Folate Receptor Alpha Autoantibodies in the Pediatric Acute-Onset Neuropsychiatric Syndrome (PANS) and Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal Infections (PANDAS) Population

The folate receptor alpha autoantibodies (FRAAs) are associated with cerebral folate deficiency (CFD) and autism spectrum disorder (ASD). Both of these syndromes have overlapping characteristics with Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal Infections (PANDAS) and Pediatric Acute-Onset Neuropsychiatric Syndrome (PANS). Thus, we propose that the FRAAs may contribute to the symptomatology of PANS/PANDAS. To test this hypothesis, 1 mL of serum from 47 patients (age range = 6–18 years old) clinically diagnosed with PANS/PANDAS was sent to Vascular Strategies (Plymouth Meeting, PA, USA) for analysis of FRAAs. Moreover, 63.8% of PANS/PANDAS patients (male = 15; female = 15) were found to have either the blocking and/or blinding FRAAs, with 25 (83.3%; male = 14; female = 11) having binding FRAAs, two (6.7%; all female = 2) having blocking FRAAs, and 3 (10%; male = 1; female = 2) having both binding and blocking. Furthermore, surprisingly, ASD was associated with a 0.76 lower binding titer (p = 0.02), and severe tics were associated with a 0.90 higher binding titer (p = 0.01). A case of a FRAA-positive patient is provided to illustrate that a treatment plan including leucovorin can result in symptom improvement in patients with PANS/PANDAS who are FRAA-positive. These data, for the first time, demonstrate that PANS/PANDAS is associated with FRAAs and suggest folate metabolism abnormalities may contribute to PANS/PANDAS symptomatology. Further studies investigating the therapeutic nature of leucovorin in the treatment of PANS/PANDAS are needed. Such studies may open up an alternative, safe, and well-tolerated treatment for those with the PANS/PANDAS diagnosis.


Introduction
Neuropsychiatric Disorders Associated with Streptococcal Infections (PANDAS) is an autoimmune disorder impacting neurological function in prepubescent children [1].It presents with an acute and dramatic onset of obsessive-compulsive disorder (OCD) and/or tic disorder, neurologic abnormalities with prepubescent onset, and an episodic waxing and waning course associated with Group A streptococcal (GAS) infection [2].In PANDAS, the GAS infection induces encephalitis that interferes with brain function in areas such as the basal ganglia [3].Pediatric Acute-Onset Neuropsychiatric Syndrome (PANS) is a similar disorder defined as a sudden dramatic overnight onset of OCD and/or severe restrictive eating with at least two comorbid symptoms such as anxiety, emotional lability, developmental regression, sensory and motor abnormalities, urinary symptoms, sleep disturbance, tics, and/or a decline in academic performance.Although the criteria do not specify a specific etiological trigger, it is speculated in many cases that infectious agents such as Mycoplasma pneumoniae and influenza, as well as other viruses, act as triggers [4].It has been speculated that infectious agents with prolonged colonization periods are likely to cause PANS [4].PANS encapsulates disorders that cannot be explained by a known neurological or medical disorder [1].
Determination of PANS/PANDAS requires a clinical diagnosis based on thorough consideration of family, medical, and psychiatric history, physical and psychiatric exams, and infectious and autoimmune disease evaluations [1,4].Laboratory work can support diagnosis; however, as the name suggests, diagnosis of PANS/PANDAS is determined by the acute onset of symptoms.Therefore, in the face of a clear acute onset of symptoms and negative laboratory findings that rule out other potential diagnoses, PANS or PAN-DAS is entertained.PANS/PANDAS treatment follows a three-pronged approach.First, antimicrobials are used to treat any underlying infections.Second, cognitive-behavioral therapy and pharmacological interventions are used for psychiatric and behavioral support.Lastly, anti-inflammatory agents, corticosteroids, intravenous immunoglobulin, and nutrient supplementation are used for immunomodulatory support [5].
Not all patients respond adequately to this three-pronged approach to the treatment of PANS/PANDAS, suggesting that their symptoms may be either driven by another etiology or that there is a comorbid condition that also requires treatment.One disorder that is being increasingly recognized is cerebral folate deficiency (CFD).CFD has been associated with many symptoms that overlap PAN/PANDAS, such as acute onset, movement disorders such as tics, cognitive impairment, developmental delays, and autism spectrum disorder (ASD) [6,7].CFD has been reported in disorders that overlap PANS/PANDAS symptomatology, such as refractory depression and suicidal ideations [8,9] and schizophrenia [10].Many individuals with CFD have ASD, a disorder that includes OCD and repetitive behaviors similar to PANS/PANDAS symptomatology [11].Thus, it may be possible that individuals with PAN/PANDAS may be affected by the same mechanism that causes CFD.This has never been investigated before.
Folate is a water-soluble B-vitamin necessary for cerebral metabolism and function throughout development, adolescence, and adulthood [12][13][14].In patients with CFD, normal levels of 5-methyltetrahydrofolate (5MTHF) are found in serum despite belownormal concentrations of 5-methyltetrahydrofolate (5MTHF) in the cerebrospinal fluid (CSF) [15,16].This is caused by dysfunction of the folate receptor alpha (FR severe restrictive eating with at leas lability, developmental regression, sleep disturbance, tics, and/or a de do not specify a specific etiological agents such as Mycoplasma pneumon gers [4].It has been speculated that i are likely to cause PANS [4].PANS known neurological or medical diso Determination of PANS/PAND consideration of family, medical, an and infectious and autoimmune dis diagnosis; however, as the name sug the acute onset of symptoms.Theref negative laboratory findings that ru entertained.PANS/PANDAS treatm crobials are used to treat any underl and pharmacological interventions Lastly, anti-inflammatory agents, co trient supplementation are used for Not all patients respond adequ of PANS/PANDAS, suggesting that ology or that there is a comorbid con is being increasingly recognized is c ated with many symptoms that ove disorders such as tics, cognitive imp disorder (ASD) [6,7].CFD has been symptomatology, such as refractor phrenia [10].Many individuals wit repetitive behaviors similar to PANS sible that individuals with PAN/PA causes CFD.This has never been inv Folate is a water-soluble B-vita throughout development, adolescen mal levels of 5-methyltetrahydrofol mal concentrations of 5-methyltetra [15,16].This is caused by dysfuncti porter of folate into the brain [16].then undergoes endocytosis to activ One of the main causes of FRɑ bind to the FRɑ and inhibit folate t described, i.e., the blocking and bin the site where folate binds to the FR while the binding FRAA binds to o to function optimally.
To determine the presence of measure the 5MTHF concentration and generally requires a general an ure FRAAs using the folate recept found the serum titers of FRAAs in concentration of 5MTHF, validating in the brain [12,15,18].The drawba with only serum FRAA titers.Howe leucovorin, is very safe, the FRAAs have a lower risk than performing a ), the main transporter of folate into the brain [16].In healthy conditions, folate binds to the FR severe restrictive eating with at lability, developmental regressi sleep disturbance, tics, and/or a do not specify a specific etiolog agents such as Mycoplasma pneu gers [4].It has been speculated t are likely to cause PANS [4].PA known neurological or medical Determination of PANS/PA consideration of family, medica and infectious and autoimmune diagnosis; however, as the name the acute onset of symptoms.Th negative laboratory findings tha entertained.PANS/PANDAS tre crobials are used to treat any un and pharmacological intervent Lastly, anti-inflammatory agent trient supplementation are used Not all patients respond ad of PANS/PANDAS, suggesting ology or that there is a comorbid is being increasingly recognized ated with many symptoms that disorders such as tics, cognitive disorder (ASD) [6,7].CFD has symptomatology, such as refra phrenia [10].Many individuals repetitive behaviors similar to PA sible that individuals with PAN causes CFD.This has never been Folate is a water-soluble B throughout development, adole mal levels of 5-methyltetrahydr mal concentrations of 5-methylt [15,16].This is caused by dysfu porter of folate into the brain [1 then undergoes endocytosis to a One of the main causes of bind to the FRɑ and inhibit fola described, i.e., the blocking and the site where folate binds to th while the binding FRAA binds to function optimally.
To determine the presence measure the 5MTHF concentrat and generally requires a genera ure FRAAs using the folate re found the serum titers of FRAA concentration of 5MTHF, valida in the brain [12,15,18].The draw with only serum FRAA titers.Ho leucovorin, is very safe, the FRA have a lower risk than performi , which then undergoes endocytosis to actively transport folate across the blood-brain barrier.
One of the main causes of FR severe restrictive eating with at least two comorbid symptoms such as anxiety, lability, developmental regression, sensory and motor abnormalities, urinary sleep disturbance, tics, and/or a decline in academic performance.Although do not specify a specific etiological trigger, it is speculated in many cases that agents such as Mycoplasma pneumoniae and influenza, as well as other viruses, gers [4].It has been speculated that infectious agents with prolonged colonizati are likely to cause PANS [4].PANS encapsulates disorders that cannot be exp known neurological or medical disorder [1].Determination of PANS/PANDAS requires a clinical diagnosis based on consideration of family, medical, and psychiatric history, physical and psychia and infectious and autoimmune disease evaluations [1,4].Laboratory work c diagnosis; however, as the name suggests, diagnosis of PANS/PANDAS is dete the acute onset of symptoms.Therefore, in the face of a clear acute onset of sym negative laboratory findings that rule out other potential diagnoses, PANS or P entertained.PANS/PANDAS treatment follows a three-pronged approach.Fi crobials are used to treat any underlying infections.Second, cognitive-behavio and pharmacological interventions are used for psychiatric and behaviora Lastly, anti-inflammatory agents, corticosteroids, intravenous immunoglobuli trient supplementation are used for immunomodulatory support [5].
Not all patients respond adequately to this three-pronged approach to the of PANS/PANDAS, suggesting that their symptoms may be either driven by a ology or that there is a comorbid condition that also requires treatment.One di is being increasingly recognized is cerebral folate deficiency (CFD).CFD has b ated with many symptoms that overlap PAN/PANDAS, such as acute onset, disorders such as tics, cognitive impairment, developmental delays, and autism disorder (ASD) [6,7].CFD has been reported in disorders that overlap PANS symptomatology, such as refractory depression and suicidal ideations [8,9] a phrenia [10].Many individuals with CFD have ASD, a disorder that includes repetitive behaviors similar to PANS/PANDAS symptomatology [11].Thus, it m sible that individuals with PAN/PANDAS may be affected by the same mech causes CFD.This has never been investigated before.
Folate is a water-soluble B-vitamin necessary for cerebral metabolism an throughout development, adolescence, and adulthood [12][13][14].In patients with mal levels of 5-methyltetrahydrofolate (5MTHF) are found in serum despite mal concentrations of 5-methyltetrahydrofolate (5MTHF) in the cerebrospinal [15,16].This is caused by dysfunction of the folate receptor alpha (FRɑ), the m porter of folate into the brain [16].In healthy conditions, folate binds to the F then undergoes endocytosis to actively transport folate across the blood-brain One of the main causes of FRɑ dysfunction are FRɑ autoantibodies (FRA bind to the FRɑ and inhibit folate transport into the CSF [6,17].Two FRAAs described, i.e., the blocking and binding FRAAs.The blocking FRAA binds spe the site where folate binds to the FRα, thereby preventing folate from binding while the binding FRAA binds to other regions of the FRɑ and interferes with to function optimally. To determine the presence of CFD, standard practice utilizes lumbar pu measure the 5MTHF concentration in the CSF.Since the lumbar puncture is in and generally requires a general anesthetic [14], it has become common practi ure FRAAs using the folate receptor autoantibody test (FRAT).Previous st found the serum titers of FRAAs in patients with CFD and ASD correlate wi concentration of 5MTHF, validating the use of FRAAs as a proxy for folate abn in the brain [12,15,18].The drawback to this approach is that CFD cannot be with only serum FRAA titers.However, since the treatment of central folate abn leucovorin, is very safe, the FRAAs in conjunction with a treatment trial are have a lower risk than performing a lumbar puncture [12].dysfunction are FR J. Pers.Med.2024, 14, x FOR PEER REVIEW severe restrictive eating with at least two comorbid symp lability, developmental regression, sensory and motor ab sleep disturbance, tics, and/or a decline in academic per do not specify a specific etiological trigger, it is speculate agents such as Mycoplasma pneumoniae and influenza, as w gers [4].It has been speculated that infectious agents with are likely to cause PANS [4].PANS encapsulates disorde known neurological or medical disorder [1].
Determination of PANS/PANDAS requires a clinica consideration of family, medical, and psychiatric history, and infectious and autoimmune disease evaluations [1,4 diagnosis; however, as the name suggests, diagnosis of PA the acute onset of symptoms.Therefore, in the face of a cle negative laboratory findings that rule out other potential entertained.PANS/PANDAS treatment follows a three-p crobials are used to treat any underlying infections.Secon and pharmacological interventions are used for psych Lastly, anti-inflammatory agents, corticosteroids, intrave trient supplementation are used for immunomodulatory Not all patients respond adequately to this three-pro of PANS/PANDAS, suggesting that their symptoms may ology or that there is a comorbid condition that also requi is being increasingly recognized is cerebral folate deficien ated with many symptoms that overlap PAN/PANDAS, disorders such as tics, cognitive impairment, developmen disorder (ASD) [6,7].CFD has been reported in disorde symptomatology, such as refractory depression and suic phrenia [10].Many individuals with CFD have ASD, a d repetitive behaviors similar to PANS/PANDAS symptoma sible that individuals with PAN/PANDAS may be affect causes CFD.This has never been investigated before.
Folate is a water-soluble B-vitamin necessary for ce throughout development, adolescence, and adulthood [12 mal levels of 5-methyltetrahydrofolate (5MTHF) are foun mal concentrations of 5-methyltetrahydrofolate (5MTHF) [15,16].This is caused by dysfunction of the folate recep porter of folate into the brain [16].In healthy conditions then undergoes endocytosis to actively transport folate ac One of the main causes of FRɑ dysfunction are FRɑ bind to the FRɑ and inhibit folate transport into the CSF described, i.e., the blocking and binding FRAAs.The bloc the site where folate binds to the FRα, thereby preventing while the binding FRAA binds to other regions of the FR to function optimally.
To determine the presence of CFD, standard practi measure the 5MTHF concentration in the CSF.Since the l and generally requires a general anesthetic [14], it has bec ure FRAAs using the folate receptor autoantibody test found the serum titers of FRAAs in patients with CFD a concentration of 5MTHF, validating the use of FRAAs as in the brain [12,15,18].The drawback to this approach is with only serum FRAA titers.However, since the treatmen leucovorin, is very safe, the FRAAs in conjunction with have a lower risk than performing a lumbar puncture [12 autoantibodies (FRAAs), which bind to the FR severe restrictive eating with at least two comorbid symptoms such as anxiety, emotional lability, developmental regression, sensory and motor abnormalities, urinary symptoms, sleep disturbance, tics, and/or a decline in academic performance.Although the criteria do not specify a specific etiological trigger, it is speculated in many cases that infectious agents such as Mycoplasma pneumoniae and influenza, as well as other viruses, act as triggers [4].It has been speculated that infectious agents with prolonged colonization periods are likely to cause PANS [4].PANS encapsulates disorders that cannot be explained by a known neurological or medical disorder [1].
Determination of PANS/PANDAS requires a clinical diagnosis based on thorough consideration of family, medical, and psychiatric history, physical and psychiatric exams, and infectious and autoimmune disease evaluations [1,4].Laboratory work can support diagnosis; however, as the name suggests, diagnosis of PANS/PANDAS is determined by the acute onset of symptoms.Therefore, in the face of a clear acute onset of symptoms and negative laboratory findings that rule out other potential diagnoses, PANS or PANDAS is entertained.PANS/PANDAS treatment follows a three-pronged approach.First, antimicrobials are used to treat any underlying infections.Second, cognitive-behavioral therapy and pharmacological interventions are used for psychiatric and behavioral support.Lastly, anti-inflammatory agents, corticosteroids, intravenous immunoglobulin, and nutrient supplementation are used for immunomodulatory support [5].
Not all patients respond adequately to this three-pronged approach to the treatment of PANS/PANDAS, suggesting that their symptoms may be either driven by another etiology or that there is a comorbid condition that also requires treatment.One disorder that is being increasingly recognized is cerebral folate deficiency (CFD).CFD has been associated with many symptoms that overlap PAN/PANDAS, such as acute onset, movement disorders such as tics, cognitive impairment, developmental delays, and autism spectrum disorder (ASD) [6,7].CFD has been reported in disorders that overlap PANS/PANDAS symptomatology, such as refractory depression and suicidal ideations [8,9] and schizophrenia [10].Many individuals with CFD have ASD, a disorder that includes OCD and repetitive behaviors similar to PANS/PANDAS symptomatology [11].Thus, it may be possible that individuals with PAN/PANDAS may be affected by the same mechanism that causes CFD.This has never been investigated before.
Folate is a water-soluble B-vitamin necessary for cerebral metabolism and function throughout development, adolescence, and adulthood [12][13][14].In patients with CFD, normal levels of 5-methyltetrahydrofolate (5MTHF) are found in serum despite below-normal concentrations of 5-methyltetrahydrofolate (5MTHF) in the cerebrospinal fluid (CSF) [15,16].This is caused by dysfunction of the folate receptor alpha (FRɑ), the main transporter of folate into the brain [16].In healthy conditions, folate binds to the FRɑ, which then undergoes endocytosis to actively transport folate across the blood-brain barrier.
One of the main causes of FRɑ dysfunction are FRɑ autoantibodies (FRAAs), which bind to the FRɑ and inhibit folate transport into the CSF [6,17].Two FRAAs have been described, i.e., the blocking and binding FRAAs.The blocking FRAA binds specifically to the site where folate binds to the FRα, thereby preventing folate from binding to the FRɑ, while the binding FRAA binds to other regions of the FRɑ and interferes with its ability to function optimally.
To determine the presence of CFD, standard practice utilizes lumbar punctures to measure the 5MTHF concentration in the CSF.Since the lumbar puncture is invasive [12] and generally requires a general anesthetic [14], it has become common practice to measure FRAAs using the folate receptor autoantibody test (FRAT).Previous studies have found the serum titers of FRAAs in patients with CFD and ASD correlate with the CSF concentration of 5MTHF, validating the use of FRAAs as a proxy for folate abnormalities in the brain [12,15,18].The drawback to this approach is that CFD cannot be diagnosed with only serum FRAA titers.However, since the treatment of central folate abnormalities, leucovorin, is very safe, the FRAAs in conjunction with a treatment trial are believed to have a lower risk than performing a lumbar puncture [12].
and inhibit folate transport into the CSF [6,17].Two FRAAs have been described, i.e., the blocking and binding FRAAs.The blocking FRAA binds specifically to the site where folate binds to the FRα, thereby preventing folate from binding to the FR J. Pers.Med.2024, 14, x FOR PEER REVIEW severe restrictive eating w lability, developmental r sleep disturbance, tics, a do not specify a specific agents such as Mycoplasm gers [4].It has been specu are likely to cause PANS known neurological or m Determination of PA consideration of family, m and infectious and autoi diagnosis; however, as th the acute onset of sympto negative laboratory findi entertained.PANS/PAND crobials are used to treat and pharmacological in Lastly, anti-inflammatory trient supplementation a Not all patients resp of PANS/PANDAS, sugg ology or that there is a co is being increasingly reco ated with many symptom disorders such as tics, cog disorder (ASD) [6,7].CF symptomatology, such a phrenia [10].Many indiv repetitive behaviors simil sible that individuals wi causes CFD.This has nev Folate is a water-sol throughout development mal levels of 5-methyltet mal concentrations of 5-m [15,16].This is caused by porter of folate into the b then undergoes endocyto One of the main cau bind to the FRɑ and inh described, i.e., the blockin the site where folate bind while the binding FRAA to function optimally.
To determine the pr measure the 5MTHF con and generally requires a ure FRAAs using the fo found the serum titers o concentration of 5MTHF in the brain [12,15,18].T with only serum FRAA ti leucovorin, is very safe, have a lower risk than pe , while the binding FRAA binds to other regions of the FR J. Pers.Med.2024, 14, x FOR PEER REVIEW severe restrictive eating with at least two comorbid symp lability, developmental regression, sensory and motor a sleep disturbance, tics, and/or a decline in academic pe do not specify a specific etiological trigger, it is speculat agents such as Mycoplasma pneumoniae and influenza, as gers [4].It has been speculated that infectious agents with are likely to cause PANS [4].PANS encapsulates disorde known neurological or medical disorder [1].
Determination of PANS/PANDAS requires a clinic consideration of family, medical, and psychiatric history and infectious and autoimmune disease evaluations [1,4 diagnosis; however, as the name suggests, diagnosis of P the acute onset of symptoms.Therefore, in the face of a cl negative laboratory findings that rule out other potential entertained.PANS/PANDAS treatment follows a threecrobials are used to treat any underlying infections.Seco and pharmacological interventions are used for psyc Lastly, anti-inflammatory agents, corticosteroids, intrav trient supplementation are used for immunomodulatory Not all patients respond adequately to this three-pr of PANS/PANDAS, suggesting that their symptoms may ology or that there is a comorbid condition that also requ is being increasingly recognized is cerebral folate deficie ated with many symptoms that overlap PAN/PANDAS disorders such as tics, cognitive impairment, developmen disorder (ASD) [6,7].CFD has been reported in disorde symptomatology, such as refractory depression and su phrenia [10].Many individuals with CFD have ASD, a repetitive behaviors similar to PANS/PANDAS symptom sible that individuals with PAN/PANDAS may be affec causes CFD.This has never been investigated before.
Folate is a water-soluble B-vitamin necessary for ce throughout development, adolescence, and adulthood [1 mal levels of 5-methyltetrahydrofolate (5MTHF) are fou mal concentrations of 5-methyltetrahydrofolate (5MTHF [15,16].This is caused by dysfunction of the folate recep porter of folate into the brain [16].In healthy condition then undergoes endocytosis to actively transport folate a One of the main causes of FRɑ dysfunction are FRɑ bind to the FRɑ and inhibit folate transport into the CS described, i.e., the blocking and binding FRAAs.The blo the site where folate binds to the FRα, thereby preventin while the binding FRAA binds to other regions of the F to function optimally.
To determine the presence of CFD, standard pract measure the 5MTHF concentration in the CSF.Since the and generally requires a general anesthetic [14], it has be ure FRAAs using the folate receptor autoantibody test found the serum titers of FRAAs in patients with CFD concentration of 5MTHF, validating the use of FRAAs as in the brain [12,15,18].The drawback to this approach i with only serum FRAA titers.However, since the treatme leucovorin, is very safe, the FRAAs in conjunction with have a lower risk than performing a lumbar puncture [12 and interferes with its ability to function optimally.
To determine the presence of CFD, standard practice utilizes lumbar punctures to measure the 5MTHF concentration in the CSF.Since the lumbar puncture is invasive [12] and generally requires a general anesthetic [14], it has become common practice to measure FRAAs using the folate receptor autoantibody test (FRAT).Previous studies have found the serum titers of FRAAs in patients with CFD and ASD correlate with the CSF concentration of 5MTHF, validating the use of FRAAs as a proxy for folate abnormalities in the brain [12,15,18].The drawback to this approach is that CFD cannot be diagnosed with only serum FRAA titers.However, since the treatment of central folate abnormalities, leucovorin, is very safe, the FRAAs in conjunction with a treatment trial are believed to have a lower risk than performing a lumbar puncture [12].
Studies suggest that central folate abnormalities play an essential role in the pathogenesis and exacerbation of ASD [12,17].Successful treatment of CFD and FRAA-positive ASD patients provides a basis for believing that central folate abnormalities are a treatable condition.Similar to ASD, it is believed that the etiology of the majority of PANS/PANDAS cases includes triggers from environmental factors, including factors that induce the production of FRAAs [17].A meta-analysis estimates the prevalence of FRAAs in ASD to be 71% [11].Leucovorin treatment (folinic acid) in FRAA-positive ASD patients results in improvements in ASD symptoms, including speech, communication, and repetitive behaviors [18].
Based on the overlap between PANS/PANDAS symptoms and CFD/ASD, such as repetitive behaviors, tics, depression, neurodevelopmental regression, and speech disfluency, we propose that FRAAs may play a role in the symptomatology of PANS/PANDAS.If FRAAs were found in patients with PANS/PANDAS, leucovorin or other reduced folate may provide symptomatic relief for many of the symptoms that often remain refractory to treatment.This is the first study to address this question.

Subjects
In total, 47 children and adolescents (age range = 6 to 18; age mean = 11 years; male = 29; female = 18) that met the criteria for PANS/PANDAS were included in this study.PANDAS is defined by the prepubescent acute and dramatic onset of OCD and/or tic disorder, along with neurologic abnormalities and an episodic waxing and waning course associated with GAS infection [2].PANS is defined as the sudden dramatic overnight onset of OCD and/or severe restrictive eating with at least two comorbid symptoms such as anxiety, emotional lability, developmental regression, sensory and motor abnormalities, urinary symptoms, sleep disturbance, tics, and/or a decline in academic performance [1].
These patients were evaluated, tested, and diagnosed by Lindsey Wells, ND, and Nancy O'Hara, MD, after a thorough workup to eliminate other cases.Associated symptoms such as OCD and ASD were diagnosed by a developmental-behavioral pediatrician, neurologist, or psychiatrist.Laboratory values and symptomatology were abstracted from records by the treating physicians into a deidentified database for analysis.This procedure was determined to be exempt under 45 CFR § 46.104(d)(4) by the WCG IRB (Puyallup, WA, USA).Symptoms of anxiety, OCD, tics, depression, and attention deficit-hyperactivity disorder (ADHD) were rated on four levels: none, mild, moderate, and severe.ASD was rated as present or not present.

Folate Receptor Alpha Autoantibody Assay
Approximately 2-4 mL of blood was drawn into a serum-separating tube, and the serum was separated by centrifugation.Moreover, 1 mL of serum was transferred to a transport tube and shipped.The FRAA assay was performed by Vascular Strategies (Plymouth Meeting, PA, USA) through their clinical laboratory improvement amendmentscertified pathway.The assay provides three results, i.e., the binding and blocking titers in the serum and whether the soluble folate-binding proteins are present.
Soluble folate-binding proteins are proteins in the blood that bind folate, thereby making folate less available.None of the patients manifested soluble folate-binding proteins in their serum; this is not discussed further.
The binding titer measures the binding of IgG antibodies to the FR Not all patients respond adequately t of PANS/PANDAS, suggesting that their s ology or that there is a comorbid condition is being increasingly recognized is cerebra ated with many symptoms that overlap P disorders such as tics, cognitive impairmen disorder (ASD) [6,7].CFD has been repor symptomatology, such as refractory depr phrenia [10].Many individuals with CFD repetitive behaviors similar to PANS/PAND sible that individuals with PAN/PANDAS causes CFD.This has never been investiga Folate is a water-soluble B-vitamin n throughout development, adolescence, and mal levels of 5-methyltetrahydrofolate (5M mal concentrations of 5-methyltetrahydrof [15,16].This is caused by dysfunction of t porter of folate into the brain [16].In heal then undergoes endocytosis to actively tra One of the main causes of FRɑ dysfu bind to the FRɑ and inhibit folate transpo described, i.e., the blocking and binding FR the site where folate binds to the FRα, ther while the binding FRAA binds to other re to function optimally. To determine the presence of CFD, s measure the 5MTHF concentration in the C and generally requires a general anesthetic ure FRAAs using the folate receptor aut found the serum titers of FRAAs in patien concentration of 5MTHF, validating the us in the brain [12,15,18].The drawback to th with only serum FRAA titers.However, sin leucovorin, is very safe, the FRAAs in con have a lower risk than performing a lumba using an enzymelinked immunosorbent assay (ELISA) as previously described [19].Both positive and negative controls are run with the assay, and the titer is reported in optical density units.
The blocking assay specific for binding IgG is used to measure binding FRAAs as previously described [19].The blocking assay uses an in vitro assay to measure the amount of autoantibody that is specifically blocking the binding site for folate in the FR J. Pers.Med.2024, 14, x FOR PEER REVIEW severe restrictive eating with at l lability, developmental regressio sleep disturbance, tics, and/or a do not specify a specific etiologi agents such as Mycoplasma pneum gers [4].It has been speculated th are likely to cause PANS [4].PA known neurological or medical d Determination of PANS/PA consideration of family, medical and infectious and autoimmune diagnosis; however, as the name the acute onset of symptoms.Th negative laboratory findings that entertained.PANS/PANDAS tre crobials are used to treat any und and pharmacological interventi Lastly, anti-inflammatory agents trient supplementation are used Not all patients respond ad of PANS/PANDAS, suggesting t ology or that there is a comorbid is being increasingly recognized ated with many symptoms that disorders such as tics, cognitive i disorder (ASD) [6,7].CFD has b symptomatology, such as refrac phrenia [10].Many individuals repetitive behaviors similar to PA sible that individuals with PAN causes CFD.This has never been Folate is a water-soluble Bthroughout development, adoles mal levels of 5-methyltetrahydro mal concentrations of 5-methylte [15,16].This is caused by dysfun porter of folate into the brain [1 then undergoes endocytosis to a One of the main causes of F bind to the FRɑ and inhibit fola described, i.e., the blocking and the site where folate binds to the while the binding FRAA binds t to function optimally.
To determine the presence measure the 5MTHF concentrati and generally requires a general ure FRAAs using the folate rec found the serum titers of FRAA concentration of 5MTHF, validat .In this assay, the amount of radiolabeled folate displaced from the FR J. Pers.Med.2024, 14, x FOR PEER REVIEW severe restrictive eating with at least two comorbid lability, developmental regression, sensory and m sleep disturbance, tics, and/or a decline in academ do not specify a specific etiological trigger, it is sp agents such as Mycoplasma pneumoniae and influen gers [4].It has been speculated that infectious agen are likely to cause PANS [4].PANS encapsulates d known neurological or medical disorder [1].
Determination of PANS/PANDAS requires a consideration of family, medical, and psychiatric h and infectious and autoimmune disease evaluatio diagnosis; however, as the name suggests, diagnos the acute onset of symptoms.Therefore, in the face negative laboratory findings that rule out other pot entertained.PANS/PANDAS treatment follows a crobials are used to treat any underlying infections and pharmacological interventions are used for Lastly, anti-inflammatory agents, corticosteroids, trient supplementation are used for immunomodu Not all patients respond adequately to this th of PANS/PANDAS, suggesting that their symptom ology or that there is a comorbid condition that also is being increasingly recognized is cerebral folate d ated with many symptoms that overlap PAN/PAN disorders such as tics, cognitive impairment, devel disorder (ASD) [6,7].CFD has been reported in d symptomatology, such as refractory depression a phrenia [10].Many individuals with CFD have A repetitive behaviors similar to PANS/PANDAS sym sible that individuals with PAN/PANDAS may be causes CFD.This has never been investigated befo Folate is a water-soluble B-vitamin necessary throughout development, adolescence, and adulth mal levels of 5-methyltetrahydrofolate (5MTHF) a mal concentrations of 5-methyltetrahydrofolate (5M [15,16].This is caused by dysfunction of the folate porter of folate into the brain [16].In healthy con then undergoes endocytosis to actively transport fo One of the main causes of FRɑ dysfunction ar bind to the FRɑ and inhibit folate transport into t described, i.e., the blocking and binding FRAAs.T the site where folate binds to the FRα, thereby prev while the binding FRAA binds to other regions of to function optimally.
To determine the presence of CFD, standard measure the 5MTHF concentration in the CSF.Sin and generally requires a general anesthetic [14], it ure FRAAs using the folate receptor autoantibod found the serum titers of FRAAs in patients with when the patient's serum is added is measured and reported in pmol blocked per ml as previously described [19].
The blocking titer has been shown to correlate with CSF levels of 5MTHF [12], and patients with blocking and/or binding titers may have unique behavioral and biochem-ical characteristics [18].The presence or absence of the blocking and binding FRAAs is predictive of the response to leucovorin, at least in patients with ASD [11].

Statistical Analysis
To determine the relationship between symptomatology and FRAA titers, first a multivariable binary logistic regression (LR) model was used for dichotomous variables such as ASD (ASD/no ASD), and a multivariable ordinal (multinomal) LR was used for symptomatic variables with multiple levels (none, mild, moderate, or severe).Symptomatic variables include ASD, anxiety, OCD, tics, depression, and ADHD symptoms.Demographic variables, including sex and age, were initially included in the analyses but were found not to be significant and were subsequently removed.In these analyses, binding and blocking FRAA titers were predictor variables.Once a relationship between the symptomatology and FRAA titers was found, a linear regression equation was developed with the blocking or binding FRAA titers as the dependent variable and the symptoms identified as related to the FRAAs as the predictor variables.This provided an analysis of the relative influences of each symptom on the FRAA titer as well as testing for interactions between the symptoms.This two-step approach was used to limit the number of variables in the models to prevent overfitting.An alpha of 5% was used, and all models were simplified to remove nonsignificant, non-dependent variables.The odds ratio (OR) and linear regression coefficients (β) are presented with their 95% confidence interval.

Patient Characteristics
Table 1 provides the demographics of the patients.The majority of patients were either positive for only the binding FRAA (53%), or they were negative for both FRAAs (40%).The patients were of similar ages, although those that were positive for both the binding and blocking FRAAs were slightly younger.Those positive for FRAAs tended to be more female than those negative for FRAAs.The population was mostly white across all groups.Those that were positive for both the binding and blocking FRAAs had a higher rate of being prescribed ADHD medications and having allergies.
To examine the effect of ASD and tics simultaneously on the blocking FRAA, the tic variable was dichotomized as to whether severe tics were present or absent.ASD was found to be significantly related to binding titers [X 2 (1) = 5.40, p = 0.02; β = −0.76(−0.10, −1.41)], with those with ASD demonstrating a binding FRAA titer that was 0.76 OD lower than those without ASD.Tics were also significantly related to binding FRAA titers [X 2 (1) = 6.30, p = 0.01; β = 0.90 (0.18, 1.62)], with those having severe tics having a binding FRAA titer that was 0.90 OD higher than those not having severe tics.Nine patients had ASD, with three of them having tics, but none of them having severe tics.

Case Study of a FRAA-Positive PANS Patient
An 18-year-old male was previously diagnosed with PANS and autoimmune encephalitis at 13 years of age due to a sudden onset of anxiety, refusing to go to school, frequent urination, OCD, and repeated strep infections.Antibiotic treatment for GAS resulted in a 30% improvement in symptoms.IVIG treatment resulted in a 60-70% improvement in symptoms.Symptoms were manageable for several years with prophylactic antibiotics.The patient then developed mycoplasma pneumonia, resulting in an exacerbation of anxiety and OCD.Plasmapheresis decreased mycoplasma titers, but the titers remained elevated.Two years later, he developed an Epstein-Barr virus (EBV) infection, which was treated with antivirals and other supplements.Since then, he has been unable to tolerate interventions such as antibiotics, herbals, and supplements due to negative reactions and continued to experience severe anxiety and OCD.Other interventions included cognitive behavioral therapy, antipsychotics, and selective serotonin reuptake inhibitor medications.Previous medical and surgical history also included a tonsillectomy and adenoidectomy at 2 years of age, frequent GAS infections, influenza, and a ruptured appendix at 11 years of age.Due to sensitivities, he eliminated dairy and gluten from his diet.The patient had trouble leaving the house, taking care of personal hygiene, and began having insomnia and obsessive thoughts that mainly occurred in his dreams.
Upon presentation to our clinic, medications included propranolol (10 mg/day) and fluvoxamine (50 mg/day).The diagnosis of PANS was made due to the acute onset of symptoms at 13 years of age, including anxiety, OCD, sleep disturbances, recurrent GAS infections, improvement with antibiotics/plasmapheresis/IVIG in the past, lab evidence of mycoplasma and viruses, and low immunoglobulins.Continued therapy was recommended, and he began working with a cognitive-behavioral therapist three times a week.Further recommendations were made based on medical history and laboratory work.
Important findings from laboratory work included persistent mycoplasma IgG, IgM, and MTHFR C677T homozygous defects, as well as low normal levels of vitamin D (32) and vitamin A (30), IgG subclass 2 (118), red blood cell (RBC), magnesium (3.8), and RBC zinc (7.67).Interventions tried without improvements in symptoms included antivirals, immune, mitochondrial, and metabolic support.Vitamins D and A, zinc, and magnesium were added to treat mild deficiencies.The patient also had a positive binding FRAA autoantibody.To address this, leucovorin (5 mg/day) was added.The patient was instructed to increase leucovorin weekly by 1 pill (5 mg) up to 30 mg per day (6 pills per day) as tolerated and helpful.Additionally, further antimicrobials were prescribed to treat mycoplasma.However, the patient did not take them due to a past history of symptom exacerbations.
Upon implementation of the above, OCD and anxiety improved by 50%, and daily function improved by 70%.Dreams were no longer obsessive, his ability to take care of hygiene improved, and his personality started to come back, but social anxiety persisted.After further implementation of leucovorin and despite persistent mycoplasma titers and no antimicrobial therapy, the patient's symptoms improved by 90%.He noted that when he neglected to take his leucovorin, his anxiety and OCD symptoms worsened.With leucovorin treatment, the patient is currently excelling academically at a four-year university with honors, has friends, enjoys multiple hobbies, lives independently, and no longer experiences significant social anxiety or OCD.

Discussion
FRAT was conducted on 47 children and adolescents clinically diagnosed with PANS/ PANDAS to determine the presence of serum FRAAs.An FRAA prevalence of 63.8% was found, similar to the prevalence found in ASD [11].Patients with CFD and/or ASD positive for FRAAs respond well to leucovorin, a folate that can circumvent the block of the FR J. Pers.Med.2024, 14, x FOR PEER REVIEW 7 of 10 hygiene improved, and his personality started to come back, but social anxiety persisted.
After further implementation of leucovorin and despite persistent mycoplasma titers and no antimicrobial therapy, the patient's symptoms improved by 90%.He noted that when he neglected to take his leucovorin, his anxiety and OCD symptoms worsened.With leucovorin treatment, the patient is currently excelling academically at a four-year university with honors, has friends, enjoys multiple hobbies, lives independently, and no longer experiences significant social anxiety or OCD.

Discussion
FRAT was conducted on 47 children and adolescents clinically diagnosed with PANS/PANDAS to determine the presence of serum FRAAs.An FRAA prevalence of 63.8% was found, similar to the prevalence found in ASD [11].Patients with CFD and/or ASD positive for FRAAs respond well to leucovorin, a folate that can circumvent the block of the FRᾳ.Thus, similarly, it is possible that FRAA-positive PANS/PANDAS patients may benefit from the same treatment.We provided a case study of one patient that showed significant improvement in OCD and anxiety with leucovorin, but we have found that other PANS/PANDAS patients also do very well when treated with leucovorin or 5-methyltetrahydrofolate (5-MTHF).
Blocking FRAA titers has been shown to correlate with CSF folate concentrations in CFD [17] and ASD [13].In a study, FRAAs were found in 89% of children with CFD but were not found in any children without neurological or developmental disorders [20].However, FRAAs have been detected in unaffected siblings and parents of children with ASD [21].In contrast, FRAAs have only been found in 15% of normal children without ASD siblings [11].Therefore, we can assume that the presence of FRAAs in non-PANS/PANDAS patients is possible and emphasize that the FRAAs are not diagnostic of any specific disorder but instead serve as an adjunctive measure to direct a treatment approach.
The FRAT differentiates between binding and blocking autoantibodies.In this study, 83.3% of PANS/PANDAS patients that tested positive for FRAAs in their serum had binding autoantibodies, and 6.7% had blocking autoantibodies.While there is no research on the effect of autoantibody type in PANS/PANDAS, binding and blocking FRAAs have been associated with different physiologies and phenotypes in children with ASD [18].It .Thus, similarly, it is possible that FRAA-positive PANS/PANDAS patients may benefit from the same treatment.We provided a case study of one patient that showed significant improvement in OCD and anxiety with leucovorin, but we have found that other PANS/PANDAS patients also do very well when treated with leucovorin or 5-methyltetrahydrofolate (5-MTHF).
Blocking FRAA titers has been shown to correlate with CSF folate concentrations in CFD [17] and ASD [13].In a study, FRAAs were found in 89% of children with CFD but were not found in any children without neurological or developmental disorders [20].However, FRAAs have been detected in unaffected siblings and parents of children with ASD [21].In contrast, FRAAs have only been found in 15% of normal children without ASD siblings [11].Therefore, we can assume that the presence of FRAAs in non-PANS/PANDAS patients is possible and emphasize that the FRAAs are not diagnostic of any specific disorder but instead serve as an adjunctive measure to direct a treatment approach.
The FRAT differentiates between binding and blocking autoantibodies.In this study, 83.3% of PANS/PANDAS patients that tested positive for FRAAs in their serum had binding autoantibodies, and 6.7% had blocking autoantibodies.While there is no research on the effect of autoantibody type in PANS/PANDAS, binding and blocking FRAAs have been associated with different physiologies and phenotypes in children with ASD [18].It was found that children with ASD that were positive for the binding FRAA had significantly higher B12 concentrations in their serum compared to children with ASD that were negative for the binding FRAA [18].High B12 levels in the serum may indicate inadequate cellular uptake of B12, which may exacerbate ASD symptoms.This assumption supports the finding that ASD patients with the binding FRAA had poorer social skills than those with the blocking FRAA [18].Children with ASD who were positive for the blocking FRAA had better physiological and behavioral profiles compared to children with ASD who were positive for the binding FRAA and or who were FRAA negative.The blocking FRAA was also associated with better redox metabolism and inflammation markers as compared to those negative for the blocking FRAA [18].More research is necessary to see if the type of FRAAs, binding or blocking, impacts PANS/PANDAS cellular mechanisms and symptoms.
When bioavailable folate concentrations are low, folate enters the CSF by binding to the FR J. Pers.Med.2024, 14, x FOR PEER REVIEW 7 of 10 hygiene improved, and his personality started to come back, but social anxiety persisted.After further implementation of leucovorin and despite persistent mycoplasma titers and no antimicrobial therapy, the patient's symptoms improved by 90%.He noted that when he neglected to take his leucovorin, his anxiety and OCD symptoms worsened.With leucovorin treatment, the patient is currently excelling academically at a four-year university with honors, has friends, enjoys multiple hobbies, lives independently, and no longer experiences significant social anxiety or OCD.

Discussion
FRAT was conducted on 47 children and adolescents clinically diagnosed with PANS/PANDAS to determine the presence of serum FRAAs.An FRAA prevalence of 63.8% was found, similar to the prevalence found in ASD [11].Patients with CFD and/or ASD positive for FRAAs respond well to leucovorin, a folate that can circumvent the block of the FRᾳ.Thus, similarly, it is possible that FRAA-positive PANS/PANDAS patients may benefit from the same treatment.We provided a case study of one patient that showed significant improvement in OCD and anxiety with leucovorin, but we have found that other PANS/PANDAS patients also do very well when treated with leucovorin or 5-methyltetrahydrofolate (5-MTHF).
Blocking FRAA titers has been shown to correlate with CSF folate concentrations in CFD [17] and ASD [13].In a study, FRAAs were found in 89% of children with CFD but were not found in any children without neurological or developmental disorders [20].However, FRAAs have been detected in unaffected siblings and parents of children with ASD [21].In contrast, FRAAs have only been found in 15% of normal children without ASD siblings [11].Therefore, we can assume that the presence of FRAAs in non-PANS/PANDAS patients is possible and emphasize that the FRAAs are not diagnostic of any specific disorder but instead serve as an adjunctive measure to direct a treatment approach.
The FRAT differentiates between binding and blocking autoantibodies.In this study, 83.3% of PANS/PANDAS patients that tested positive for FRAAs in their serum had binding autoantibodies, and 6.7% had blocking autoantibodies.While there is no research on the effect of autoantibody type in PANS/PANDAS, binding and blocking FRAAs have been associated with different physiologies and phenotypes in children with ASD [18].It was found that children with ASD that were positive for the binding FRAA had significantly higher B12 concentrations in their serum compared to children with ASD that were negative for the binding FRAA [18].High B12 levels in the serum may indicate inadequate cellular uptake of B12, which may exacerbate ASD symptoms.This assumption supports the finding that ASD patients with the binding FRAA had poorer social skills than those with the blocking FRAA [18].Children with ASD who were positive for the blocking FRAA had better physiological and behavioral profiles compared to children with ASD who were positive for the binding FRAA and or who were FRAA negative.The blocking FRAA was also associated with better redox metabolism and inflammation markers as compared to those negative for the blocking FRAA [18].More research is necessary to see if the type of FRAAs, binding or blocking, impacts PANS/PANDAS cellular mechanisms and symptoms.
When bioavailable folate concentrations are low, folate enters the CSF by binding to the FRᾳ on the epithelium of the choroid plexus and undergoes endocytosis.FRᾳ has a high affinity for folic acid and 5-MTHF [12,22].However, when folate concentrations are high, folate can also cross the blood-brain barrier using the reduced folate carrier (RFC) [22], which is a transmembrane protein found on the basolateral and apical sides of the epithelium of the choroid plexus [23].The RFC only transports reduced folates such as 5-MTHF and leucovorin but does not transport oxidized folates such as folic acid [19].The RFC provides another mechanism for folate to enter the CSF.In CFD, when the FRᾳ is dysfunctional, high doses of leucovorin are needed because of the low affinity of the RFC for folate [18].This is also reflected in our case study, in which leucovorin doses were on the epithelium of the choroid plexus and undergoes endocytosis.FR J. Pers.Med.2024, 14, x FOR PEER REVIEW hygiene improved, and After further implemen no antimicrobial therap he neglected to take hi covorin treatment, the with honors, has friend periences significant so
The FRAT differen 83.3% of PANS/PANDA ing autoantibodies, an the effect of autoantib been associated with d was found that childre cantly higher B12 conc negative for the bindin cellular uptake of B12, the finding that ASD p with the blocking FRA FRAA had better phys who were positive for FRAA was also associ compared to those neg if the type of FRAAs, b and symptoms.
When bioavailabl the FRᾳ on the epithel high affinity for folic a high, folate can also cr [22], which is a transm epithelium of the choro MTHF and leucovorin RFC provides another dysfunctional, high do for folate [18].This is has a high affinity for folic acid and 5-MTHF [12,22].However, when folate concentrations are high, folate can also cross the blood-brain barrier using the reduced folate carrier (RFC) [22], which is a transmembrane protein found on the basolateral and apical sides of the epithelium of the choroid plexus [23].The RFC only transports reduced folates such as 5-MTHF and leucovorin but does not transport oxidized folates such as folic acid [19].The RFC provides another mechanism for folate to enter the CSF.In CFD, when the FR J. Pers.Med.2024, 14, x FOR PEER REVIEW hygiene improved After further imple no antimicrobial th he neglected to tak covorin treatment, with honors, has fr periences significa

Discussion
FRAT was co PANS/PANDAS to 63.8% was found, ASD positive for FR of the FRᾳ.Thus, si benefit from the sa significant improv other PANS/PAND thyltetrahydrofola Blocking FRA CFD [17] and ASD were not found in However, FRAAs ASD [21].In contr ASD siblings [11] PANS/PANDAS p any specific disord proach.
The FRAT diff 83.3% of PANS/PA ing autoantibodies the effect of autoa been associated wi was found that ch cantly higher B12 c negative for the bin cellular uptake of B the finding that AS with the blocking FRAA had better p who were positive FRAA was also as compared to those if the type of FRAA and symptoms.
When bioavai the FRᾳ on the ep high affinity for fo high, folate can als [22], which is a tra is dysfunctional, high doses of leucovorin are needed because of the low affinity of the RFC for folate [18].This is also reflected in our case study, in which leucovorin doses were increased according to patient tolerance and resulted in improved symptoms.While further research on the effects of leucovorin in PANS/PANDAS patients is necessary, it should still be considered as mood support for those patients due to evidence that it helps with depression, cognitive function, anxiety, and OCD.
Methylenetetrahydrofolate reductase (MTHFR) is necessary for folate metabolism [24].Polymorphisms of the MTHFR gene result in a decrease in MTHFR enzyme activity and have been associated with psychiatric disorders such as ADHD, ASD, depression, schizophrenia, and bipolar disorder.The clinical presentation of MTHFR deficiency includes gait disorder, cognitive decline, epileptic syndromes, encephalopathy, and psychotic symptoms [25].Metabolic treatment has been shown to stabilize patients or improve their symptoms.In a study with patients clinically diagnosed with depression and no improvement after using three different antidepressants at maximum dosage for adequate duration, it was found that 36% of patients had CFD [26].When their antidepressant medication was supplemented with leucovorin for at least 6 weeks, symptoms improved, ranging from marginal to substantial improvement.
In the case study, the patient showed improvement in OCD symptoms with leucovorin supplementation and noted worsening of symptoms when the dose was missed.There is conflicting evidence suggesting that folate deficiency underlies OCD [27][28][29].When looking at individuals with OCD compared to neurotypical controls, OCD individuals had lower serum folate levels and higher homocysteine levels [27].However, B12 deficiency is known to exacerbate OCD symptoms [28,29].As previously stated, the binding FRAA in children with ASD (the case study was positive for binding autoantibodies) was associated with higher serum B12 levels; therefore, the OCD-like symptoms in PANS/PANDAS may be associated with B12 deficiency as well, considering that B12 and folate are involved in the same metabolic pathway.Because ASD and PANS/PANDAS have different overlapping but distinct symptomatology, it could be that CFD manifests differently in the two disorders.Our case also showed improved anxiety.Folate deficiency has been seen to cause an increase in anxiety in a rat model.Rats that were given FRAAs during gestation and pre-weaning displayed anxiety behaviors as adults, suggesting that folate is implicated in anxiety-like behaviors as well [30].Taken together, these findings show that supplementing with a reduced folate, like leucovorin or 5-MTHF, in children diagnosed with PANS/PANDAS may improve symptoms.
This study has several limitations, including the limited sample size, the retrospective nature of the data collection, the use of a sample for convenience, and the lack of blinding by the assessor.However, the physicians who diagnosed the children were not aware of their FRAA status before the test was performed, providing some blinding to the outcome variables.Clearly, a prospective study with blinded assessments is needed to confirm this compelling data.

Figure 1 .
Figure 1.Prevalence of folate receptor alpha antibodies in patients.(a) Positive or negative status of all study participants.(b) Prevalence of specific FRAA for those that were FRAA positive.Double+ signifies positivity for both blocking and binding FRAA.

Figure 1 .
Figure 1.Prevalence of folate receptor alpha antibodies in patients.(a) Positive or negative status of all study participants.(b) Prevalence of specific FRAA for those that were FRAA positive.Double+ signifies positivity for both blocking and binding FRAA.