Open Access Case Report Volume 1 Issue 1 Journal of Clinical Case Reports and Clinical Research

Tendencies in Changes of Blood Parameters and in Autistic Symptoms Improvements as a Result of Antiviral Treatment: Descriptive Case Series of 11 Children from Eastern Europe and Central Asia Kenneth Alibek1,2*, Sean Farmer1,2, Albina Tskhay2, Alibek Moldakozhayev2, Kira Alibek2and Terence Isakov3 1FLAASK, LLC, 30500 Aurora Road, Suite 120, Solon, 44139, OH, USA 2Locus Fermentation Solutions, LLC, 30500 Aurora Road Suite 180, Solon, 44139, OH, USA 3New Family Physicians Associates, Inc, Lyndhurst, OH, USA *Corresponding Author: Kenneth Alibek, FLAASK, LLC, Senior Vice President, R&D at Locus Fermentation Solutions, Locus Fermentation Solutions, LLC Ohio, USA. E-mail: Citation: Kenneth Alibek, Sean Farmer, Albina Tskhay, Alibek Moldakozhayev and Kira Alibek., et al (2019) Tendencies in Changes of Blood Parameters and in Autistic Symptoms Improvements as a Result of Antiviral Treatment: Descriptive Case Series of 11 Children from Eastern Europe and Central Asia. J Clin Case Rep Clin Res 1(1): 102

Abstract In this descriptive series 11 ASD cases with detected chronic viral infection and inflammation were treated by standard anti-viral regimen for 6-12 months. It was observed that in all 11 children the antiviral treatment resulted in normalization of blood parameters which indicate infection and inflammation. At the same time the decrease in the CARS-2 scores and positive behavioral changes observed by parents had a tendency to occur after the normalization of these parameters. Further studies are needed to make a clear conclusions about this effect of antiviral treatment on autism. Keywords: Autistic Disorder; Behavior; Immune System; Infection; Inflammation; Nutritional supplements; Treatment; Valacyclovir Abbreviations I: ASD: Autism Spectrum Disorder; HSV: Herpes Simplex Virus; EBV: Epstein - Barr virus; CMV: Cytomegalovirus; HHV: Human Herpes Virus;

Introduction Autism spectrum disorder (ASD) is a neurodevelopmental pathology characterized by a number of signs and symptoms including, but not limited to: impairment of social communication skills, repetitive and restricted behavior and sometimes by language skills development delay [1]. Despite a long history of autism research, which started when Hans Asperger described the first observed cases in 1938 [2], and then Leo Kanner gave the first definition and described symptoms in 1943 [3], ASD as a disorder is still thought to be untreatable. One of the reasons for the lack of effective treatment is the absence of consensus regarding the etiology of the disorder. While there is a universal agreement on the role of genetic changes, the environmental causes are still unknown. With dozens of theories speculating the etiology of ASD: the damaging effects of heavy metals, maternal metabolic conditions, and parental age, etc., infection and inflammation are the factors that have the strongest evidence in terms of their role in ASD [4]. Several large studies conducted in Sweden, Denmark and Israel showed the statistical correlation between maternal infection during pregnancy and the following ASD diagnosis in a child [5-7]. Along side of that, numerous animal studies showed that the exposure of pregnant animals to viral infections or to substances induced a similar immune response that resulted in autistic behavior in the offspring [8-12]. Infections that were found to be statistically significant in children with ASD include: herpes simplex virus 1/2 (HSV) [13], Epstein-Barr virus (EBV) [14], Cytomegalovirus (CMV) [15,16], human herpesvirus 6 (HHV 6) [17], rubella virus [18], Chlamydia spp. and Mycoplasma spp [17]. Inflammation is an important indicator of both infection and autism. According to [19], at least 69% of children

© All copy rights are reserved by Kenneth Alibek www.scienceinquest.com Journal of Clinical Case Reports and Clinical Research

with ASD have detectable neuroinflammation. In a review article by [20], the authors summarized the evidence of inflammation in the brain of ASD children published in the years from 2005 to 2013. As a summary of the reviewed studies it was shown that ASD children have: activation of microglia and astroglia in the middle frontal gyrus, anterior cingulate gyrus and cerebellum, increased levels of anti-inflammatory cytokine tumor growth factor–1 and pro- inflammatory macrophage chemoattractant protein-1, increased levels of IFN-gamma, MCP-1, TGF-beta2, and IL-8 in cerebrospinal fluid (CSF), decreased quinolinic acid and neopterin, an increased level of biopterin in CSF, increased TNF-alpha level in CSF, elevated expression levels of some of the immune-related genes in the superior temporal gyrus, reactive gliosis in BA22, BA44, and BA39, increased proinflammatory cytokines in the frontal cortex, elevated NF-κB expression in the orbitofrontal cortex and increased 3-chlorotyrosine concentrations in the cerebellum and temporal cortex. Additionally, it was found that the levels of cytokines IL-1β, IL-6, IL-17, and TNF-α are increased in the brains of autistic children [21]; all of these parameters are indicators of neuroinflammation. Despite this, blood tests that assess levels of infection and inflammation are not included to the routine tests conducted for children with ASD, and antiviral and anti-inflammatory treatment is not part of the treatment regimen for children afflicted with ASD. Based on this, the group of children, whose blood tests indicated the presence of chronic viral infections and markers of inflammation were treated with an approved antiviral drug, Valacyclovir, and by supplements which are shown by studies to be effective against viral infections and inflammation and safe for children. The aim of this approach was the treatment of detected viral infections and inflammation, not ASD per se. However, as a result of this treatment, there were pronounced, observable positive changes in regards to both signs and symptoms. In this paper, we describe 11 cases of children who underwent long-term antiviral treatment, and as a result, showed substantial positive changes in terms of their ASD signs and symptoms [22]. Treatment Rationale Valacyclovir is an antiviral drug reported to be effective in the inhibition of herpes viruses: HHV-1 [22], HHV-2, HHV-3/VZV [23,24], and HHV-4/EBV [25,26], HHV-5/CMV [27]. In a 2002 clinical trial, Valacyclovir was tolerated well by children with the only side effects being headache in 8%, and gastrointestinal disturbances in 16% [28]. L-lysine is an amino acid that is essential to the human organism, due to its ability to aid in the processes of proper growth, muscle protein building and metabolism [29]. L-lysine can inhibit herpes simplex virus by reducing the cyto- pathogenicity of the HHV-1 [30]. It was also reported that L-lysine wasn’t toxic and didn’t cause any side effects [30]. Elderberry extract in a nutritional supplement extracted from the black elder (Sambucus nigra L.). This supplement demonstrated antiviral (influenza A and B, RSV and parainfluenza), anti-inflammatory, immunomodulatory, and antioxidant activities. Multiple clinical trials showed that elderberry root extract was effective against influenza A and B virus infections [31-33], respiratory syncytial virus (RSV), parainfluenza [31] and HHV-1 [34]. Furthermore, it was proven that there are no side or adverse effects to taking elderberry extract for all age groups including infants [33]. Olive leaf extract has been reported to possess hypoglycemic, antioxidant, antiviral and antimicrobial effects [35]. A double-blinded placebo-controlled trial [36] showed that olive leaf extract stimulates downregulation of genes that participate in inflammatory processes, (EGR1, COX-2 and ID3), and another study by [37] demonstrated a significant reduction in the production of cytokines participating in the inflammatory processes; these findings were supported by other studies [38,39]. Olive leaf extract has also demonstrated antiviral and anti-oxidant activities [40,41], such as decreasing the viral infectivity of viral haemorrhagic septicaemia virus (VHSV) by 10-30% [41]. Astragalus root extract is extracted from Astragalus membranaceus Moench herb, and has demonstrated the ability to stimulate the immune system [42]. It was observed that intake of astragalus root extract enhances the production of macrophages and lymphocytes [43,44], these cells being key players in the immune system. Studies done by [42] and [45] showed that Astragalus root extract has antiviral, anti-inflammatory, and antibacterial properties as well. Bacillus coagulans probiotic was shown to improve digestive health by allowing for more effective digestive absorption and modulation of gut microbiota [46,47]. This probiotic's property is especially important to children who have ASD, because 70% of children who had ASD also report to having various problems of the gastrointestinal tract [48]. Intake of this probiotic demonstrated positive influence on ASD children because its able to target two vital issues at once: microbiota-gut-brain axis, through balancing the microbiota. And the inhibition of the toxic effects of some compounds that influence the brain, which leads to a decrease in ASD symptoms [49]. Several studies also indicated that Bacillus Coagulans probiotic intake increases the immune response to viral agents via increased production of

Volume 1 Issue 1 www.scienceinquest.com | Page 2 of 13 Journal of Clinical Case Reports and Clinical Research

T-cells as a response to viral infections [50] and it was also reported that the Bacillus coagulans probiotic has the ability to inhibit 3 viruses of the herpes viruses’ group: HHV-1, HHV-2 and HHV-3 [51]. The toxicological assessment of Bacillus Coagulans probiotic showed that the supplement had no toxic or other side effects [52]. Method Participants All children were self-referred by their parents. The study included 11 children, 10 males and 1 female, ages ranging from 2.5 to 9 years old, all from different cities of Ukraine and Kazakhstan. The study was not prospective, and there were no exclusion criteria; the only inclusion criteria being that the parents of the children confirmed the ASD diagnosis, and that the parents gave written consent, allowing their child to be included in the study. With this consent, the child and their parents are guaranteed a non-disclosure agreement, which in the study, we will not disclose them or their child's identities. Confirmation of diagnoses This study was conducted using both written questionnaires and a tele-medicine approach. The telecommunications application, Skype®, was used for the consultations and general communication. All participants completed a questionnaire CARS-2, via email-based communication, which assessed the presence of ASD symptoms and their severity, to confirm the diagnosis of ASD and the prevalence of signs/symptoms. It was explained to parents that this study did not undertake the treatment of autism, but rather the study would focus on the diagnosis and treatment of chronic/latent infections, reduction of the child’s inflammatory state, and the modulation of the child’s immune system. Parents were asked to have laboratory tests done of their child’s: presence of antibodies to viral and bacterial infections, and red/white blood cell counts. If the parents agreed, the tests were performed by local clinical diagnostic centers, which parents had the decision to choose where the tests were conducted. Treatment groups and regimens When the presence of viral/bacterial infection were confirmed by blood test results, parents received the following recommendations for treatment. All children were divided into three groups by age and treatment regimens Ages up to 3 years: 3 cycles of Valacyclovir treatment (125 mg, twice a day). Each cycle continues for 3 weeks with a 9-day break, along with the sustained intake of 125 mg of each supplement and 0.5 bln CFUs of probiotic per day without break. Ages 3 to 7: 3 cycles of Valacyclovir treatment (250 mg. twice a day). Each cycle continues for 3 weeks with a 9-day break, along with the continuous intake of 250 mg of each supplement and 1 bln CFUs of probiotic per day without break. Parents were advised to not administer any other drugs or supplements, unless there were extraneous circumstances that made the intake of other medications necessary (e.g. painkillers for headache, antibiotics for bacterial infection, etc.). Assessment of the results Assessments were based on bi-monthly or monthly test results that recorded antibody levels to infections in blood samples, and red and white blood cell count tests. Assessments also relied on the parent’s observation of their child’s ASD symptoms and other ASD-related disorders. With the analysis of the changes in blood parameters, we mainly focused on white and red blood cell counts (erythrocyte sedimentation rate (ESD), neutrophil, lymphocyte, erythrocyte and monocyte counts) and antibodies levels to viral infections detected in relation to the reference ranges established at the local diagnostic centers. Assessments of treatment results was conducted via video-conferences Parents were asked to fill out CARS-2 (Childhood Autism Rating Scale-2) [53], which is the upgraded version of the CARS, the instrument for the assessment of the severity of the autistic symptoms in children. The questionnaire consisted of 15 questions about autistic symptoms, which were rated on a 4-point scale with an option of choosing 0.5 interval points. The sum of the points was interpreted in the following way: 0-30 – no autism; 30-36 – mild to

Volume 1 Issue 1 www.scienceinquest.com | Page 3 of 13 Journal of Clinical Case Reports and Clinical Research

moderate autism; 36 to 60 – severe autism. This questionnaire was not intended for diagnostic purposes, in this study it was used only for the evaluation of the dynamics of the treatment results. In order to avoid false-positive results, the parents were asked to score the child both at the moment of treatment initiation and 3 months before the initiation of the treatment. Due to the method of conducting the study, parents had the choice not to do some of the blood works. That is why there are some missing values at some points of the treatment. Results and Discussion The results are represented as tables for each case. The tables show how the main parameters of blood, IgG to viruses detected and CARS scores were changing over time. The decrease of CARS score means that the severity of autistic symptoms also declines, while the CARS scores below 30 may suggest the absence of autistic symptoms. For all of the 11 children the CARS score at the moment of the treatment was equal to the CARS score 3 months before the initiation of the treatment. Abbreviations II: CARS: The score in accordance with Childhood Autism Rating Scale-2. 36 and above – severe autism, 30 to 36- mild to moderate autism, below 30 – no autism; RBC: Red blood cells count. The reference ranges vary in different countries and clinics. Increase in the red blood cells count may indicate inflammation, polythetemia, or hypoxia and oxidative stress [54,55]. NEU: Neutrophil count. The reference ranges vary in different countries and clinics. Decrease in the neutrophil count usually indicates a viral infection [56], inflammation and a disrupted immune system [57]. MON: Monocyte count. The reference ranges vary in different countries and clinics. Increase in monocyte count points to the viral or bacterial infection, often monocyte level increase is caused by herpetic infection [58]. LYM: lymphocyte count. The reference ranges vary in different countries and clinics. Lymphocytosis may be a sign of chronic infection and in particular often a sign of chronic herpetic infection [59,60]. ESD: erythrocyte sedimentation rate. The reference ranges vary in different countries and clinics. Elevated ESD is a sign of infection [61], and inflammation [62]. Rubella: IgG to rubella virus CMV: IgG to cytomegalovirus EBV: IgG to Epstein-Barr virus VCA IgG antibodies to infection demonstrate the presence of latent infection, while IgM shows current or recent acute infection [63]. As well, the IgG level can show the severity of cognitive decline [64]. The reference ranges for the red and white blood cell parameters differ in different countries and for different ages and sexes. In the table 1, the reference ranges for Ukraine, Kazakhstan and USA are shown.

USA Parameters Pediatric Manual, Ukraine Kazakhstan 2005 2-6 y.o 3-6 y.o 3-17 y.o Erythrocytes, 10^12/L M [3.92-4.72] [3.7-4.9] [3.5-5] F [3.89-4.67] 1-4 y.o 2-6 y.o 2-5 y.o [35-55] Neutrophils, % M [31.7-75.4] [32-55] 4-10 y.o F [33.6-77.5] [40-60] 1-4 y.o 2-6 y.o 2-5 y.o [40-50] Lymphocytes, % M [11-54] [33-55] 4-6 y.o F [11-59] [32-42] 1-4 y.o 2-6 y.o 2-15 y.o [2-7] Monocytes, % M [4-9] [3-9] 4-10 y.o F [4-8] [1-6] 0-10 y.o 0-13 y.o F [0-12] Sed rate, mm/h M [0-10] [0-10] M [0-8] F [0-20] Table 1: Reference ranges for the blood parameters in different countries.

Volume 1 Issue 1 www.scienceinquest.com | Page 4 of 13 Journal of Clinical Case Reports and Clinical Research

Case: Case #1 Male, 5 y.o. In this case, as it can be seen from the Table 2, CARS score decreased from 40 to 31 indicating significant improvements. It also can be observed that (apart from IgG to rubella and erythrocytes count they were within the normal range, but bordering the higher end of the range) all blood and immune parameters have improved. In particular, SDR normalized from 12 to 2, IgG to CMV and EBV were constantly decreasing, the neutrophils count were increasing, and lymphocytes and monocytes counts were decreasing. After the 3rd cycle, IgG to rubella sharply increased, which indicated the reactivation of the virus. A month after that, the lymphocytes count increased and the neutrophil count decreased. At the same time, the CARS score stopped decreasing and remained at 31. Another month later, IgG to EBV slightly increased, as well as the monocyte count. This did not affect other blood parameters, as they continued to improve, but possibly indicates that after the reactivation of the rubella virus, the immune system could not suppress EBV. The parameter that matters in this case is the IgG to rubella virus. The reactivation it seem to cause a slight change in neutrophils and lymphocytes, and was most likely the cause in the plateau of improvements. In terms of behavioral changes, he became more communicative, found a friend in his kindergarten class, his speech became clearer, he became more attention, perceived speech addressed to him better, became able to comprehend questions and requests, began asking his own questions, and was able to reply to more abstract questions

CARS CMV Rubella EBV RBC NEU MON LYM ESD Before treatment 39 107 38 21.5 4.8 22.7 11 56.4 12 After the 1st cycle 36 100 40 17 4.8 30 10.7 52 12 After the 2nd cycle 34 80 100 13 4.8 40 10.5 48 12 After the 3rd cycle 33 72 130 11.5 4.96 41.8 10 46 12 After the 4th cycle 31 76.5 27 12 4.63 31.8 8.6 57 2 After the 5th cycle 31 73 94 12 5.0 42 11.1 44.4 4 After the 6th cycle 30.5 - - 15 4.86 48.6 8.2 40 2 Table 2: Case #1. The changes of the blood parameters and CARS score during the treatment. Case # 2. Male, 4 y.o. It is seen from the Table 3, that as the treatment began, both blood and behavioral parameters started to improve. The blood test before treatment indicated the presence of both IgM and IgG to EBV, and after the 1st cycle only IgG were present, this explains the rise of monocytes during the first two months (after the EBV reactivation). During the first two cycles erythrocytes were increasing (this may be the consequence of active EBV). After the third cycle we see sharp changes in every blood parameter: IgG to rubella virus significantly decreased, IgG to EBV started increasing (most likely due to EBV reactivation at the beginning of the treatment), lymphocytes started to increase, neutrophils and monocytes started to decrease. During this time, the rate of change slowed down. This table indicates that there might be undetected virus (not EBV, not rubella, and not CMV, because monocytes were decreasing), which was not affected by Valacyclovir and supplements and persisted for quite a long time indicting the chronic nature of this viral infection (but sedimentation rate did not change, so this was not reactivation but the result of persistent chronic course).

CARS Rubella EBV RBC NEU MON LYM ESD Before treatment 34 61 11.6 4.64 40 10.1 48 4 After the 1st cycle 32 55 11 4.8 41 11.3 47 2 After the 2nd cycle 27 51 10 5.19 43 12 46 2 After the 3rd cycle 25 30 11 5.1 40 10 50 2 After the 4th cycle 23 14 11.5 5.0 38 8 54 2 After the 5th cycle 23 16 8 4.65 60 4 32 12 After the 7th cycle 19 4.4 30 6 62 4 Table 3: Case #2. The changes of the blood parameters and CARS score during the treatment.

Volume 1 Issue 1 www.scienceinquest.com | Page 5 of 13 Journal of Clinical Case Reports and Clinical Research

So, EBV and rubella virus seem to be very important players because successful control of these viruses affected behavior and blood parameters, however there might be at least one more important player (chronic viral infection, not CMV) which is not affected by the treatment and does not allow to further sustain the fast rate of the improvements. In terms of behavioral changes, he became less hyperactive, his vocabulary has increased and continues to increase, started to communicate with other children, started to answer questions, and performs writing and oral tasks in his kindergarten class. Case # 3. Male, 3 y.o. The child did not show such significant improvements (Table 4). After the first cycle, the score was 2 points lower, after every other cycle the score was 1 point lower, and after the 4th cycle there were no changes, but the accumulated skills that the child acquired through this line of treatment remained. After the third cycle, SDR rapidly increased from 7 to 27 and continued to rise by the end of the fourth cycle. All other blood parameters and IgG to infection improved or did not change, but the erythrocytes count increased. After the fourth cycle, the blood test explains this rapid SDR increase: IgG to CMV increased from 60 to 150. Since we did not have IgM and PCR tests done, we can assume that by the end of the third cycle severe reactivation of CMV took place. During this time, along with the rise of IgG, monocyte started to increase and stagnation in behavioral improvement could be observed. This immune stress gave rise to IgG to rubella, lymphocytes started to increase, and neutrophils started to decrease by the end of the 5th cycle. It can be concluded that CMV reactivation caused stagnation, but after the reactivation it was strongly inhibited by antiviral treatment. However, immune stress allowed for rubella to IgG to increase. In terms of behavioral changes, the child began to communicate with his parents, during the first couple of cycles he began to communicate using gestures/body language, and then eventually eased into speaking and using words for communication. He also began to understand addressed speech, and his spectrum of emotions have notably expanded.

CARS Rubella CMV EBV RBC NEU MON LYM ESD Before treatment 36 121 103 18.5 5.05 21 7.25 67 9 After the 1st cycle 34 146 104 19 4.8 28 8.9 57 7 After the 2nd cycle 33 146 104 19 4.8 28 8.7 57 7 After the 3rd cycle 31 103 62 16 5.0 41 8.7 44 27 After the 4th cycle 30 118 153 18 5.5 43 8.7 42 33 After the 5th cycle 30 125 93 17 4.9 40 14 42 13 After the 6th cycle 30 75 15 5.2 24 7.2 65 8 Table 4: Case #3. The changes of the blood parameters and CARS score during the treatment. Case # 4 Male, 7 y.o. As it was shown in the Table 5, on the 3rd cycle similar to the cases before, reactivation of EBV took place (IgM to EBV were positive). However, monocytes, erythrocytes, lymphocytes, and SDR levels did not correlate with the EBV course. CMV was successfully suppressed by antiviral treatment, and IgB to CMV continued to decrease. The conclusion made here is that these two viruses were not the key factors in this case. Instead, the graph shows that neutrophil counts and lymphocyte counts are dependent on the rubella course. As the IgG increases, neutrophils counts decreases and lymphocytes count increases. Vice versa, as IgG decreases, neutrophils count starts to increase, and lymphocytes count starts to decrease. It was also observed that when IgG to rubella started to increase, the SDR rate also slightly increased. During this time, after the 5th cycle, ribavirin was added to the regimen (only in breaks between Valacyclovir courses). The only blood test result that we received back after the ribavirin addition was after the 7th cycle. In this test, IgG to rubella was reduced significantly for the first time, positively affecting lymphocytes and neutrophils count. It was during this time that the mother first saw behavioral improvements. Our conclusion is that rubella was the main player in this case. Until rubella was suppressed, there were no changes that were observed. Along side of this, all blood parameters were dependent only on this virus, not on CMV or EBV. Considering these factors, it might prove more useful of ribavirin was adminstered for longer cycles (not only in the nine day breaks between Valacyclovir cycles).

Volume 1 Issue 1 www.scienceinquest.com | Page 6 of 13 Journal of Clinical Case Reports and Clinical Research

If our conclusion relies on the rubella virus playing the main role in this disorder, we might consider the actual treatment began only after the 5th cycle. It typical for our patients to observe improvements two months after the implementation of ribavirin into their treatment regimen.

CARS Rubella CMV EBV RBC NEU MON ESD Before treatment 41 48 71 21 4.76 56 11 4 After 3rd Cycle 41 50 57 25 4.3 48 9 4 After 4th Cycle 41 56 53 20 4.1 44.5 9.5 5 After 7th Cycle 36 45 48 26 3.9 55 8.7 5 Table 5: Case #4: The changes of the blood parameters and CARS score during the treatment. Case # 5: Male, 2.5 y.o. In this case, the child was consistently improving with substantial progress after each cycle, as it can be seen from the Table 6. During 6 cycles his CARS score decreased from 44 to 23.5 indicating absence of autistic behavior. (30 is a minimal score for autism). At the same time, we see that the trend of changing in CARS score almost identical with the change of the level of IgG to rubella virus; SDR and erythrocytes levels also had aligned in regards to movement with the IgB to rubella value. IgG to CMV and EBV were both increasing until the 5th cycle, and then IgG to EBV continued to increase, while IgG to CMV started to decrease. However, the monocytes level were decreasing while IgG to the viruses were increasing. A possible explanation for this is that prior to treatment, the child experienced reactivation of these herpetic viruses, and an IgG increase was a consequence of the reactivation, while monocytes level decreased since the peak of infection was gone. The lymphocytes level seem to follow IgG to CMV level, while neutrophils have constantly decreased without connection to any of the detected viruses. It seems that CMV and EBV do not seriously affect autistic behavior, and instead it depends on the rubella virus. In this case, we see that inhibition of rubella virus allowed to observe positive changes without stagnation, even with the rise of IgG to EBV and CMV. SDR and erythrocytes level also depend on rubella virus. In terms of behavioral changes, the child started to speak and by the end of the 8th cycle could already build short sentences and participate in dialogues. The mother noticed that the child became interested in things around, constantly asks questions about everything and awaits for the answers, in the kindergarten explains other children if he wants to share toys, plays games. After the 6th cycle the psychiatrist who was observing the child from the moment of diagnosis said that the child does not have autistic symptoms.

CARS Rubella EBV CMV RBC NEU MON LYM ESD Before treatment 44 50 21 16 5.1 31 11 46 15 After the 3rd cycle 33 45 40 25 5.2 29 5.8 60 8 After the 5th cycle 24.5 35 33 16 4.6 27 6.3 58 2 After the 6th cycle 23.5 33 45 11.5 4.6 25 6.3 59 3 After the 8th cycle 16 32 61.5 19 4.85 46 1.15 40 29 Table 6: Case #5. The changes of the blood parameters and CARS score during the treatment.. Case # 6: Male, 5 y.o. While there were positive results that were obtained from both blood parameters and in terms of our patient’s behavior, it’s been made clear from the Table 7 that neither rubella or EBV are the culprits in this case. We can conclude this because the levels of antibodies did not correlate with the other blood parameters. Using the blood parameters illustrated in the graph, we can say that the reactivation of infection, virus unknown, occurred after both the 3rd cycle and the 9th cycle (decrease of neutrophil count, increase of lymphocyte, erythrocyte and monocyte counts, and increase of ESD). During this time, the rate of decrease for the CARS score slowed down, but none of the detected viruses did not reactivate. The etiological factor in this case that being the unknown virus, was being affected by our treatment. In terms of behavioral changes, the child started to speak, started to say “hello” and “bye” in the appropriate social situations, expressed his needs verbally, started understanding and reacting to speech addressed to him, and tried to communicate with other children.

Volume 1 Issue 1 www.scienceinquest.com | Page 7 of 13 Journal of Clinical Case Reports and Clinical Research

CARS Rubella EBV RBC NEU MON LYM ESD Before Treatment 48 14 13 4.97 49.6 8.3 38.5 8 After the 2nd cycle 46 10 11 5.1 58.2 7 32 9 After the 3rd cycle 38 6 10 4.87 53.4 8.3 34.5 24 After the 5th cycle 34 8 11 5.27 58.3 5.6 33.6 13 After the 6th cycle 31 7 10 5.2 53.1 9 34.6 8 After the 7th cycle 29 17 22 5.06 63.8 8.2 25.5 11 After the 9th cycle 29 5 14 4.84 40.6 9.3 48 7 After the 11th cycle 28 0 11.93 5.23 45.5 4.1 31.4 21 After the 12th cycle 28 0 16.22 5.14 60 6.8 28.5 11 Table 7: Case #6: The changes of the blood parameters and CARS score during the treatment. Case #7. Female, 6 y.o. As a result of treatment, IgG to CMV values decreased into their appropriate reference ranges, and lymphocyte and neutrophil counts also normalized (Table 8). Within the first five cycles, the CARS score significantly decreased as well. Although EBV and Rubella were not affected by treatment, the improvements in blood parameters and autistic symptoms suggest that CMV played the main role in this case. During the course of this treatment, the girl learned to express her thoughts verbally, learned to solve math problems, began playing a musical instrument, started playing with other children, and started to understand jokes. The medical commission which was observing this girl from when she was first diagnosed concluded that there were no autistic symptoms, and allowed her entry into a regular school for neurotypical children. There was a 2 month long period after the 7th cycle of Valacyclovir where she didn’t take any Valacyclovir. During that period, there was no more positive changes that were observed, and the reactivation of CMV, EBV, and rubella took place. Along with the reactivation of CMV, the neutrophil count fell below the reference ranges, while the lymphocyte count became elevated. This did not significantly alter or affect her behavior or skills, but continuation of treatment was recommended in order to avoid regression of symptoms.

CARS Rubella EBV CMV RBC NEU MON LYM ESD Before Treatment 36 1.5 7 2 4.7 41 9 43 5 After the 1st cycle 31 6 4 2 4.3 65 6.6 25 18 After the 2nd cycle 28 12 5 1 4.5 37 10.6 46 8 After the 4th cycle 25 6 7 0 4.4 39 9.5 44.5 8 After the 5th cycle 23 23 7.5 0 4.7 40 8.3 45 10 After the 9th cycle 23 39 15 68 4.3 23 9 63.5 2 Table 8: Case #7. The changes of the blood parameters and CARS score during the treatment. Case #8. Male, 4 y.o In this case, as it is seen from Table 9, the child did not show significant improvement in his CARS score as other children did. While standard antiviral therapy was used, EBV was not affected, and there was reactivation after the 6th cycle. The reactivation resulted in an increase in monocyte and lymphocyte counts, and stagnation in his CARS score. During this 9 month period, the child said his first word, learned how to count, started to eat almost all types of food, began to observe other children (before he showed no interest in them), started trying to communicate with others by holding their hand, etc., and started playing with toys. On the other hand, the mother did observe that the child’s stimming became more severe.

CARS CMV EBV Rubella RBC NEU MON LYM ESD Before Treatment 40 11.7 106 9.77 5.1 39 6 52 10 After 3rd Cycle 39 4.06 119 16.43 5.14 37.4 9.8 50.2 10 After 6th Cycle 36 6.29 109 16.03 5.21 27.2 14.1 56.7 5 After 9th Cycle 35 4.79 180 20.94 5.17 33.3 6.2 57.1 8 Table 9: Case #8. The changes of the blood parameters and CARS score during the treatment.

Volume 1 Issue 1 www.scienceinquest.com | Page 8 of 13 Journal of Clinical Case Reports and Clinical Research

Case #9: Male, 6 y.o. In this case, it is seen that the CARS score was most likely dependent on the level of IgG to rubella virus (Table 10). As the antibodies were decreasing the CARS score was gradually declining as well, but when the treatment stopped affecting the virus, the CARS score stopped changing too. Neutrophil count was also negatively correlating with this virus. Two other viruses detected – CMV and EBV did not affect any of the blood parameters which remained within the reference ranges during the entire course of the treatment. In terms of autistic symptoms, the child was actively acquiring new vocabulary during the treatment, induced speech appeared, stimming stopped, his learning skills have improved, he learned to write, became less hyperactive and more attentive. The reactivation of the CMV on the 7th cycle did not affect any of the blood parameters, but during the talk with the patient’s mother, it was revealed that it affected the child negatively – stimming that stopped during the first 5 cycles, returned. However, after the 8th cycle when the reactivation was stopped, the stimming was gone, and the positive dynamics continued.

CARS Rubella EBV CMV RBC NEU MON LYM ESD Before Treatment 28 106 9.9 8.87 5.3 40.5 6.8 47.6 1 After 2nd Cycle 28 63.7 10.2 10.15 5.3 37.5 6.7 48.6 3 After 3rd Cycle 25 53.6 7.9 12.48 5.3 30 4 59 7 After 5th Cycle 23 31.8 10 5.81 5.6 41 7 44 8 After 6th Cycle 23 36.6 11.3 6.61 5.53 31.3 5.9 50 4 After 7th Cycle 21 27 7.9 108 5.3 36.3 5.8 50.2 3 After 8th Cycle 18 24.6 11.9 98.8 5.0 38.1 5.8 49.6 6 After 9th Cycle 18 26.8 12.8 100 5.7 27 6 60 1 After 10th Cycle 18 27 10.2 106 5.7 33 7 51 3 After 11th Cycle 18 23.2 13.2 100 5.6 41.5 6.4 45.6 2 Table 10: Case #9 The changes of the blood parameters and CARS score during the treatment.

Case #10. Male, 4 y.o. In this case, as it is seen from the Table 11, although the CARS score could be decreased from 41.5 to 35, the most pronounced changes occurred during the first 3 cycles, and after this the score did not change significantly. A group of three values repeat this pattern: EBV, and erythrocyte and lymphocyte counts. Neutrophil count is also affected by EBV changes, as the level of antibodies increases: neutrophil count decreases suggesting immune response. CMV seems to be affected by this, because the rise in IgG to CMV increases after a sharp decrease only after EBV increase. Although ESD seems to be affected by rubella virus, it is not clear in this case what is the role of this virus because neither other blood counts nor CARS score are affected by it. It may be assumed that it is an additional factor affecting immune system, which weakening it allows reactivation of EBV that causes a cascade of inflammatory response and effect on CARS score by the latter virus. In terms of behavioral changes, during the first five cycles of treatment the child began to speak (before he only pronounced syllables), now has learned about 15 new words, social communication improved, started to look into eyes when wants attention, started reacting when his name was called, he started to imitate parents and teachers, began to use toilet by himself, the spectrum of food preferences significantly increaed (before he agreed only to eat dairy), now eats vegetables, soups, noodles, etc.,

CARS EBV Rubella CMV RBC NEU MON LYM ESD Before Treatment 41.5 14.75 29.08 46.99 4.9 47.6 6.2 39.2 12 After 1st Cycle 39.5 12.72 33.14 37.76 4.73 55 6.32 33.9 15 After 3rd Cycle 36 13.77 34.57 31.32 4.97 48.5 5.8 41.6 17 After 5th Cycle 35.5 19.73 31.92 28.88 4.93 45.2 7.72 41.47 14 After 7th Cycle 35.5 18.46 31.49 34.59 5.1 50 5 41 10 After 8th Cycle 35 11.44 26.54 33.94 4.87 45.1 9.1 41.23 15 After 11th Cycle 35 19.58 33.81 35.99 5.39 44.7 9.03 42.45 22 Table 11: Case #10: The changes of the blood parameters and CARS score during the treatment.

Volume 1 Issue 1 www.scienceinquest.com | Page 9 of 13 Journal of Clinical Case Reports and Clinical Research

Case #11. Male, 9 y.o. As indicated in the Table 12, the CARS score has decreased from 40.5 to 27 during 10 months of antiviral treatment. Until the 6th cycle the score was gradually decreasing. It was quickly declining during the first four cycles, which was followed by more slow decrease until the 7th cycle. However, at the 7th cycle there was a slight increase in the score. As well, during this time and at the 4th cycle when the rate of improvements slowed down, the sharp increase in the IgG to EBV is observed along with decrease in neutrophils count, increase in lymphocyte and monocyte counts and in ESD. After this reactivation of EBV, increase in IgG to rubella virus and CMV is seen. This may be a result of weakened immune system after the reactivation of the EBV. It may be concluded for this case, that the main player here is EBV, which affects all of the blood parameters and CARS score. In terms of behavioral changes, the parents noted increase of active vocabulary, appearance of induced speech, development of motor skills, the child started eating all types of food although before his ration was highly limited. The child also became calmer (for example, it became possible to go to cinema theater and spend there 2.5 hours without behavioral problems), before treatment the parents said that it was not possible to keep social contacts because of the behavioral problems. He also became more communicative, began to play with siblings, although before he did not communicate with other children.

CARS Rubella CMV EBV RBC NEU MON LYM ESD Before Treatment 40.5 2.47 3.71 0.5 4.7 59 7 31 25 After the 2nd Cycle 35.5 1.27 6.96 0.5 4.3 68 6 27 17 After the 4th Cycle 30 22.54 19.43 93.1 4.15 54.7 8.43 33.27 6 After the 5th Cycle 28 19.36 24.32 81.6 4.37 51.5 9 36.7 7 After the 6th Cycle 28 14.87 22.99 79.8 4.66 60.8 8.1 27.6 11 After the 7th Cycle 30 6.59 17.33 99.7 4.46 48.3 8.7 36.2 15 After the 9th Cycle 29 11.18 14.09 137 4.33 57.9 8.34 28.15 13 After the 10th Cycle 27 80.83 54.99 18.69 4.51 61.2 8.78 27.2 10 Table 11: Case #11: The changes of the blood parameters and CARS score during the treatment. Summary of the Results: Eleven cases that were reviewed in this paper are obviously not enough to make any conclusions regarding the ASD in general and the effect of the antiviral treatment on ASD. However, there are several inferences that can be done in relation to the described cases:

Figure 1: The mean CARS score value. Each child’s score is represented in dotted line. The red line shows the average score at each point of treatment. There is a general tendency towards decrease of the score.

Volume 1 Issue 1 www.scienceinquest.com | Page 10 of 13 Journal of Clinical Case Reports and Clinical Research

1) In every of the eleven cases there was a virus which played the most significant role and inhibition of which might have resulted in normalization of blood parameters and decrease of autistic signs and symptoms. There was no a single virus for all of these cases, but in every case it was one of three: CMV, EBV or rubella virus. 2) In some cases the initiation of the treatment results in the reactivation of the existing viruses. It can be assumed that it is the mechanism of adaptation to negative environmental conditions via the quorum sensing [65]. 3) In all eleven cases CARS scores were decreasing with treatment initiation (Figure 1). It cannot be said whether this decrease was a result of the antiviral treatment. Although all of the parents reported that no changes were observed before treatment, further studies are needed to confirm the correlation. 4) In these particular cases the decrease in the CARS scores which represent the relieve of autistic symptoms tend to occur after the decrease in the levels of antibodies and consequent normalization of blood parameters, while the decrease in CARS tend to stop after the detected viruses reactivation or other acute infectious diseases. 5) CARS scores tend to decrease not when the blood parameters reach the reference ranges, but when they start shifting towards reference ranges. In case if the correlation between these parameters and the severity of autism symptoms is confirmed, despite the fact that most of the viruses discussed are incurable, their inhibition and even partial consequent regulation of blood parameters will significantly improve ASD children’s quality of life. Limitations of the Study This study has several limitations that prevent us from coming up with other more generalized conclusions. In particular, this was not a clinical study, the number of children observed was too small, the CARS scores were reported by parents, which could be biased, the blood parameters were measured by different clinics in different cities. Conclusion In this descriptive series of 11 ASD cases it was observed that in these children the antiviral treatment resulted in normalization of blood parameters indicating infection and inflammation. This normalization of the parameters tend to coincide with the decrease in the CARS-2 score which represents the severity of autism in children. Further studies are needed to make a clear conclusions about this effect of antiviral treatment on autism. Compliance with Ethical Standards The study was funded by FLAASK, LLC. The authors declare that they have no conflict of interest. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent was obtained from parents of all individual participants included in the study. Acknowledgements We are grateful to Andrew Lefkowitz, CEO and chairman of FLAASK, LLC, for his financial, administrative, and moral support provided for this work. As well, we thank Alla Dianova for organizing communications with the parents of the ASD children and for collecting and summarizing the results of the children’s tests and symptom descriptions. References 1. American Psychiological Association (APA). (2016). What Is Autism Spectrum Disorder? 2. Asperger H (1938). Das psychisch abnormale Kind. Wiener Klinische Wochenschrift, 51, 1314-7. 3. Kanner L (1943). Autistic disturbances of affective contact. Nervous Child 2, 217-250. 4. Rossignol DA and Frye RE (2011). A review of research trends in physiological abnormalities in autism spectrum disorders: immune dysregulation, inflammation, oxidative stress, mitochondrial dysfunction and environmental toxicant exposures. Mol Psychiatry. 17(4): 389- 401. 5. Al-Haddad BJS, Jacobsson B, Chabra S, Modzelewska D and Olson EM., et al (2019). Long-term Risk of Neuropsychiatric Disease After Exposure to Infection In Utero. JAMA Psychiatry. 6. Atladóttir H, Thorsen P, stergaard LO, Schendel D, Lemcke S., et al. Maternal Infection Requiring Hospitalization During Pregnancy and Autism Spectrum Disorders. J Autism Dev Disord. 40(12): 1423-30. 7. Kumar P, Malhorta S, Kaur N, Madan P, Patil V., et al (2014). Correlation between viral infections and autism: an overview. Delhi Psych J 17:2 8. Bauman MD, Iosif AM, Smith SEP, Bregere C, Amaral DG., et al (2014). Activation of the Maternal Immune System During Pregnancy Alters Behavioral Development of Rhesus Monkey Offspring. Biol Psychiatry. 75(4): 332-41.

Volume 1 Issue 1 www.scienceinquest.com | Page 11 of 13 Journal of Clinical Case Reports and Clinical Research

9. Choi GB, Yim YS, Wong H, Kim S and Kim H., et al (2016). The maternal interleukin-17a pathway in mice promotes autism-like phenotypes in offspring. Science 351: 933-9. 10. Garbett K, Hsiao E, Kálmán S, Patterson P and Mirnics K., et al (2012). Effects of maternal immune activation on gene expression patterns in the fetal brain. Transl Psychiatry. 2012 Apr 3;2:e98. 11. Patterson PH (2011). Modeling autistic features in animals. Pediatr Res 69(5 Pt 2): 34R-40R. 12. Ratajczak H (2011). Theoretical aspects of autism: Causes—A review. J Immunotoxicol. 8(1): 68-79. 13. Mahic M, Mjaaland S, Bovelstad HM, Gunnes N and Susser E., et al. (2017). Maternal Immunoreactivity to Herpes Simplex Virus 2 and Risk of Autism Spectrum Disorder in Male Offspring. mSphere. 22;2(1). 14. Valayi S, Eftekharian MM, Taheri M and Alikhani MY (2017). Evaluation of antibodies to cytomegalovirus and Epstein-Barr virus in patients with autism spectrum disorder. Hum Antibodies. 26(3): 165-9. 15. Binda S, Bubba L, Pellegrinelli L, Primache V, Gentile I, Zappulo E, et al (2016). DNA detection of herpetic viruses in dried blood spots in children with autism spectrum disorders. J Clinical 9(82): S137-8. 16. Slawinski BL, Talge N, Ingersoll B, Smith A and Glazier A., et al (2018). Maternal cytomegalovirus sero-positivity and autism symptoms in children. Am J Reprod Immunol. 79(5): e12840. 17. Nicolson GL, Gan R, Nicolson NL, Haier J (2007). Evidence for Mycoplasma ssp., Chlamydia pneunomiae, and human herpes virus-6 coinfections in the blood of patients with autistic spectrum disorders. J Neurosci Res 85(5): 1143-8. 18. Chess S (1977). Follow-up report on autism in congenital rubella. J Autism Child Schizophr. 7(1): 69-81. 19. Kern JK, Geier DA, Sykes LK and Geier MR (2016). Relevance of Neuroinflammation and Encephalitis in Autism. Front Cell Neurosci 9: 519. 20. Rossignol DA and Frye RE (2014). Evidence linking oxidative stress, mitochondrial dysfunction, and inflammation in the brain of individuals with autism. Front Physiol. 5: 150. 21. Siniscalco D, Schultz S, Brigida AL, Antonucci N (2018). Inflammation and Neuro-immune Dysregulations in Autism Spectrum Disorders. Pharmaceuticals (Basel). 4: 11(2). 22. Schlossberg D and Rafik S (2017). Valtrex (Valacyclovir). Antibiotics Manual: A Guide to Commonly Used Antimicrobials. ISBN: 978-1- 119-22075-6. 23. Baker DA (2002). Valacyclovir in the treatment of genital herpes and herpes zoster. J Cutan Med Surg. 7(5): 372-81. 24. Cunha B and Baron J (2016). The pharmacokinetic basis of oral valacyclovir treatment of herpes simplex virus (HSV) or varicella zoster virus (VZV) meningitis, meningoencephalitis or encephalitis in adults. J Chemother 29(2): 122-125. 25. Sunde PT, Olsen I, Enersen M and Grinde B. (2008). Patient with severe periodontitis and subgingival Epstein-Barr virus treated with antiviral therapy. J Clin Virol. 42(2):176-8. 26. Vezina HE, Balfour HH, Weller DR, Anderson BJ and Brundage RC (2010). Valacyclovir Pharmacokinetics and Exploratory Pharmacodynamics in Young Adults With Epstein‐Barr Virus Infectious Mononucleosis. J Clin Pharmacol. 50(7): 734-42. 27. Kline J, Pollyea DA, Stock W, Artz A., Rich E., et al (2006). Pre-transplant ganciclovir and post transplant high-dose valacyclovir reduce CMV infections after alemtuzumab-based conditioning. Bone Marrow Transplant. 37(3):307-10. 28. Simon MW, Fish DN, Deeter RG (2002). Pharmacokinetics and Safety of Valaciclovir in Children with Epstein-Barr Virus Illness. Drugs R D 3(6):365-73. 29. Singh M. (2016). Medicinal Uses of L-Lysine: Past and Future. Int. J. Res. Pharm. Sci 2(4), 637-64. 30. Griffith RS, Walsh DE, Myrmel KH, Thompson RW and Behforooz A., et al (1987). Success of L-Lysine Therapy in Frequently Recurrent Herpes simplex Infection. Dermatologica. 175(4): 183-90. 31. Barak V, Halperin T and Kalickman I (2001). The effect of Sambucol, a black elderberry-based, natural product, on the production of human cytokines: I. Inflammatory cytokines. Eur Cytokine Netw. 12(2): 290-6. 32. Zakay-Rones Z, Varsano N, Zlotnik M, Manor O, Regev L., et al (1995). Inhibition of several strains of influenza virus in vitro and reduction of symptoms by an elderberry extract (Sambucus nigra L.) during an outbreak of influenza B Panama. J Altern Complement Med. 1(4): 361-9. 33. Zakay-Rones Z, Thom E, Wollan T, Wadstein J (2004). Randomized study of the efficacy and safety of oral elderberry extract in the treatment of influenza A and B virus infections. J Int Med Res. 32(2): 132-40. 34. Morag AM, Mumcuoglu M, Baybikov T, Schlesinger M and Zakay-Rones Z., et al (1997). Inhibition of several strains of influenza virus in vitro and reduction of symptoms by an elderberry extract (Sambucus nigra L.) during an outbreak of influenza B Panama. J Altern Complement Med. 1(4): 361-9. 35. Sedef N E and Karakaya S (2009). Olive tree (Olea europaea) leaves: potential beneficial effects on human health. Nutr Rev 67(11): 632-8. 36. Boss A, Kao C H, Murray PM, Marlow G and Barnett MP., et al (2016). Human Intervention Study to Assess the Effects of Supplementation with Olive Leaf Extract on Peripheral Blood Mononuclear Cell Gene Expression. Int J Mol Sci 2;17(12). pii: E2019. 37. Lockyer S, Corona G, Yaqoob P, Spencer JP and RowlandI (2015). Secoiridoids delivered as olive leaf extract induce acute improvements in human vascular function and reduction of an inflammatory cytokine: a randomised, double-blind, placebo-controlled, cross-over trial. Br J Nutr 114(1): 75-83. 38. Rosignoli P, Fuccelli R, Fabiani R, Servili M and Morozzi G., et al (2013). Effect of olive oil phenols on the production of inflammatory mediators in freshly isolated human monocytes. J. Nutr. Biochem, 24: 1513-9. 39. Ryu SJ, Choi HS, Yoon KY, Lee OH, Kim KJ and Lee BY., et al (2015). Oleuropein suppresses LPS-induced inflammatory responses in RAW 264.7 cell and zebrafish. J Agric Food Chem. 63(7): 2098-105.

Volume 1 Issue 1 www.scienceinquest.com | Page 12 of 13 Journal of Clinical Case Reports and Clinical Research

40. Benavente-Garcia J, Castillo J, Lorente A, Ortuno A, Del Rio JA., et al (2000). Antioxidant activity of phenolics extracted from Olea europaea L. leaves. Food Chem 68:457-62. 41. Micol V, Caturla N, Pérez-Fons L, Más V, Pérez L, Estepa A (2005). The olive leaf extract exhibits antiviral activity against viral haemorrhagic septicaemia rhabdovirus (VHSV). Antiviral Res 66(2-3):129-36. 42. Block KI and Mead MN (2003). Immune system effects of Echinacea, ginseng and astragalus: a review. Integr Cancer Ther. 2(3): 247-67. 43. Das S, Bordoloi R and Newar N (2014). A Review on immune modulatory effect of some traditional medicinal herbs. J Pharmace Chem Biol Sci 2(1): 33-42. 44. Sun Y, Hersh EM, Lee SL, McLaughlin M, Loo TL et al (1983). Preliminary observations on the effects of the Chinese medicinal herbs Astragalus membranaceus and Ligustrum lucidum on lymphocyte blastogenic responses. J Biol Response Mod. 2(3): 227-37. 45. Kwok‐Kin LP, Chan JY, Cheng L, Lau CP., et al (2013). Isolation of Anti‐Inflammatory Fractions and Compounds from the Root of Astragalus membranaceus. Phytother Res. 27(4): 581-7. 46. Jäger R, Purpura M, Farmer S, Cash HA, Keller D (2017). Probiotic Bacillus coagulans probioticGBI-30, 6086 Improves Protein Absorption and Utilization. Probiotics Antimicrob Proteins 10(4): 611-615. 47. Nyangale EP, Farmer S, Keller D, Chernoff D and Gibson GR., et al (2014). Effect of prebiotics on the fecal microbiota of elderly volunteers after dietary supplementation of Bacillus coagulans probioticGBI-30, 6086. Anaerobe. 30: 75-81. 48. Omura Y, Lu D, Jones MK, Nihrane A and Duvvi Het al (2015). Early Detection of Autism (ASD) by a Non-invasive Quick Measurement of Markedly Reduced Acetylcholine & DHEA and Increased β-Amyloid (1-42), Asbestos (Chrysotile), Titanium Dioxide, Al, Hg & often Coexisting Virus Infections (CMV, HPV 16 and 18), Bacterial Infections etc. in the Brain and Corresponding Safe Individualized Effective Treatment. Acupunct Electrother Res 40(3): 157-87. 49. Li Q and Zhou JM (2016). The microbiota-gut-brain axis and its potential therapeutic role in autism spectrum disorder. Neuroscience 324: 131-9. 50. Baron MA (2009). Patented strain of Bacillus coagulans probiotic increased immune response to viral challenge. Postgrad Med 121(2): 114-8. 51. Farmer S (1997). Topical Compositions Containing Bacillus coagulans probioticExtracellular Products and Uses Thereof. Us7541042b2. 52. Endres JR, Qureshi I, Farber T, Hauswirth J and Hirka G et al (2011). One-year chronic oral toxicity with combined reproduction toxicity study of a novel probiotic, Bacillus coagulans, as a food ingredient. Food Chem Toxicol 49(5): 1174-82. 53. Schopler E, Van Bourgondien ME, Wellman GJ, Love SR (2010). Childhood Autism Rating Scale – 2nd Edition. Los Angeles: Western Psychological Services. 54. Faquin WC, Schneider TJ, Goldberg MA (1992). Effect of inflammatory cytokines on hypoxia-induced erythropoietin production. Blood 79(8):1987-94. 55. Pierson and David J (2000). Pathophysiology and Clinical Effects of Chronic Hypoxia. Respir Care 45(1):39-51; discussion 51-3. 56. Kimberlin D, Lin C, Jacobs R, Powell D, Corey L and Gruber W., et al (2001). Safety and Efficacy of High-Dose Intravenous Acyclovir in the Management of Neonatal Herpes Simplex Virus Infections. Pediatrics 108(2): 230-8. 57. Kumar V. and Sharma A (2010). Neutrophils: Cinderella of innate immune system. Int Immunopharmacol. 10(11): 1325-34. 58. Shi C, and Pamer EG (2011). Monocyte recruitment during infection and inflammation. Nat Rev Immunol 11(11): 762-74. 59. Kimura H, Hoshino Y, Kanegane H, Tsuge I and Okamura T (2001). Clinical and virologic characteristics of chronic active Epstein-Barr virus infection. Blood. 98(2): 280-6. 60. Yotis F, Tsaparas ML, Brigden RM, Thomas E and Raboud J., et al (2000). Proportion Positive for Epstein-Barr Virus, Cytomegalovirus, Human Herpesvirus 6, Toxoplasma, and Human Immunodeficiency Virus Types 1 and 2 in Heterophile-Negative Patients With an Absolute Lymphocytosis or an Instrument-Generated Atypical Lymphocyte Flag. Arch Pathol Lab Med 124(9): 1324-30. 61. Mayoclinic.org. Sed rate (erythrocyte sedimentation rate). 62. Elwood E, Lim Z, Naveed H and Galea I (2017). The effect of systemic inflammation on human brain barrier function. Brain Behav Immun 62: 35-40. 63. Stevens JG and Cook ML (1974). Maintenance of Latent Herpetic Infection: An Apparent Role for Anti-Viral IgG. J Immunol 113(6): 1685- 93. 64. Aiello A, Haan M, Blythe L, Moore K and Gonzalez J., et al. (2006). The Influence of Latent Viral Infection on Rate of Cognitive Decline over 4 Years. J Am Geriatr Soc 54(7): 1046-54. 65. Erez Z, Steinberger-Levy I, Shamir M, Doron S and Stokar-Avihail., et al (2017). Communication between viruses guides lysis–lysogeny decisions. Nature 541(7638): 488-93.

Volume 1 Issue 1 www.scienceinquest.com | Page 13 of 13