INTRODUCTION:

Autism spectrum disorder (ASD) is an urgent challenge affecting at least 1% of the global pediatric population.¹ Despite the increasing incidence of this condition, there are no FDA-registered drug products for managing childhood ASD or sustaining recovery. The latest meta-analyses and systematic reviews argue that ASD is associated with genetic folate cycle disruptions (GFCDs).^2-6

The folate cycle defects are not a feature present in all children with ASD even though it is an important line of research and an association has been identified in recent studies.

It sheds light on the pathogenetic pathways of transmethylation disorder,⁷ persistent oxidative stress,⁸ immunodeficiency and related immune dysregulation,⁹ and reactivation of opportunistic infections ^10,11 – the important pathways of brain damage in ASD patients. According to some researchers, ASD children have a disturbed cytokine balance, some signs of persistent neuroglial inflammation in the CNS, adaptive and innate immune system defects, altered serum immunoglobulin levels, and autoimmune responses to myelin and extracerebral autoantigens.⁹

Due to immune dysfunction, ASD patients may have reduced resistance to some microorganisms. There are clinical reports and controlled studies that show how opportunistic microbial agents cause severe infections in children with ASD. One of the possible causes is the GFCD-induced damage to the immune system. Among the key intracellular opportunistic agents undergoing reactivation in ASD patients are herpes viruses, such as HSV-1 (10), EBV,¹¹ CMV,¹² and HHV-6.¹³ Herpes infections have been repeatedly reported among ASD children.^10-13 It must be emphasized that the idea of herpesvirus reactivation in ASD remains more of a scientific hypothesis than a firm fact.

The clinical phenotype of ASD can develop after an HSV-1-induced episode of temporal encephalitis.¹⁰ The first such case was reported by Mohammad Ghaziuddin in 1992.¹⁰ Subsequently, researchers detected a relatively high prevalence of congenital cytomegalovirus neuroinfections among ASD children.¹² This type of infection can increase the severity of neuropsychiatric disorders and acts as an unfavourable prognosis factor in ASD.¹² Some other researchers detected abnormally frequent cases of EBV and HHV-6 reactivation in ASD children by blood leukocyte PCR and IgM serologic tests.^11,13 Yet, little attention was paid to reactivated HHV-7 infections, even though HHV-7 is a widespread viral agent in the pediatric population.

Reactivated herpes virus agents can affect ASD children through a variety of mechanisms, causing virus-induced encephalitis¹⁰ and/or neurodegenerative processes in the limbic region of the temporal lobe.¹⁴ These conditions can enhance the GFCD-induced methylation disorders,¹⁵ systemic inflammation and oxidative stress¹⁶ as well as modulate the allergic¹⁷ and autoimmune¹⁸ pathways of brain damage. Yet, no clinical studies are testing the effect of acyclic nucleoside analogues (valacyclovir and valganciclovir) versus artesunate that were recently described as highly effective against herpesvirus infections in ASD patients.¹⁹ The suppression of these reactivated opportunistic agents can reasonably be assumed as beneficial for the clinical status of ASD children, for it reduces the negative impact of viral factors on the brain damage pathways. It helps to achieve a better clinical outcome and expand the social adaptation range of children with ASD. Therefore, there is an urgent need to study the safety and efficacy of different antiviral drug products used in the treatment of reactivated herpesvirus infections in children with ASD in order to assess the potential impact of such treatment on their mental status.

This study aims to investigate the efficacy of valaciclovir, valganciclovir, and artesunate in treating reactivated Epstein-Barr virus (EBV), herpes virus type 6(HHV-6) and herpes virus type 7(HHV-7) infections in children with an autism spectrum disorder. Here, the ASD diagnoses were associated with genetic folate cycle disruptions (GFCDs). The laboratory markers of brain damage suggest a neuroprotective effect. The secondary objectives of the study are (1) to determine which antiviral drug is most effective in treating chronic reactivated EBV, HHV‐6 and HHV‐7 infections in ASD patients with GFCDs and (2) to link clinical outcomes with the serum levels of brain damage markers, specifically neuron-specific enolase (NSE) and S-100 protein.

MATERIALS AND METHODS:

Sampling:

An investigator reviewed medical records of 225 children with genetic folate cycle disruptions (GFCDs) and an autism spectrum disorder (ASD) who were between the ages of 2 to 9 years (treatment group); 183 were boys and 42 were girls (average age: 4.26±0.31 years). The difference in the number of boys and girls in observation groups is associated with gender differences in the prevalence of ASD in children, since boys are affected 3-4 times more often than girls.

All patients were treated in the Vivere clinic (Kyiv, Ukraine; registration No. 10/2212-М, dated 12/22/2018) between 2019 and 2022. The clinical materials were examined under Cooperation Agreement No. 150221 (dated February 15, 2021) at the Research Institute of Experimental and Clinical Medicine affiliated with the O’Bogomolets National Medical University (Kyiv, Ukraine). The clinical analysis protocol was issued by the Commission on Biotic Expertise (protocol No. 140, dated 21.12.2020). The children were assessed according to DSM-IV-TR criteria by experienced pediatric psychiatrists. All participants in the study who had already been diagnosed with ASD by local specialists had their ASD diagnosis reconfirmed by the same group of highly qualified child psychiatrists with the appropriate experience in such assessments. The ability to assemble such a large group of ASD patients over the 4-year study period was facilitated by funding from the Ministry of Health of Ukraine, enabling coordination with regional hospitals. Additionally, the Vivere clinic, being the sole neuroimmunology centre in Ukraine with a population of 40 million, played a crucial role.

Research Design:

This is a single-center prospective controlled non-randomized interventional clinical study.

Ethical Statement:

The research complies with the guidelines for human studies and was conducted ethically in accordance with the World Medical Association Declaration of Helsinki. All patients were treated in the Vivere clinic (Kyiv, Ukraine; registration No. 10/2212-М, dated 12/22/2018) between 2019 and 2022. The clinical materials were examined under Cooperation Agreement No. 150221 (dated February 15, 2021) at the Research Institute of Experimental and Clinical Medicine affiliated with the O’Bogomolets National Medical University (Kyiv, Ukraine). The clinical analysis protocol was issued by the Commission on Biotic Expertise (protocol No. 140, dated 21.12.2020). The written informed consent was obtained from participants.

Methods (Procedures):

The diagnosis of GFCD was established by detecting pathogenic polymorphic variants in the genes of folate cycle enzymes. Identification was performed in the Synevo laboratory (Ukraine) using the polymerase chain reaction (PCR) method. MTHFR C677T mutations were present alone (n = 68, 30%) and in concert with other variants, including MTHFR A1298C, MTR A2756G and/or MTRR A66G (n = 157, 70%, Figure 1, table 1). The prevalence of compound MTHFR variations was as follows: MTHFR C677T+MTHFR A1298C was detected in 26 patients (12.5%); MTHFR C677T+ MTRR A66G was present in 19 patients (8.5%); and 25 children were positive for MTHFR C677T+MTR A2756G mutations (11%). There were also cases of a triple-variant mutation, namely MTHFR C677T+ MTRR A66G+MTR A2756G (n = 23, 10.5%), MTHFR C677T+MTHFR A1298C+MTR A2756G (22, 9.5%), and MTHFR C677T + MTHFR A1298C + MTRR A66G (21, 9%). Some 21 (9%) children had a quadripartite MTHFR C677T + MTHFR A1298C + MTR A2756G + MTRR A66G mutation.

Figure 1. The percentage of patients with GFCDs based on PCR findings (n = 225)

The diagnosis of reactivated EBV, HHV-6, and HHV-7 infections was performed using Biokom’s reagents for PCR (Russia) with species-specific primers (Romodanov Institute of Neurosurgery, National Academy of Sciences of Ukraine). The study identified 133 patients with EBV reactivation (59%), 153 patients with HHV-6 reactivation (68%), and 178 patients with HHV-7 reactivation (79%). Mixed infection occurred in 135 children (60%).

Serum levels of NSE and S-100 protein were measured by Electrochemiluminescence assay (ECLIA) with a Cobas 6000 analyzer (Roche Diagnostics, Switzerland) in Sinevo (Ukraine). The cut-off values for these brain damage markers were <16.3ng/ml and <0.105μg/l, respectively. Patients appeared to have elevated serum NSE and S-100 concentrations at diagnosis, which suggests the presence of encephalopathy and some psychiatric symptoms. The mean NSE concentration was 25.38±3.56ng/ml, and the mean serum S-100 concentration was 131.94±12.46μg/L in the studied group, whereas in the control group, these values were 15.17±2.61ng/ml and 102.71±9.48μg/L respectively.

Research Protocol:

The parents of each child participating in the study signed informed consent for participation in the research the day before inclusion in the trial.

Patients in the SG were asked to take one of the study drugs against herpesviruses in case of detection of reactivated herpesvirus infections without randomization on the basis of a voluntary agreement with the researcher, taking into account the cost of the drug, the availability of the drug on the pharmaceutical market at the time the patient entered the study, the history of tolerability of antiviral drugs in the child, individual concerns of the child's parents about any drug. The study antiviral agents were valacyclovir (Valtrex; GlaxoSmithKline, UK), valganciclovir (Valcyte; Hoffmann-La Roche, Switzerland) and artesunate (Artesunat; Mekophar Chemical Pharmaceuticals Joint‐Stock Company, Vietnam). The substances were administrated orally for 3 consecutive months. The dosage varied depending on the patient's age and body weight. Patients received valacyclovir 500 to 1000mg thrice daily (n = 60: 41 – EBV, 52 – HHV-6, 59 – HHV-7), valganciclovir 225 to 450mg twice a day (n = 59: 45 – EBV, 49 – HHV-6, 56 – HHV-7), and artesunate 25 to 50 mg twice daily (n = 59; 47 – EBV, 52 – HHV-6, 63 – HHV-7). The viral activity was monthly monitored via blood leukocyte PCR tests. The patients abstained from the intake of other medications to avoid pharmacological interactions.

The control group included 52 children diagnosed with ASD who also had GFCDs; 37 were boys and 15 were girls aged 2 to 8 years. The study group and the control group had similar proportions of reactivated EBV, HHV-6- and HHV-7 infections. Patients in the control group received no antiviral medication during the observation period (3 months) and were monthly monitored through blood leukocyte PCR testing to assess viral activity.

Statistical Analysis:

Clinical outcomes were analyzed by means of comparative and structural analyses. The distribution of quantitative values in the studied sample for compliance with the normal distribution was checked using the Shapiro-Wilk test. For parameters that did not demonstrate a normal distribution pattern, the results in the observation groups were compared using the Kruskal-Wallis rank one-way analysis, and for pairwise comparisons, the Dunn or Mann–Whitney test with Bonferroni correction was applied. A Student's T-test was applied to assess the probability of differences between groups; the Z-values were determined according to V. Yu. Urbakh.²⁰ Differences were considered probable at p<0.05 and Z<Z_0.05.

The difference between groups was considered significant if the null hypothesis was not confirmed (Z < Z_0,05). The main steps of calculation based on the criterion of signs include the following:

1. Defining the tendency in groups under comparison; dynamics is marked with corresponding signs +, –, =; results with no dynamics are excluded from further calculations (=).

2. Calculating the number of observations with positive and negative outcomes.

3. Calculating the number of characters that are less likely to occur.

4. Comparing a smaller number of signs (Z‐criterion) with the tabulated critical value for the corresponding number of observations.

The odds ratio (OR) and 95% confidence interval (95% CI) were used to examine associations between antiviral treatment outcomes and brain damage markers. Statistical calculations were done in Microsoft Excel (Redmond, WA).

RESULTS:

Based on PCR findings, all antiviral agents under analysis inhibited viral reproduction in blood leukocytes regardless of the infection scenario. This affected the course of herpes virus infections, as indicated by between-group differences at all time points (p<0.05; Z<Z_0.05; Figures 2-4). The antiviral agents in point demonstrated equivalent efficacy with mono-infections and some mixed infections, such as EBV + HHV-6, EBV + HHV-7, HHV-6 + HHV-7, and EBV + HHV-6 + HHV-7. There were no differences in treatment effectiveness between viruses in mono- and mixed infections (р<0.05; Z<Z_0.05). The most likely factor to influence the effectiveness of antiviral treatment was the antiviral drug resistance of the virus strain, rather than the total viral load. Each virus responded separately to the treatment, and their combination with another virus or virus generally did not change the response pattern.

All antiviral agents under analysis demonstrated the same therapeutic action in all infection scenarios. Specifically, the number of responders was progressively increasing, while the number of negative PCR results was gradually reducing (Figure 3). At the same time, these drugs were not equally potent in suppressing herpes virus reproduction in blood; the therapeutic effect varied significantly between species.

Valacyclovir treatment achieved undetectable EBV DNA in 25% of treated patients in the first month after the treatment began. This percentage later increased to 36 and 39% at 2 and 3 months. In PCR assays with species-specific EBV primers, some 34% of treated patients who received valgancyclovir yielded negative results over one month of treatment. After 2 and 3 months, the negative rate reached 42 and 47%, respectively. Artesunate was more effective in comparison. Of all patients receiving this agent, 41% achieved undetectable EBV DNA after one month of treatment. At 2 and 3 months, EBV DNA was undetectable in 55 and 62% of patients, respectively (Figure 2).

After valacyclovir, 19% of treated patients had HHV-6 DNA undetected at 1 month, 26% at 2 months, and 29% at 3 months. By PCR with species-specific HHV-6 primers, 25% of treated patients with valganciclovir yielded negative results at 1 month. After 2 and 3 months, the negative rate reached 29 and 32%, respectively. Artesunate was more effective once again, with HHV-6 DNA being undetected in 34, 47 and 57% of treated patients at 1, 2 and 3 months, respectively (Figure 3).

After 1 month, patients with undetectable HHV-7 DNA in the valacyclovir group accounted for 14% of cases examined. This percentage was higher at 2 and 3 months (19 and 24%, respectively). The PCR tests with species-specific HHV-7 primers revealed negative rates of 22, 31 and 35% at 1, 2 and 3 months of valganciclovir treatment, respectively. Artesunate, was more effective, with HHV-7 DNA undetected in 31, 39 and 44%, respectively (Figure 4, table 2). Differences in the effectiveness of valacyclovir, valganciclovir, and artesunate in infections caused by various herpesviruses after each month of the treatment course are presented in Figures 5, 6, and 7.

Figure 2. The overall response rates for EBV treatments over time

Figure 3. The overall response rates for HHV-6 treatments over time

Figure 4. The overall response rates for HHV-7 treatments over time

Figure 5. Complete response to valacyclovir in infections caused by herpesviruses of different types in the treatment group (n=225) during treatment.

Figure 6. Complete response to valganciclovir in infections caused by herpesviruses of different types in the treatment group (n=225) during treatment.

Figure 7. Complete response to artesunate in infections caused by herpesviruses of different types in the treatment group (n=225) during treatment.

Table 2. The proportion of complete responders to various antiviral drugs among patients in the treatment group (n=225) after 1, 2, and 3 months of the therapeutic course.

Medication	EBV			HHV-6			HHV-7
Medication	1 mo	2 mo	3 mo	1 mo	2 mo	3 mo	1 mo	2 mo	3 mo
Valacyclovir	25%	36%	39%	19%	26%	29%	14%	19%	24%
Valganciclovir	34%	42%	47%	25%	29%	32%	22%	31%	35%
Artesunate	41%	55%	62%	34%	47%	57%	31%	39%	44%

As can be seen, there are associations between negativization of the PCR tests and normalization of the brain damage markers over the 3-month treatment period. Note that the strength of these associations corresponds to the antiviral drug resistance of herpes viruses. As the least resistant virus, EBV had the closest association with NSE and S-100 protein, and the most resistant virus, HHV-7, had the most distant association, by comparison. In addition, the normalized NSE concentration was more closely associated with effective antiviral treatment than the normalized S-100 level, regardless of the antiviral agent used. These findings suggest the potential neuroprotective effect of antiviral treatment in children with ASD who have GFCDs. More studies are needed to validate this assumption.

No gender differences in the effectiveness of the tested antiviral drugs were found in all investigated reactivated herpesvirus infections in the treatment group.

The antiviral drugs under analysis showed good tolerability. Valacyclovir caused a slight increase in hepatic transaminase levels, but only 7% of patients were affected. With valganciclovir, this percentage was 19%. It also caused neutropenia (16%). Finally, the adverse effects of artesunate included mild anaemia and neutropenia, which were observed in 8 and 11% of treated patients. The said side effects were mild and did not disrupt the course of treatment.

The limitations of this study include the absence of randomization, placebo control, and participant blinding, as well as the restriction to investigating associations solely through the examination of laboratory biomarkers of cerebral damage without employing clinical criteria for mental status throughout the treatment course.

DISCUSSION:

Therefore, the results of this controlled clinical study indicate that valacyclovir, valganciclovir, and artesunate exert certain antiviral effects in reactivated EBV, HHV-6, and HHV-7 infections in children with ASD associated with GDFC. It has been demonstrated that the number of complete responders to antiviral therapy increases with each month of therapy in a diminishing manner when using each of the tested agents. Artesunate has been established as the most effective, valganciclovir exhibits moderate efficacy, and valacyclovir is the least effective antiviral drug among the three tested medications. Furthermore, EBV is the most sensitive, HHV-6 demonstrates moderate sensitivity, and HHV-7 is the least sensitive agent to the tested antiviral drugs. However, regardless of the agent used, a considerable number of non-responders are observed following the completion of the three-month therapy course for the reactivation of each of the three herpesviruses, indicating a significant prevalence of resistant cases and necessitating the exploration of additional methods for antiviral treatment.

Many studies exist on ASD and its association with reactivated herpes virus infections. The mechanisms whereby these viral agents affect encephalopathy have also been substantiated. It is known that the increased risk of developing encephalopathies due to the reactivation of latent viral infections can be determined by various factors, such as gender, genetic background and immune dysfunction^22,23 Herpevirus infections are reactivated with the participation of cellular immunity, and occur 20-100 times more often in patients who have reduced cellular immunity.^24,25 Such patients are, in particular, people with autism spectrum disorders.²⁶ Even so, it is difficult to find controlled clinical trials that would examine the therapeutic efficacy of certain antivirals in cases such as this. With this in mind, the present study seems to be of scientific value. It provides evidence that different herpes viruses have different resistance to antiviral drugs and that certain antivirals are better than others in treating reactivated infections caused by these herpes viruses. This information can be used to optimize antiviral treatment outcomes in children with ASD who also have GFCDs. The study also shows a supposed connection between negative PCR results and normalized NSE and S-100 protein levels, suggesting the potential neuroprotective effect of antiviral treatment among children with ASD who also have GFCDs. These biomarkers were chosen specifically because they were rather informative for assessing encephalopathy in previous clinical studies.²⁷ A meta-analysis of 10 randomized clinical trials established that the serum level of S-100 protein is likely to be higher among children with ASD than among healthy individuals;²¹ therefore, it can be used as a brain damage biomarker in cases such as this. A controlled clinical study with 80 patients revealed a significantly higher NSE serum concentration in children with ASD as compared with mentally healthy individuals.²⁸

The detection of immunodeficiency and immune dysregulation in ASD children helps to understand what causes the opportunistic herpesvirus to reactivate.⁹ Binstock was the first person to reveal the selectively reduced resistance of ASD children to opportunistic and conditionally pathogenic agents. The researcher distinguished a special category of patients prone to infections caused by intra-monocyte pathogens, namely measles virus, CMV, HHV-6, and Yersinia enterocolitica.²⁹ In such children, diminished hematopoiesis, immune impairments, and altered blood-brain barrier function may be seen. These clinical and laboratory signs were reported as common for GFCD.³⁰ Another clinical study with blood leukocyte PCR as its main research method found a high prevalence of Mycoplasma, Chlamydia pneumoniae and HHV-6 infections among ASD patients.¹³ Some researchers report that ASD children are more likely to have anti-EBV IgM antibodies in their serum than healthy individuals.¹¹ Such children are also more prone to congenital cytomegalovirus neuroinfection (7.4% vs 0.31%, respectively, p=0.004), as evidenced by real-time PCR of dried blood and umbilical-cord specimens.¹² Another controlled clinical study argues that there is an association between ASD and reactivation of EBV, HHV-6 and HHV-7.¹⁹ The blood serum PCR is less sensitive than blood leukocyte PCR and detects fewer cases of herpes virus reactivation in ASD children.³¹

Scientists currently discuss whether herpes virus agents have an active role in ASD patients’ pathogenesis of encephalopathy. Herpes viruses are capable of exerting both direct damaging effects on the brain parenchyma, manifesting their neurotropic cytopathic effect, and realizing CNS damage through some indirect immune-mediated mechanisms, such as by modulating systemic and/or intracerebral inflammation or by inducing anti-brain autoimmunity.

The direct brain damage from herpes viruses manifests itself in the induction of encephalitis and some neurodegenerative processes. The infectious process covers both the brain and spinal cord, as well as the meninges.³² There are several known cases where an HSV-1-induced episode of temporal necrotic-hemorrhagic encephalitis triggers the acute development of autism.^10,33 This process exemplifies the direct (encephalitic) damaging effect that herpes viruses have on CNS in ASD patients. HHV-6 is capable of travelling from the upper respiratory tract into the brain using the olfactory pathway,³⁴ and it can affect the limbic structures. At the same time, the virus induces a specific neurodegenerative process called mesial temporal sclerosis (MTS),¹⁴ the clinical and radiological signs of which can be observed in ASD patients.³⁵ The association between HHV-6 and MTS was recently confirmed by a meta-analysis of randomized controlled clinical trials.¹⁴ This may be the second way in which the direct damage of herpes viruses manifests itself. It is most likely an important component in the pathogenesis of encephalopathy in ASD children with GFCDs.

Regarding the potential indirect effects of herpes viruses on the brain, there are two main pathways to distinguish. First, herpetic agents can act as triggers for anti-brain autoimmunity. ASD patients with serologic evidence of HHV-6 infection were reported to also have laboratory signs of anti-brain autoimmunity.³⁶ According to the current reports, ASD children with autoimmune limbic encephalitis respond positively to recommended immunomodulators.^37,38 In such cases, however, herpes viruses, including HHV-7, provoke a loss of immune tolerance to certain autoantigens.¹⁸ On the other hand, herpes viruses, especially HHV-6, can modulate systemic inflammatory responses with hypercytokinemia in patients with immune dysregulation.¹⁶ Systemic hypercytokinemia with an aberrant proinflammatory profile is common in ASD, as evidenced by recent meta-analyses and systematic reviews.^39,40 When reactivated, HHV-6 can induce the hyperactivation of macrophages, causing the subsequent development of hypercytokinemia.¹⁶

In ASD, inflammatory reactions associated with herpes virus-induced brain damage take place along the microbiota-gut-brain functional axis. This means that by increasing the intensity of inflammation in the intestinal wall, herpes viruses can provoke further inflammation, extending the inflammatory site to the blood-brain barrier and the brain.⁴¹

The concept of the negative impact of viral infections on the mental health of children with ASD is supported by the results of an observational cross-sectional study by Amorim R. et al. on the psychiatric consequences of COVID-19 in ASD.⁴² Microbe-mediated mechanisms altering social behaviour have been demonstrated in experimental animal models inducing genetic, environmental, and idiopathic forms of ASD in mice.⁴³ Findings from the literature review by Shuid AN et al., covering data from 141 clinical studies, indicate that viral infections during pregnancy in mothers and during early childhood in the child increase the risk of ASD development.⁴⁴ Such influences can modify or potentially increase the severity of the initial genetic mechanisms of disease development.⁴⁵ Al-Beltagi M. et al. in a review analyzing the results of 158 clinical studies identified a bi-mutual effect of viral infections in ASD children. On one hand, viral agent infections increase the risk of ASD development, and, on the other hand, children with ASD exhibit an abnormally elevated frequency of viral infection episodes due to a state of persistent immune dysregulation.⁴⁶ The recent review by Abib RT et al. confirms early concepts formed by Binstock T. in 2001 that ASD is primarily associated with intracellular (intramonocytic) infectious agents, including herpesviruses, which play a direct or indirect role in sustaining chronic intracerebral neuroglial inflammation in the brains of children with ASD.⁴⁷

A similar comparative study investigates the efficacy of valacyclovir, valganciclovir, and artesunate in treating reactivated HHV-6 and HHV-7 infections associated with chronic fatigue syndrome/myalgic encephalomyelitis.¹⁹ Valacyclovir and valganciclovir are inhibitors of viral DNA replication, while effects of artesunate still remain unclear.⁴⁸^-50 Note that despite the similar therapeutic effects of these antivirals, ASD children were significantly less responsive to antiviral treatment than adults with chronic fatigue syndrome/myalgic encephalomyelitis. The likely explanation for this difference is the low intestinal absorption of antiviral drugs associated with specific inflammatory lesions of the small intestine in ASD children.⁵^1-53 This necessitates the need to study the pharmacokinetics and pharmacodynamics of antiherpetic drugs in ASD. Another possible explanation is the potentiating effect of GFCD on the reproduction of herpes viruses.¹⁵ The herpes virus agents can also use human pro-inflammatory cytokines as a stimulator for DNA replication.⁵^4-56 Therefore, systemic and neuroglial inflammation with persistent hypercytokinemia that was seen in ASD children⁵^7-59 can be an important factor behind the antiviral treatment efficacy.

CONCLUSION:

Based on the PCR findings, the antiviral drugs under analysis intervened with the natural course of chronic reactivated EBV, HHV-6, and HHV-7 infections in ASD children, reducing the viral DNA content in their blood leukocytes. Artesunate appeared to be the most effective drug, with the highest number of complete responses at each follow-up. Valacyclovir was the least effective one compared to the other two antiviral options. EBV is more sensitive to these antiviral drugs than HHV-7. The elimination of herpes virus agents in blood leukocytes is associated with the normalization of brain damage markers, which indicates a potential neuroprotective effect of antiviral treatment in children with ASD. Due to the large number of non-responders with different herpesvirus infections, it seems reasonable to search for ways to improve the effectiveness of antiviral treatment in ASD. The present findings can be used by pediatric psychiatrists, clinical immunologists and infectious disease specialists who manage ASD children to plan antiviral procedures in the event of reactivated herpesvirus infection. Up to this point, no clinical trials have been conducted to assess the efficacy of antiviral therapy in children with ASD associated with GDFC. Therefore, the data from this study elucidate the effectiveness of antiviral drugs and demonstrate the advantages of certain medications over others in this patient cohort. The results of this study can also be utilized in further clinical trials to examine the virological aspects of ASD. Additional research is needed to study mental and neurological changes after antiviral therapy in children with ASD to clarify whether the phenomenon of normalization of laboratory biomarkers of brain damage identified in this work has clinical significance.

CONFLICT OF INTEREST:

The author declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

AUTHOR CONTRIBUTIONS:

Dmytro Maltsev: conceptualization, methodology, software, validation, formal analysis, investigation, resources, data curation, writing—original draft preparation, writing-review and editing, visualization, supervision, project administration, funding acquisition.

FUNDING:

This clinical study was supported by research funds allocated in the state budget (No. 0121U107940).

DATA AVAILABILITY STATEMENT:

Data will be made available on request.

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Received on 09.01.2024 Modified on 01.05.2024

Research J. Pharm. and Tech 2024; 17(9):4177-4186.

DOI: 10.52711/0974-360X.2024.00646

Variables	EBV		HHV-6		HHV-7
Variables	OR crude; 95% CI	OR adjusted*; 95% CI	OR crude; 95% CI	OR adjusted; 95% CI	OR crude; 95% CI	OR adjusted; 95% CI
NSE	8.32; 3.85- 17.97	8,31; 3,82-17,7	4.58; 2.27-9.22	4,58; 2,26-9,22	5.37; 2.78-10.34	5,35; 2,74-10,31
S-100 protein	5.38; 2.58-11.21	5,39; 2,59-11,23	4.13; 2.04-8.35	4,11; 2,01-8,31	3.70; 1.94-7.05	3,68; 1,91-7,02

Genotype	Both Genders		Boys		Girls
Genotype	ABS	%	ABS	%	ABS	%
MTHFR C677T	68	30%	56	24,7%	12	5,3%
MTHFR C677T + MTHFR A1298C	26	12.5%	21	10,5%	5	2%
MTHFR C677T + MTRR A66G	19	8.5%	16	7,2%	3	1,3%
MTHFR C677T + MTR A2756G	25	11%	20	9%	5	2%
MTHFR C677T + MTRR A66G + MTR A2756G	23	10.5%	17	7,5%	6	3%
MTHFR C677T + MTHFR A1298C + MTR A2756G	22	9.5%	18	7,8%	4	1,7%
MTHFR C677T + MTHFR A1298C + MTRR A66G	21	9%	17	7,3%	4	1,7%
MTHFR C677T + MTHFR A1298C + MTR A2756G + MTRR	21	9%	18	7,7%	3	1,3%