, thus generating anatomic abnormalities of neural connections, could be helpful in the understanding of such multi-faceted disorders.
The aim of the present study was to evaluate some specific viral exposure markers, notably the prevalence and the titre of anti-CMV and anti-EBV IgG antibodies, in a cohort of patients with ASD and in healthy controls.
Patients and Methods
Patients. Patients were recruited at the Child and Adolescent Neuropsychiatry Unit at the Second University of Naples and at the Department of Pediatrics of the University of Naples “Federico II”, Italy, between January 2010 and January 2013. Informed consent was obtained from all children's parents or legally-authorized representatives and identifying information was removed from each sample. The Ethics Committee of the University of Naples “Federico II” approved the study (protocol number: 85/09). Inclusion criteria for cases were diagnosis of ASD [according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth edition, Text Revision (DSM-IV-TR)(50)] and informed consent signed by parents/guardians; the only exclusion criterion was the inability to sign an informed consent form.
Controls were recruited at the Division of Pediatric Surgery of the University of Naples “Federico II”, Italy, where they entered for minor surgical treatments. They went through an interview to exclude the presence of a possible ASD, which consequently disallowed their participation at the study.
In order to validate the diagnosis of autism, cases underwent the Autism Diagnostic Interview, Revised version (51) and were examined by means of the Childhood Autism Rating Scales (CARS) (52), the Autism Diagnostic Observation Schedule (ADOS)-Generic (53), the Griffith Mental Developmental Scales (GMDS) (54) and the Vineland Adaptive Behavior Scales (VABS) (55).
Virological tests. In order to detect the specific IgG antibodies to Epstein-Barr viral capsid antigens (VCA) and to CMV in human serum a chemiluminescent immunoassay (CLIA) technology has been performed (LIAISON EBV IgG assay, LIAISON CMV IgG II assay, DiaSorin S.p.A. - Saluggia (VC), Italy).
In detail, for the determination of specific IgG to VCA or to CMV, the p18 synthetic peptide or a CMV antigen, respectively, were used for coating magnetic particles (solid-phase), then mouse monoclonal antibodies were linked to isoluminol derivatives (isoluminol-antibody conjugates). During the first incubation, VCA antibodies or CMV antibodies present in calibrator, samples or controls binded to the solid phase. During the second incubation, the antibody conjugates reacted with VCA- or CMV-IgGs already bound to the solid phase. After each incubation, the unbound material was removed with a wash cycle.
Subsequently, the starter reagents were added, hence inducing flash chemiluminescence reactions. The light signal, thus the amount of isoluminol-antibody conjugate, was measured by a photomultiplier as relative light units (RLU) and was indicative of VCA- or CMV-IgG concentration present in calibrators, samples or controls. The analyzer automatically calculated EBV or CMV antibody concentrations expressed as U/mL and graded the results.
Demographic and clinical features of children with Autism Spectrum Disorders.
With reference to EBV antibodies detection: samples with VCA-IgG concentrations below 20 U/mL have been interpreted as negative while samples with VCA-IgG concentrations equal to or above 20 U/mL have been interpreted as positive.
With reference to CMV antibodies detection: samples with CMV-IgG concentrations below 12.0 U/mL have been interpreted as negative; samples with CMV-IgG concentrations ranging between 12.0 and 14.0 U/mL, that are considered equivocal to the manufacturer, have been interpreted as negative in the present study and samples with CMV-IgG concentrations equal to or above 14.0 U/mL have been interpreted as positive.
Statistical analysis. Kolmogorov-Smirnov test was applied to quantitative variables to check for Gaussian distribution. Data are presented as mean±standard deviation (SD) or as median and interquartile range (IQR) in case of Gaussian and non-Gaussian distribution, respectively. For comparisons between quantitative variables, the Student's t-test for unpaired variables or the Mann-Whitney U-test was used in case of Gaussian or non-Gaussian distribution respectively. For comparisons of quantitative variables among three groups (e.g. controls, AD, and non-AD ASD), ANOVA or Kruskal-Wallis test were used in case of Gaussian or non-Gaussian distribution, respectively. Age and sex were added to a logistic regression analysis model together with antibody titre or antibody prevalence, considering health status (ASD vs. control) as the dependent variable. For categorical variables the χ2 test with Yates correction (Fisher's exact test where appropriate) was used. Rates of scales (VINELAND and GMDS) considered as continuous variables were correlated to antibody titre using Spearman's rho test. For ADOS and CARS the cutoffs provided by the manufacturer were used to classify patients in AD or non-AD ASD.
For all tests, p-value <5%, at two-sided tests was considered statistically significant. All statistical analyses were carried out using the Statistical Package for the Social Sciences version 18.0 (SPSS Inc. Chicago, IL, USA).
Rate of seropositivity to VCA-IgG and CMV-IgG in cases and controls and in Autistic Disorder and non-Autistic Disorder Autism Spectrum Disorders.
VCA-IgG and CMV-IgG titres in cases and controls and in Autistic Disorder and non-Autistic Disorder Autism Spectrum Disorders.
Results
We enrolled 100 children in our study, 54 with ASD (19 with AD and 35 with non-AD ASD) and 46 controls. None of the controls was found to be affected by a neuropsychiatric disorder. The mean age was 6.1 years for cases (SD=2.5 years) and 5.9 years for controls (SD=2.8 years) (p=0.852). Males out-numbered females, both among cases (41/54, 75.9%) and controls (39/46, 84.8%, p=0.270). The neuropsychiatric and clinical features of the children with ASD are shown in Table I.
We assessed the rate of seropositivity for the two herpes viruses in cases and controls. As shown in Table II, the rate of seropositivity was similar in cases and controls.
We measured and compared antibody titres for EBV (VCA-IgG) and CMV in cases and controls. As shown in Table III, the titres of antibodies against the two herpes viruses did not differ between cases and controls.
In a logistic regression analysis model, which included age and gender, it was confirmed that the presence or the title of anti-CMV or anti-EBV antibody was not an independent predictor for health status (ASD condition or health).
We also evaluated the levels and seropositivity rate of the two antibodies in three specific categories (children with AD, children with non-AD ASD and controls). As shown in Table III, neither the seropositivity rate for the two viruses nor antibody titres differed significantly among the three groups.
Therefore, neither antibody level nor seropositivity rate was associated with ASD status.
We correlated severity of GMDS and VABS scales with CMV and EBV antibody levels using Spearman's Rho test. None of them showed a significant correlation with the antibody levels. For CMV, p-values were −0.130 (p=0.353) and - 0.091 (p=0.518) and for EBV were 0.14 (p=0.922) and 0.047 (p=0.737) for GMDS and VABS, respectively.
Finally, we evaluated the median values of severity scales in seropositive and seronegative children with ASD for the two antibodies. GMDS was 47 (IQR: 42.75-54.25) vs. 54 (IQR: 43-68, p=0.073) and VABS was 45 (IQR: 34.75-53.50) vs. 52 (IQR: 38-64, p=0.054) in anti-CMV seropositive and seronegative patients, respectively. GMDS was 50 (IQR: 44-60) vs. 50.5 (IQR: 42.25-58, p=0.823) and VABS was 52 (IQR: 36.5-59) vs. 47 (IQR:40.25-61.25, p=0.761) in anti-EBV seropositive and seronegative patients, respectively.
Discussion and Conclusion
Our study shows that patients with ASD have similar rates of CMV and EBV contact markers compared to healthy controls. This finding even corroborated by the results of a multivariate model that included potential confounding variables such as age and gender.
Herpes viruses are a family of DNA viruses characterized by the ability to engender acute infection, survive in latency status or recurrently reactivate in response to different kinds of triggers. They support a complex interaction with the immune system, that can explain their suggested role as factors or co-factors of autoimmune diseases (56, 57).
In particular, EBV is a candidate for several autoimmune disorders involving the central nervous system such as multiple sclerosis or autoimmune encephalomyelitis. To the best of our knowledge, ours is among the few studies to evaluate the exposure to EBV in the setting of ASD. Results of our study are not in favour of a role of EBV as an etiological factor or co-factor of ASD and confirm those obtained by Singh et al. who found a similar titre of anti-EBV antibodies in a cohort of 44 patients with ASD compared to healthy controls and those by Mora et al. who documented no significant differences for IgG and IgM antibodies for EBV in a cohort of 40 patients with ASD compared to healthy controls (29, 58). These results are in contrast to those achieved in the setting of multiple sclerosis, where a higher prevalence and a higher titre of anti-EBV antibodies in patients compared to controls were reported (45).
With respect to CMV, this virus has been repeatedly invoked as a potential etiological agent of ASD (3, 11). In fact, several clinical cases reported an association between CMV infections and ASD onset (36-41, 43). A well-documented study described the occurrence of symptomatic CMV infection at birth, diagnosed also by the mean of high viremia (CMV-DNA=6.0×106 copies/ml) and virus isolation from placenta, decidua villi and amnion, followed by ASD onset (35). Other researchers retrospectively diagnosed a congenital CMV infection in a 4-year boy with AD, by the identification of viral genome in preserved umbilical cord blood by polymerase chain reaction (42).
The results of our study are in agreement with a previous study by Singh et al. and by Mora et al. that found no difference in antibody titre nor in seroprevalence of CMV antibody in similar-sized cohorts of patients (29, 58). Our results are in contrast with those obtained in an Egyptian study that revealed a significantly higher rate of anti-CMV antibodies in patients with ASD compared to healthy children (43.3% vs. 7%) (44).
However, the role of CMV as an etiological factor of ASD cannot be completely ruled-out based on the results of the present study for two reasons: i) considering only patients with ASD, those with anti-CMV antibodies tended to perform worse on severity scales compared to those without anti-CMV antibodies. This trend is at the limit of statistical significance, but it is noteworthy that it has been observed for both tested scales (GMDS and VABS) and it has not been found for EBV; ii) the present study evaluated only one of the possible etiological factors for ASD (viral exposure). However, nothing is known in our sample cohort regarding other co-factors that can contribute to ASD etio-pathogenesis (such as genetic predisposition to autoimmune disease, vitamin D levels) and that could be critical, in a sub-group of exposed subjects, for the development of the disorder.
In conclusion, the rate of seroprevalence and titre of anti-CMV and EBV antibodies are similar in subjects with ASD and same-aged healthy controls.
Footnotes
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Conflicts of Interest
Each Author certifies that he or she has no commercial associations that might pose a conflict of interest in connection with the submitted article.
- Received January 7, 2014.
- Revision received March 26, 2014.
- Accepted March 27, 2014.
- Copyright © 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
