Асд 2 от цитомегаловируса

Препарат АСД относится к группе биогенных стимуляторов, он обладает широким спектром лечебного и профилактического действия и применяется при довольно большом числе заболеваний с различной этиологией.

Сейчас препарат хорошо известен ветеринарным врачам и активно применяется при лечении животных, но раньше его область применения не ограничивалась ветеринарией.

В пятидесятых годах двадцатого века проводилось множество исследований препарата в лечении различных заболеваний людей. Положительные результаты были получены в лечении кожных заболеваний, профилактике и лечении туберкулеза, лечении простатита и ряда гинекологических заболеваний. В онкологии даже в наше время лучшие препараты (витурид, кошачий коготь, акулий хрящ) до сих пор не могут сравниться по эффективности с АСД.

История препарата

В середине 20 века препарат АСД был широко известен и популярен в СССР, но в наше время он вдруг стал сенсацией, так, словно его не существовало до этого. Как это могло получиться? Почему АСД широко применяется в ветеринарии медицине, но полностью забыт в "человеческой" медицине, для которой создавался?

В разное время исследования Дорогова то блокировались властью, то поддерживались. Препарат несколько раз засекречивали, ограничивали область применения, несмотря на высокую эффективность АСД и широкий спектр действия.

Изначально препарат разрабатывался как средство, защищающее людей и животных от радиации. Стояла задача создать дешевое и недифицитное средство, существенно повышающее иммунитет организма. Над разработкой такого препарата трудились десятки лабораторий при различных научных учреждениях по всему Союзу, но успеха добилась только лаборатория Всесоюзного института экспериментальной ветеринарии (ВИЭВ). Руководителем этой лаборатории был А. В. Дорогов.

Созданный в лаборатории Дорогова препарат обладал антисептическими, стимулирующими, ранозаживляющими свойствами и был назван АСД (антисептик-стимулятор Дорогова). Впоследствии препарат разделили на две фракции - вторую и третью

Вторая и третья фракции

Фракция имеет светло-желтый цвет с коричневым оттенком, легко смешивается с водой, имеет сильный запах. Предназначена для внутреннего и наружного применения.

АСД-2 (-2Ф) может применяться при сердечных и легочных заболеваниях, заболеваниях нервной системы, для профилактики и лечения туберкулеза, при язве желудка и 12-перстной кишки, лечении предраковых форм. При лечении раковых заболеваний АСД-2 останаливает развитие заболевания, снимает боль.

Кроме того, фракция успешно применяется для лечения гинекологических заболеваний, простатита, импотенции, кожных заболеваний, глазных и ушных заболеваний воспалительного характера.

По внешнему виду препарат представляет собой густую жидкость от темно-коричневого до черного цвета, растворимую в спирте, растительных и животных маслах и практически не растворимую в воде.

Фракция применяется для лечения ран, дерматитов, экзем, трофических язв, свищей и других поражений кожных покровов у животных.

Подробно применение препарата описано в инструкции (наставлении).

Применение АСД

Препарат АСД-2Ф назначают сельскохозяйственным животным (в том числе птице) и собакам, с лечебной и профилактической целью при болезнях желудочно-кишечного тракта, органов дыхания, мочеполовой системы, поражениях кожных покровов, нарушениях обмена веществ, для стимуляции деятельности центральной и вегетативной нервной системы, повышения естественной резистентности у ослабленных и переболевших инфекционными и инвазионными болезнями животных, а также для стимуляции роста и развития поросят, цыплят и повышения яйценоскости кур.

Биологические свойства и порядок применения подробно описаны в статье АСД-2 в ветеринарии и наставлении (инструкции) АСД-2.

Третья фракция (АСД-3Ф) предназначена только для наружного применения - для лечения ран, дерматитов, экзем, трофических язв, свищей и других поражений кожных покровов. Подробная информация представлена в Справочнике и в наставлении.

Где купить АСД

В настоящее время производством фракций АСД занимается ФГУП "Армавирская биофабрика".

Приобрести препарат можно в нашей интернет-аптеке:

ЦИТОМЕГАЛОВИРУС (CMV/ЦМВ)
• ЧТО ТАКОЕ ЦМВ?
• КАК ЛЕЧАТ ЦМВ?
• МОЖНО ЛИ ЧТО-ТО СДЕЛАТЬ ДЛЯ ПРОФИЛАКТИКИ ЦМВ?
• КАК МНЕ ВЫБРАТЬ ЛЕЧЕНИЕ ОТ ЦМВ?
• ИТОГ

Как лечат ЦМВ?
Первые методы лечения ЦМВ требовали ежедневных внутривенных инъекций. У большинства людей был постоянный медицинский катетор, вставленный в ключицу или руку. Людям приходилось принимать лекарства против ЦМВ всю жизнь.
Способы лечения ЦМВ невероятно улучшились за последние несколько лет. Сейчас существует семь видов лечения, одобренных Управлением по контролю за продуктами и лекарствами (FDA, США).
Сильные антиретровирусные препараты (АРВ) могут улучшить иммунную систему. Пациенты могут прекратить прием лекарств от ЦМВ, если уровень CD4 клеток у них поднимается от 100 до 150 и держится на этом уровне (и выше) в течение трех месяцев. Однако, есть два особых случая:
1. Синдром иммунного восстановления может вызвать воспаление в глазах людей, инфицированных ВИЧ, даже если у них раньше и не было ЦМВ. Стандартный способ лечения –добавить препарат от ЦМВ к схеме АРВ препаратов пациента.
2. Если уровень CD4 падает ниже 50, риск возникновения заболевания вызванного ЦМВ значительно возрастает.

Можно ли что-то сделать для профилактики ЦМВ?
Ганцикловир был одобрен для предупреждения (профилактики) ЦМВ. Однако, многие врачи не прописывают его. Они не хотят прибавлять до 12 капсул ежедневно к схеме своих пациентов. Кроме того, не очевидно то, что это приносит пользу. Два больших исследования пришли к разным выводам. В конце концов, АРВ препараты поддерживают уровень CD4 клеток у большинства людей на уровне, которого достаточно, чтобы не заболеть ЦМВ.

Как мне выбрать лечение от ЦМВ
Есть несколько проблем, которые необходимо рассмотреть выбирая лечение от заболевания, вызванного ЦМВ:
Ваше зрение под угрозой?
Возможно вам придется предпринять быстрые действия, чтобы спасти свое зрение
Насколько оно (лечение) эффективно?
Внутривенное введение ганцикловира –наиболее эффективное из всех видов лечения ЦМВ. Имплантанты очень эффективны для остановки воспаления сетчатки. Однако, они действуют только в том глазу, куда были имплантированы.

Какова форма применения?
Таблетки наиболее легкий способ. Внутривенные уколы подразумевают иголки или катетор, в который могут проникнуть инфекции (. ). Глазные инъекции означают введение иглы прямо в глаз. Для того чтобы вставить имплантанты, которые действуют от 6 до 8 месяцев, нужна операция длительностью примерно час.
Это местная или системная терапия?
Местная терапия действует только на глаза. При ЦМВ воспаление сетчатки может прогрессировать стремительно и привести к слепоте. Именно поэтому, при первых же признаках появления, его начинают лечить очень агрессивно. С помощью новейших инъекций и имплантантов лекарство помещается прямо в глаз и оказывает наиболее сильное воздействие на ретинит.
ЦМВ также может проявиться в других частях организма. Чтобы контролировать ЦМВ во всем организме, вам нужна системная (общая) терапия. Это могут быть внутривенные инъекции или таблетки вальганцикловира.

Каковы побочные эффекты?
Некоторые препараты против ЦМВ могут повредить вашим почкам или костному мозгу. В таком случае могут понадобиться дополнительные препараты. Они вводятся с помощью капельницы, что может занять долгое время. Обсудите побочные эффекты лечения ЦМВ со своим врачом.
А что говорят рекомендации (директивы)?
Недавно несколько изданий рекомендаций для профессионалов рекомендовали вальганцикловир в качестве предпочтительного лечения для пациентов, у которых не стоит риск немедленной потери зрения.

ИТОГ
Прием АРв препаратов, вероятно, лучший способ предотвратить появление ЦМВ. Если ваш уровень CD4 ниже 100, поговорите со своим врачом о профилактике ЦМВ и регулярном глазном тестировании. Если у вас низкий уровень CD4 и вы испытываете любые проблемы со зрением, немедленно обратитесь по этому поводу к своему врачу!
Лекарство, вводимое непосредственно в глаз позволяет контролировать развитие воспаления сетчатки, вызванного ЦМВ. С учетом появления новейших лекарств для лечения ЦМВ, вы можете избежать ежедневных уколов и вставленных катеторов.
Большинство людей могут совершенно спокойно прекратить прием препаратов от ЦМВ, если уровень их CD4 клеток поднялся со 100 до 150 при приеме АРВ –терапии.

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 χ 2 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.

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=6.0×10 6 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

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.

Abstract

Background/Aim: Autism spectrum disorders (ASD) are neurodevelopmental disorders without a definitive etiology in most cases. Environmental factors, such as viral infections, have been linked with anomalies in brain growth, neuronal development, and functional connectivity. Congenital cytomegalovirus (CMV) infection has been associated with the onset of ASD in several case reports. The aim of this study was to evaluate the prevalence of congenital CMV infection in children with ASD and in healthy controls. Patients and Methods: The CMV genome was tested by polymerase chain reaction (PCR) on dried blood spots collected at birth from 82 children (38 with ASD and 44 controls). Results: The prevalence of congenital CMV infection was 5.3% (2/38) in cases and 0% (0/44) in controls (p=0.212). Conclusion: The infection rate was about 10-fold higher in patients with ASD than in the general Italian population at birth. For this reason, detection of CMV-DNA on dried blood spots could be considered in the work-up that is usually performed at ASD diagnosis to rule-out a secondary form. Given the potential prevention and treatment of CMV infection, this study could have intriguing consequences, at least for a group of patients with ASD.

According to the fifth edition of the Diagnostic and Statistical Manual of Mental Disorders, Autism spectrum disorders (ASD) are neurodevelopmental disorders characterized by impairments in two core domains: social-communication and restricted and repetitive patterns of behavior, interest or activities (1). ASD presentation is very heterogeneous depending on the severity of impairment in the two core domains. Furthermore, clinical features vary depending on the presence/absence of intellectual disability and verbal skills.

The prevalence of ASD has considerably increased in recent decades. In fact, at the beginning of the century the prevalence of ASD was estimated at around 60-70/10,000 (2), with a male to female ratio of 4:1 (3), whereas studies conducted in the past two decades revealed an increase of 100-250/10,000 (4-6) and a decrease in male predominance (ratio 2.0-2.6:1) (7, 8). Only in about 20% of cases is a definite etiology recognized. These cases are called “secondary ASD” (9). No definitive cause is found in the remaining 80% of cases, and these are designated “primary ASD” (10-12). Primary ASD is believed to result from the interaction of environmental factors, which probably occur during intrauterine life, with the genetic profile. This interaction leads to aberrant changes in brain growth, neuronal development, and functional connectivity that finally give rise to the disorder (11, 13-15).

There are no instrumental diagnostic tests for ASD. Thus, its diagnosis is based on clinical findings. However, a series of tests are routinely performed to rule-out secondary ASD. They include genetic tests (recommended in their case of familiality for genetic diseases, intellectual disability, and malformations in various organs), metabolic tests (in case of familiality for metabolic disorders, cyclical vomit, precocious epilepsy, mental retardation, clinical evidence or suspicion of metabolic disorders) (16). Several treatments have been proposed, both pharmacological and non-pharmacological, but their efficacy is currently disappointing, because of the lack of knowledge of the etiopathogenetic mechanisms of ASD (17-20).

In some cases, ASD has been linked to infection (21-39). A viral infection may per se damage the central nervous system or it might trigger an autoimmune reaction against some cerebral regions (11). Several case reports have associated cytomegalovirus (CMV) infection with ASD (40-48). We previously demonstrated that exposure to CMV was similar in a cohort of children with ASD and in same-aged healthy controls (49). However, this type of study does not allow for a precise timing of viral exposure (11).To overcome this drawback, in the present study, as samples we used dried blood spots (DBS) collected at birth from children with ASD and healthy controls. The use of DBS for studies on ASD provides a unique picture of the clinical situation at birth, which is considered a key moment in the development of ASD. CMV-DNA detection on DBS has been widely used in the retrospective diagnosis of congenital infection when no neonatal sample was available (50, 51). This test has also been used to evaluate the prevalence of congenital CMV infection in children with cortical development disorders (such as pachigiria and leucodystrophy of unknown origin) (52). The same test revealed that 20-30% of all cases of infantile deafness were due to congenital CMV infection (53, 54).

The aim of this study was to evaluate the rate of congenital CMV infection assessed via detection of viral genome in DBS by polymerase chain reaction in children with ASD and in healthy controls.

Materials and Methods

Participants. Children with ASD were enrolled at the Child and Adolescent Neuropsychiatry Unit at the Second University of Naples, Italy, between January 2010 and January 2013. Inclusion criteria for cases were: diagnosis of primary ASD [at the time performed according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth edition, Text Revision (DSM-IV-TR)(55)]. Diagnosis was confirmed based on the criteria of the new DSM-5 classification (1). The only exclusion criterion was the inability of parents or legal guardians to sign an informed consent form.

Controls were recruited by a general pediatrician during routine examination at their medical office in Torre del Greco, Naples, Italy or at the Division of Pediatric Surgery of the Federico II University of Naples, where they were admitted for minor surgical procedures (phimosis, hernia, cryptorchidism, vesicoureteral reflux, hydrocele testis, etc.). All controls were aged between 0-8 years. They were enrolled between January 2010 and January 2013 and underwent an interview to rule out the presence of neuropsychiatric disorders. To verify the diagnosis of ASD, cases were assessed with the Autism Diagnostic Observation Schedule-Generic (ADOS-G) (56), performed by a licenced clinician. Griffiths Mental Development Scales (GMDS-ER) (57) were administered to all cases to determine the level of development. The Autism Diagnostic Interview, Revised version (ADI-R) (58), the Childhood Autism Rating Scales (CARS) (59), and the Vineland Adaptive Behavior Scales (VABS) (60) were administered to the parents of all patients. A revised algorithm of ADOS was adopted to evaluate ASD severity (61-63). Based on a standardized clinical evaluation (obtained using ADOS, GMDS and VABS), three levels of ASD severity were identified as specified by DSM-5 criteria. Level 1 refers to clinical conditions in which the patient needs support for adequate adaptation; level 2 refers to clinical conditions in which they need significant support for adequate adaptation; and level 3 refers to clinical conditions in which they need very significant support for adequate adaptation.

Samples. All newborns in Italy are screened for some congenital disorders at 2-5 days of age. The DBS obtained during this procedure are stored at room temperature and are not exposed to light.

All procedures were performed in compliance with the ethical standards of relevant laws and institutional guidelines and in accordance with the with the 1964 Helsinki declaration and its later amendments. The research was prospectively reviewed and approved by the Ethics Committee of the Federico II University of Naples and the Ethics Committee of ASL NA3 SUD approved the study (85/09). Informed consent was obtained from the children's parents or legally authorized representatives.

Procedure. A 3-mm diameter circle punched out of the DBS was used for CMV-DNA extraction, and nucleic acid was amplified as previously described (64) by an operator blinded to the characteristics of the cases. Stringent control measures were applied to prevent both carryover and contamination (65). In brief, DNA was extracted from the circle of DBS by adding 35 μl of cell culture medium (minimum essential medium) followed by thermal shock (55°C for 60 min and 100°C for 7 min, centrifugation at 11,200 rcf, and the supernatant was frozen at −80°C overnight) and amplified using a nested polymerase chain reaction (PCR) designed to amplify one region in the GP58 gene (66). The first round of CMV-DNA amplification was carried out starting with 5 μl extract in 45 μl PCR mixture (10 mM Tris – HCl, 1.5 mM MgCl2,50 mM KCl, 0.1% triton X-100, 200 mM dNTP) containing 1U Taq-polymerase (DyNAzyme™ II DNA Polymerase; Finnzymes, Thermo Fisher Scientific Inc. Ratastie, Vantaa, Finland) and the primers (1 mM) necessary for amplification. The second round of CMV-DNA amplifications consisted of 2 μl amplicons in 48 μl PCR mixtures. The amplification conditions for the first round were as follows: denaturation at 94°C for 2 min followed by 35 cycles at 94°C for 30 s, 55°C for 30 seconds and 72°C for 30 s with a 5 min final extension at 72°C. The second amplification differed from the first amplification in that 30 cycles were performed and the annealing temperature was 53°C.

To verify extraction from DBS, a house-keeping gene was amplified with a PCR designed to amplify human β-globin with primers GH20 and PCO4 (67). The final PCR mixture in a 50-μl reaction included 3 μl of extracted template and the PCR conditions were as follows: denaturation at 94°C for 2 min followed by 40 cycles at 94°C for 30 s, 55°C for 30 s, 72°C for 1 min and a 5 min final extension at 72°C. The PCR products were separated by electrophoresis on a 2% agarose gel and visualized by SYBR® Safe DNA Gel Stain (Invitrogen™ by Life Technology, Carlsbad, CA, USA) with transillumination.

Each sample was tested on three series of three DBS. All cases that tested positively in at least two out of the three tests were considered positive. New series of punches were tested from the cards of cases that were positive in only one of the triplicate amplifications (50). Negative and positive controls for CMV were included in all PCR runs and consisted of blank cards with minimum essential medium and mock DBS, respectively.

Statistical analysis. The Kolmogorov–Smirnov test was applied to determine whether distribution was Gaussian or non-Gaussian. We report the data as mean±standard deviation (SD) or as median and interquartile range (IQR), respectively. For comparisons between quantitative data, Student's t-test for unpaired variables or the Mann–Whitney U-test was applied in case of Gaussian or non-Gaussian distribution, respectively. For categorical variables, the chi-squared test with Yates correction (Fisher's exact test where appropriate) was applied. A p-value of 5% at two-sided test was considered statistically significant. Statistical analysis was carried out with the Statistical Package for the Social Sciences version 18.0 (SPSS Inc. Chicago, IL, USA).

Results

A total of 82 children were enrolled in this study, 38 with ASD and 44 controls. None of the controls was affected by a neuropsychiatric disorder. The mean age was 5 years for cases (SD=1.3 years) and 4 for controls (SD=1.7 years) (p=0.007). Boys outnumbered girls, both among cases (29/38,76.3%) and controls (30/44, 68.2%, p=0.414). The main clinical scores of patients with ASD are reported in Table I. Sixteen out of the 38 patients were diagnosed with ‘ASD with associated intellectual disability’. The rate of CMV positivity as assessed by viral genome detection on DBS was 5.3% (2/38) in cases and 0% (0/44) in controls (p=0.212).

The two patients (patient 1 and patient 2) who tested positively for CMV-DNA had a mild intellectual disability. Compared with the 38 patients without CMV-DNA, the two patients with CMV-DNA had significantly lower CARS scores (median of 30 vs. 38, p=0.034) and showed a trend towards having lower total ADOS scores (median of 12.5 versus 17, p=0.069). Patient 1 was a boy born by vaginal delivery at 38 weeks of gestation (birth weight: 3,000 g). His psychomotor development was regular, except for language and sphincter control (not yet acquired at age 3 years). At 24 months of age, because of language delay, parents requested a medical consultation. A child neuropsychiatrist diagnosed relational and communication problems. The boy underwent a hearing evaluation through auditory brainstem response audiometry which did not show any impairment. He had no history of convulsions or any other neurological disorder, and his electroencephalogram was normal. The boy was intolerant to cow's milk proteins and had some feeding problems (food selectivity). During clinical evaluations, the patient showed partially preserved communication skill and willingness to interact with his parents, but the quality of this relationship was very poor. He presented a high level of hyperactivity and anxiety at separation from his parents and had self-injurious and restricted and repetitive behaviors. His development quotient evaluated by GMDS-ER, was 54; the adaptive quotient, evaluated by VABS, was 72; his ADOS scores were indicative of autistic features, both in communication and in social interaction areas. His diagnosis, at that time made according to DSM-IV criteria, was “Pervasive Developmental Disorder not otherwise specified”. Patient 2 was also a boy. He was born by Cesarean section at 38 weeks of gestation (body weight: 3,150 g). At birth, he did not cry immediately, but other perinatal events were regular. He underwent a transfontanellar ultrasound at birth which did not show any pathological finding. He presented a mild delay in psychomotor development at about 24 months of life, and a regression of language skills. In fact, he experienced a loss of the use of language for communication. At this age, he began to show a behavior characterized by a tendency for isolation and for difficulty in interacting with peers. Consequently, the child's parents requested a medical consultation with a child neuropsychiatrist who diagnosed relational and communication problems. The boy underwent hearing evaluation through auditory brainstem response audiometry, which yielded normal results. He had no history of convulsion or any other neurological problems. His electroencephalogram was normal. During clinical evaluations, the patient showed some communication skills, presented a functional use of objects and was able to play appropriately with various objects, but the quality of communication with others was impaired and variable. His development quotient, evaluated by GMDS-ER, was 54; adaptive quotient, evaluated by VABS, was 56; the ADOS scores were indicative of autistic features, both in communication and in social interaction areas. His diagnosis, at that time made according to DSM-IV criteria, was “Pervasive Developmental Disorder not otherwise specified”.

Developmental features of children with autism spectrum disorders (n=38). Data are reported as the median and interquartile range or as percentage (for Gravity score).