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Anti-neuronal autoantibodies in the cerebrospinal fluid and serum from 106 patients with recent onset depression compared to 106 individually matched healthy controls

Open AccessPublished:October 28, 2022DOI:https://doi.org/10.1016/j.bpsgos.2022.10.007

      Abstract

      No large studies have investigated the prevalences of cerebrospinal fluid anti-neuronal autoantibodies in isolated depression. In this case-control study comparing 106 patients with isolated depression (ICD-10: F32) to 106 healthy controls, cerebrospinal fluid and serum were tested for seven immunoglobulin G autoantibodies using commercial fixed cell-based assays. To explore validity of methods, positive samples were re-tested twice by cell-based assays and once by tissue-based assays (monkey cerebellum). The prevalences of any of the anti-neuronal autoantibodies in cerebrospinal fluid were 0.0% in both groups and the seroprevalences were 0.9% in both groups, based on consistent findings in cell-based assays. However, all were negative by tissue-based assays. Evaluation of anti-neuronal autoantibodies in cerebrospinal fluid cannot be recommended routinely for patients with isolated depression of moderate severity, and future studies of isolated depression should consider much larger sample sizes and evaluation of anti-neuronal autoantibodies by other modalities than commercial kits.

      Keywords

      INTRODUCTION

      Depressed mood as part of the initial symptomatology of autoimmune encephalitis (AE) has previously been reported (
      • Herken J.
      • Prüss H.
      Red Flags: Clinical Signs for Identifying Autoimmune Encephalitis in Psychiatric Patients.
      ,
      • Restrepo-Martinez M.
      • Ramirez-Bermudez J.
      • Bayliss L.
      • Espinola-Nadurille M.
      Characterisation and outcome of neuropsychiatric symptoms in patients with anti-NMDAR encephalitis.
      ,
      • Kayser M.S.
      • Titulaer M.J.
      • Gresa-Arribas N.
      • Dalmau J.
      Frequency and characteristics of isolated psychiatric episodes in anti–N-methyl-d-aspartate receptor encephalitis.
      ) and at case report level, depression has been reported as the only phenotypical trait of anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis (
      • Moldavski A.
      • Wenz H.
      • Lange B.E.
      • Rohleder C.
      • Leweke F.M.
      Case Report: Severe Adolescent Major Depressive Syndrome Turns Out to Be an Unusual Case of Anti-NMDA Receptor Encephalitis.
      ). Despite the rarity of AE (
      • Nissen M.S.
      • Ørvik M.S.
      • Nilsson A.C.
      • Ryding M.
      • Lydolph M.
      • Blaabjerg M.
      NMDA-receptor encephalitis in Denmark from 2009 to 2019: a national cohort study.
      ,
      • Dubey D.
      • Pittock S.J.
      • Kelly C.R.
      • McKeon A.
      • Lopez-Chiriboga A.S.
      • Lennon V.A.
      • et al.
      Autoimmune encephalitis epidemiology and a comparison to infectious encephalitis.
      ), commercial kits for evaluation of several anti-neuronal autoantibodies (Abs) that can cause AE are available, including; NMDAR, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-receptor 1 and 2 (AMPAR-1/2), contactin-associated protein 2 (CASPR2), leucine-rich glioma-inactivated protein 1 (LGI1), γ-Aminobutyric acid b (GABAb) receptor B1/B2 and glutamic acid decarboxylase-65 (GAD65)-directed Abs (
      • McKeon A.
      • Tracy J.A.
      GAD65 neurological autoimmunity.
      ,
      • Prüss H.
      Autoantibodies in neurological disease.
      ,

      Euroimmune (2018): IIFT: Neurology Mosaics Instructions for the indirect immunofluorescence test. p 9.

      ). Especially measurements of anti-neuronal autoantibodies in the cerebrospinal fluid (CSF) are of interest, as anti-neuronal Abs need to reach the brain to be pathogenic (
      • Diamond B.
      • Huerta P.T.
      • Mina-Osorio P.
      • Kowal C.
      • Volpe B.T.
      Losing your nerves? Maybe it’s the antibodies.
      ).
      Case-control studies of unipolar depression and the prevalence of cerebrospinal fluid (CSF) anti-neuronal Abs are missing (
      • Hansen N.
      • Lipp M.
      • Vogelgsang J.
      • Vukovich R.
      • Zindler T.
      • Luedecke D.
      • et al.
      Autoantibody-associated psychiatric symptoms and syndromes in adults: A narrative review and proposed diagnostic approach.
      ). A study of 39 patients with depressive syndromes, found no NMDAR, AMPAR-1/2 or GABABR Abs in CSF (
      • Endres D.
      • Perlov E.
      • Dersch R.
      • Baumgartner A.
      • Hottenrott T.
      • Berger B.
      • et al.
      Evidence of cerebrospinal fluid abnormalities in patients with depressive syndromes.
      ) and a study of eight patients with depression and recent suicide attempt found no CSF anti-neuronal immunoglobulin G (IgG) isotype autoantibodies (
      • Fernström J.
      • Å Westrin
      • Grudet C.
      • Träskman-Bendz L.
      • Brundin L.
      • Lindqvist D.
      Six autoantibodies associated with autoimmune encephalitis are not detectable in the cerebrospinal fluid of suicide attempters.
      ). Seroprevalences have been more thoroughly evaluated: Among 207 patients with depression admitted to acute psychiatric care, the IgG Ab seroprevalences were estimated to 2.4% for GAD65 Abs and 0.5% for NMDAR Abs, whereas CASPR2 and AMPAR Abs were absent (
      • Schou M.
      • Sæther S.G.
      • Borowski K.
      • Teegen B.
      • Kondziella D.
      • Stoecker W.
      • et al.
      Prevalence of serum anti-neuronal autoantibodies in patients admitted to acute psychiatric care.
      ). A study of the NMDAR Ab seroprevalence found none of 99 patients with major depression to have IgG isotype NMDAR Abs, while the prevalence was 0.6% among the 357 healthy controls (
      • Steiner J.
      • Teegen B.
      • Schiltz K.
      • Bernstein H.-G.
      • Stoecker W.
      • Bogerts B.
      Prevalence of N-methyl-D-aspartate receptor autoantibodies in the peripheral blood: healthy control samples revisited.
      ). The largest study to date of healthy individuals (N = 1 703) estimated the seroprevalence of IgG isotype NMDAR Abs to be ∼1% (
      • Dahm L.
      • Ott C.
      • Steiner J.
      • Stepniak B.
      • Teegen B.
      • Saschenbrecker S.
      • et al.
      Seroprevalence of autoantibodies against brain antigens in health and disease.
      ).
      The current knowledge indicates that CSF and serum anti-neuronal Abs are rare among patients with depression; however, case-control studies comparing anti-neuronal Abs especially in CSF from patients with depression compared to healthy controls are absent and warranted (
      • Hansen N.
      • Lipp M.
      • Vogelgsang J.
      • Vukovich R.
      • Zindler T.
      • Luedecke D.
      • et al.
      Autoantibody-associated psychiatric symptoms and syndromes in adults: A narrative review and proposed diagnostic approach.
      ,
      • Lang K.
      • Prüss H.
      Frequencies of neuronal autoantibodies in healthy controls: Estimation of disease specificity.
      ). Thus, the aims of this study were to investigate the prevalences of anti-neuronal Abs in CSF or serum from patients with recent onset depression compared to healthy controls, including investigations of NMDAR, AMPAR1, AMPAR2, CASPR2, LGI1, GABABR and GAD65 Abs in CSF or serum, exploring the hypothesis that anti-neuronal IgG isotype Abs are rare, however more prevalent, among patients with depression compared to healthy controls. This specific anti-neuronal Ab panel was chosen for this study as the included anti-neuronal Abs have been reported to have the highest prevalence among patients with AE (
      • Pollak T.A.
      • Lennox B.R.
      • Müller S.
      • Benros M.E.
      • Prüss H.
      • Tebartz van Elst L.
      • et al.
      Autoimmune psychosis: an international consensus on an approach to the diagnosis and management of psychosis of suspected autoimmune origin.
      ).

      METHODS AND MATERIALS

      This study was a prospective case-control study of patients with recent onset depression (International Classification of Diseases 10th Revision (ICD-10): F32) and individually matched (age and sex) healthy controls. The study was conducted at the facility of Biological and Precision Psychiatry, Copenhagen Research Centre for Mental Health (CORE), Mental Health Centre Copenhagen, Copenhagen, Denmark. A study protocol was published prior to this paper (
      • Sørensen N.V.
      • Orlovska-Waast S.
      • Jeppesen R.
      • Christensen R.H.
      • Benros M.E.
      Neuroimmunological investigations of cerebrospinal fluid in patients with recent onset depression - a study protocol.
      ) and demographic data has previously been published (

      Sørensen NV, Orlovska-Waast S, Jeppesen R, Klein-Petersen AW, Christensen RH, Benros ME (2022): Neuroinflammatory biomarkers in the cerebrospinal fluid from 106 patients with recent onset depression compared to 106 individually matched healthy controls. Press.

      ). The study was approved by The Regional Committee on Health Research Ethics (Capital Region, j.no: H-16030985) and The Danish Data Protection Agency (j.no: RHP-2016-020, I-Suite no.: 04945).

      Outcomes

      The two co-primary outcomes were the prevalences of any anti-neuronal IgG Ab against NMDAR, AMPAR1, AMPAR2, CASPR2, LGI1, GABAb receptor B1/B2 or GAD65 in 1) CSF or 2) CSF or serum from patients with isolated depression compared to healthy controls.
      The two secondary outcomes were the specific prevalences of each of the abovementioned anti-neuronal Abs in 1) CSF or 2) CSF or serum from patients with isolated depression compared to healthy controls.

      Participants and clinical assessment

      Patients were recruited from in- and outpatient clinics of the Mental Health Services of the Capital Region and Region Zealand of Denmark from October 2018 until April 2021. Inclusion criteria for patients were: 1) Patients with a first-time diagnosis of depression (according to ICD-10: F32) diagnosed within the past year. 2) Ongoing depressive symptoms. 3) Age between 18 and 50 years. 4) Obtainment of written informed consent. Age and sex matched healthy controls were recruited mainly by internet advertisement and from the same capture area as the patients from September 2018 until July 2021. Inclusion criteria for healthy controls were: 1) Healthy individual. 2) Age between 18 and 50 years. 3) Obtainment of written informed consent. Exclusion criteria are provided in Table S1 for both groups. Clinical assessment was performed in accordance with the study protocol (
      • Sørensen N.V.
      • Orlovska-Waast S.
      • Jeppesen R.
      • Christensen R.H.
      • Benros M.E.
      Neuroimmunological investigations of cerebrospinal fluid in patients with recent onset depression - a study protocol.
      ). In brief, the initial diagnosis given in the clinical setting prior to enrolment was confirmed by SCAN interview (
      • Wing J.K.
      • Babor T.
      • Brugha T.
      • Burke J.
      • Cooper J.E.
      • Giel R.
      • et al.
      SCAN. Schedules for Clinical Assessment in Neuropsychiatry.
      ) at inclusion. Severity of depressive symptoms, anxiety and psychotic symptoms were assessed. Furthermore, cognitive screening was done by Mini-Mental State Examination (MMSE) (
      • Folstein M.F.
      • Robins L.N.
      • Helzer J.E.
      The Mini-Mental State Examination.
      ) and Montreal Cognitive Assessment (MoCA) (
      • Nasreddine Z.S.
      • Phillips N.A.
      • Bédirian V.
      • Charbonneau S.
      • Whitehead V.
      • Collin I.
      • et al.
      The Montreal Cognitive Assessment, MoCA: a brief screening tool for mild cognitive impairment.
      ), whereas further cognitive testing was done by Brief Assessment of Cognition in Schizophrenia (BACS) (
      • Keefe R.S.E.
      • Goldberg T.E.
      • Harvey P.D.
      • Gold J.M.
      • Poe M.P.
      • Coughenour L.
      The Brief Assessment of Cognition in Schizophrenia: reliability, sensitivity, and comparison with a standard neurocognitive battery.
      ). Prior to lumbar puncture, a neurological examination including Neurological Evaluation Scale for evaluation of Neurological Soft Signs (
      • Dazzan P.
      • Lloyd T.
      • Morgan K.D.
      • Zanelli J.
      • Morgan C.
      • Orr K.
      • et al.
      Neurological abnormalities and cognitive ability in first-episode psychosis.
      ) was performed.

      Anti-neuronal Ab analyses

      CSF and serum samples were collected as described in the protocol (
      • Sørensen N.V.
      • Orlovska-Waast S.
      • Jeppesen R.
      • Christensen R.H.
      • Benros M.E.
      Neuroimmunological investigations of cerebrospinal fluid in patients with recent onset depression - a study protocol.
      ) and the samples were analyzed at the national accredited laboratory for testing of anti-neuronal Abs at the Department of Clinical Immunology, Odense University Hospital. Laboratory personnel were blinded to all clinical information, including case/control status. Anti-neuronal IgG isotype Abs were detected by indirect immunofluorescence (IIF) using commercial fixed CBAs transfected with the following antigens: NMDAR NR1 subunit, GAD65, LGI1, CASPR2, AMPAR1, AMPAR2 and GABAb receptor B1/B2 (Autoimmune Encephalitis Mosaic 1, Euroimmun, Lübeck, Germany). Serum samples were analyzed in dilution 1:10, and samples positive for anti-CASPR2 were also analyzed in dilution 1:100. CSF samples were analyzed undiluted. Analyses were performed according to the manufacturer’s recommendations. Results were reported according to fluorescence intensity as negative (lack of specific fluorescence), borderline positive, weakly positive, moderately positive, and strongly positive. TBA testing was performed according to the manufacturer’s recommendations. As all TBAs were negative in screening dilution, no further titers were measured. Due to the known uncertainty regarding Abs with low titers, anti-neuronal Ab reactive samples were post-hoc re-analyzed blinded to previous results; twice by CBAs and once by a tissue-based assay (TBA) using monkey cerebellum as substrate (Euroimmun, Lübeck, Germany). Samples were considered positive in the primary analyses if consistently positive by three CBAs (borderline positive samples were not included due to the reproducibility maximum deviation of ±1 fluorescence intensity).

      Follow-up

      Participants with anti-neuronal Abs in CSF or serum (positive or borderline positive in ≥ 2 CBA runs) were followed up by same procedure as for inclusion. Participants, who had serum NMDAR Abs also at follow-up, had CSF and serum samples re-tested by IIF using a fixed CBA transfected with IgG NMDAR NR1 subunit antigens only (Anti-Glutamate receptor (type NMDA) IIFT, Euroimmun, Lübeck, Germany). Furthermore, their CSF and serum samples were sent blinded for a second opinion/third re-test by CBA NMDAR transfected cells at the manufacturer’s expert reference laboratory (Labor Stöcker, Germany).

      Statistical analyses

      Pearson χ2-tests without adjustment for continuity was applied for analyses of anti-neuronal Abs. Analyses of continuous demographic data were assessed by two-sample t-test and dichotomous data by Pearson’s χ2-test. Categorical variables are shown in absolute numbers and (%). Continuous variables are shown as mean (SD). Two-sided tests with p < 0.05 was considered significant. To further explore the seroprevalence of anti-neuronal Abs, subgroup analyses of age, sex, peripheral hs-CRP and leukocyte counts were conducted by logistic regression models. As an explorative analysis the results of first run CBA were analyzed as well. All analyses were done in R version 4.0.5 (

      R CORE Team (2018): R: A language and environment for statistical computing. R Foundation for Statistical Computing. Retrieved December 21, 2021, from https://www.r-project.org/

      ).

      RESULTS

      The study comprised 106 patients with recent onset depression and 106 age and sex matched healthy controls (71 females and 35 males in each group) (Table S2). Average age of patients was 26.0 (7.6) years and of healthy controls 26.4 (6.8) years. Of patients, 67.0% had not initiated antidepressants and 84.0% were outpatients. Mean time from diagnosis to inclusion was 4.0 (5.5) weeks. The patients suffered on average from moderate depression (HDRS-17: 20.6 (6.3)). Details of co-morbidities are provided in Table S3.

      Primary outcomes

      The prevalence of any CSF anti-neuronal Ab was 0.0% and the seroprevalence was 0.9% in both groups. All CSF and serum samples were negative by confirmatory TBAs (all p-values > 0.05) (Table 1). A full overview of individual positive test results from CBAs and TBAs is provided in Table 2.
      Table 1ǀ Prevalences of anti-neuronal autoantibodies (Abs) in CSF and serum
      Depression

      N (%)
      Healthy controls

      N (%)
      p-values
      CBA1Confirmatory TBA2CBA1Confirmatory

      TBA2
      CBA1
      Primary outcomes
      Any anti-neuronal Ab in CSF0 (0.0)0 (0.0)0 (0.0)0 (0.0)1.000
      Any anti-neuronal Ab in serum1 (0.9)0 (0.0)1 (0.9)0 (0.0)1.000
      Secondary outcomes
      CSF anti-neuronal Abs
      NMDAR0 (0.0)0 (0.0)0 (0.0)0 (0.0)1.000
      AMPAR10 (0.0)0 (0.0)0 (0.0)0 (0.0)1.000
      AMPAR20 (0.0)0 (0.0)0 (0.0)0 (0.0)1.000
      CASPR20 (0.0)0 (0.0)0 (0.0)0 (0.0)1.000
      LGI10 (0.0)0 (0.0)0 (0.0)0 (0.0)1.000
      GABAbR0 (0.0)0 (0.0)0 (0.0)0 (0.0)1.000
      GAD650 (0.0)0 (0.0)0 (0.0)0 (0.0)1.000
      Serum anti-neuronal Abs
      NMDAR1 (0.9)0 (0.0)0 (0.0)0 (0.0)0.316
      AMPAR10 (0.0)0 (0.0)0 (0.0)0 (0.0)1.000
      AMPAR20 (0.0)0 (0.0)0 (0.0)0 (0.0)1.000
      CASPR20 (0.0)0 (0.0)0 (0.0)0 (0.0)1.000
      LGI10 (0.0)0 (0.0)0 (0.0)0 (0.0)1.000
      GABAbRR0 (0.0)0 (0.0)0 (0.0)0 (0.0)1.000
      GAD650 (0.0)0 (0.0)1 (0.9)0 (0.0)0.316
      1The samples were considered positive for an anti-neuronal autoantibody if three independent tests by CBA were consistently positive (borderline positives not included).
      2Confirmatory TBA tests were only assessed on samples positive in first CBA.
      p-values are based on Pearson’s χ2
      test.
      Abbreviations: Ab: autoantibody. CBA: cell-based assay. TBA: tissue-based assay. CSF: cerebrospinal fluid. NMDAR: N-methyl-D-aspartate-receptor. AMPAR: α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-receptor. CASPR2: contactin-associated protein 2. LGI1: leucine-rich glioma-inactivated protein 1. GABA: γ-Aminobutyric acid-receptor. GAD65: glutamic acid decarboxylase-65.
      Table 2ǀ Anti-neuronal autoantibodies in CSF and serum by CBA 1, CBA 2, CBA 3, TBA and CBA at follow-up
      CBA tests
      Individuals with questionable or positive test resultsCBA test 1CBA test 2CBA test 3Positive in all 3 CBA testsTBA test (Monkey cerebellum)Follow-up1
      CSF
      Healthy control 1NMDAR +NMDAR ++NMDAR (+)NoNegativeNegative2
      Serum
      Patient 1NMDAR +NMDAR ++NMDAR ++NMDAR +/++NegativeNMDAR +2
      Healthy control 1NMDAR (+)NMDAR (+)NMDAR ++NoNegativeNMDAR +2
      Healthy control 2GAD65 ++GAD65 ++GAD65 ++GAD65 ++NegativeNegative
      Healthy control 3GAD65 ++NegativeNegativeNoNegativeNo follow-up
      Healthy control 4GAD65 (+)NegativeNegativeNoNegativeNo follow-up
      Healthy control 5CASPR2 (+)3CASPR2 +CASPR2 +NoNegativeNegative
      Healthy control 6CASPR2 (+)3CASPR2 (+)CASPR2 (+)NoNegativeNegative
      Healthy control 7CASPR2 (+)3NegativeNegativeNoNegativeNo follow-up
      Healthy control 8CASPR2 (+)3NegativeNegativeNoNegativeNo follow-up
      (+): borderline positive. +: weakly positive. ++: moderately positive. +++: strongly positive.
      1Mean follow-up time was 17.6 months. All samples were negative by TBA at follow-up.
      2CBA of transfected cells (Euroimmun) by reference laboratory (Labor Stöcker, Germany). Further information is found in Table S4.
      3All CASPR2 samples were negative when analyzed in dilution 1:100.
      Abbreviations: CSF: cerebrospinal fluid. CBA: cell-based assay. TBA: tissue-based assay. NMDAR: N-methyl-D-aspartate-receptor. CASPR2: contactin-associated protein 2. GAD65: glutamic acid decarboxylase-65.

      Secondary outcomes

      The seroprevalence of NMDAR IgG Abs was 0.9% among patients and 0.0% among healthy controls and the seroprevalence of GAD65 IgG Abs was 0.0% among patients and 0.9% among healthy controls (Table 1 and Figure S1).

      Follow-up investigations of the individuals tested positive for anti-neuronal autoantibodies

      One patient and four healthy controls were followed up within a mean time of 17.6 (7.9) months. The patient and healthy control, who were seropositive of NMDAR Abs at inclusion, remained seropositive at follow-up; both when tested by the mosaic and the specific NMDAR IgG kit. The healthy control, who additionally was CSF positive/borderline positive of NMDAR Abs at inclusion, were negative by the CBA from the reference laboratory. The healthy control had a normal neurological examination and no psychiatric symptoms neither at inclusion nor at follow-up visit. The seropositive patient suffered from moderate depression (HDRS-17 score of 23), had no neurological complaints at inclusion and had a normal neurological examination except for hyperreflexia. A full characteristic of the participants with NMDAR Abs in CSF and/or serum is found in Table S4 and Figure S1. The three remaining participants did not have serum Abs at follow-up (Table 2) and the healthy control, who had GAD65 Abs in serum, did not have diabetes mellitus.

      Explorative analyses

      We found no significant subgroup differences regarding sex, age, hs-CRP levels or peripheral leukocyte levels related to the seroprevalences of anti-neuronal Abs (Table S5) nor regarding serum CBA positive or borderline positive results of first run CBA (Table S6).

      DISCUSSION

      Comprising 212 individually matched participants (106 patients with recent onset depression and 106 healthy controls), this study evaluate anti-neuronal CSF Abs in isolated, unipolar depression. The anti-neuronal IgG isotype Abs were evaluated by a commercially available kit, and five participants were followed up due to potential abnormal test results at first visit. In both groups, the prevalences of any CSF anti-neuronal Ab were 0.0% and the seroprevalences were 0.9%. One patient was consistently seropositive of NMDAR Abs and one healthy control was consistently seropositive of GAD65 Abs in three CBAs. At follow-up, two participants were still positive in serum. However, our study emphasizes the uncertainties related to the current commercially available measurements of anti-neuronal IgG isotype Abs.
      As no previous case-control studies have systematically investigated CSF anti-neuronal Abs in a cohort of patients with isolated depression compared to age and sex matched healthy controls, such study has been warranted to explore the significance of anti-neuronal Abs in depression (
      • Hansen N.
      • Lipp M.
      • Vogelgsang J.
      • Vukovich R.
      • Zindler T.
      • Luedecke D.
      • et al.
      Autoantibody-associated psychiatric symptoms and syndromes in adults: A narrative review and proposed diagnostic approach.
      ). An informed consent was necessary for participation, why patients with decreased or altered level of consciousness—a criterion for possible AE (
      • Graus F.
      • Titulaer M.J.
      • Balu R.
      • Benseler S.
      • Bien C.G.
      • Cellucci T.
      • et al.
      A clinical approach to diagnosis of autoimmune encephalitis.
      )—were not included. Furthermore, patients were excluded if they had seizures within the past 10 years. Only one eligible participant met the second diagnostic criterion for possible AE (
      • Graus F.
      • Titulaer M.J.
      • Balu R.
      • Benseler S.
      • Bien C.G.
      • Cellucci T.
      • et al.
      A clinical approach to diagnosis of autoimmune encephalitis.
      ), namely a patient with CSF pleocytosis, detected also as part of the present study (
      • Sørensen N.V.
      • Orlovska-Waast S.
      • Jeppesen R.
      • Klein-Petersen A.W.
      • Christensen R.H.B.
      • Benros M.E.
      Neuroinflammatory Biomarkers in Cerebrospinal Fluid From 106 Patients With Recent-Onset Depression Compared With 106 Individually Matched Healthy Control Subjects.
      ). The patient underwent thorough neurological examination including re-lumbar puncture and broad evaluation of CSF anti-neuronal Abs, but no cause was found (
      • Sørensen N.V.
      • Orlovska-Waast S.
      • Jeppesen R.
      • Klein-Petersen A.W.
      • Christensen R.H.B.
      • Benros M.E.
      Neuroinflammatory Biomarkers in Cerebrospinal Fluid From 106 Patients With Recent-Onset Depression Compared With 106 Individually Matched Healthy Control Subjects.
      ). Additionally, the patient had no anti-neuronal Abs in CSF and serum when re-tested for the present study. Thus, in this study of patients with recent onset depression, who were mainly outpatients and who did not fulfill the criteria for possible AE, CSF anti-neuronal Abs were absent, indicating that anti-neuronal Abs are very rare contributors to depression pathophysiology among patients with isolated depression of moderate severity. Furthermore, regardless of symptomatology, AE is a very rare condition with low prevalence and incidence rates (
      • Nissen M.S.
      • Ørvik M.S.
      • Nilsson A.C.
      • Ryding M.
      • Lydolph M.
      • Blaabjerg M.
      NMDA-receptor encephalitis in Denmark from 2009 to 2019: a national cohort study.
      ,
      • Dubey D.
      • Pittock S.J.
      • Kelly C.R.
      • McKeon A.
      • Lopez-Chiriboga A.S.
      • Lennon V.A.
      • et al.
      Autoimmune encephalitis epidemiology and a comparison to infectious encephalitis.
      ) indicating that estimates of the prevalence of AE presenting as isolated depression will most likely require a much larger sample size than of our study.
      The seroprevalence of any IgG anti-neuronal Ab was estimated to be 0.9% among patients with depression. Previous studies have reported that one of 207 (
      • Schou M.
      • Sæther S.G.
      • Borowski K.
      • Teegen B.
      • Kondziella D.
      • Stoecker W.
      • et al.
      Prevalence of serum anti-neuronal autoantibodies in patients admitted to acute psychiatric care.
      ) and none of 99 (
      • Steiner J.
      • Teegen B.
      • Schiltz K.
      • Bernstein H.-G.
      • Stoecker W.
      • Bogerts B.
      Prevalence of N-methyl-D-aspartate receptor autoantibodies in the peripheral blood: healthy control samples revisited.
      ) patients with depression had NMDAR IgG isotype Abs in serum, corroborating the low seroprevalence found in our study and emphasizing that serum IgG Abs are rare among patients with depression. Furthermore, we estimated the seroprevalence of anti-neuronal Abs to be 0.9% among the healthy controls, as one healthy control had consistent GAD65 Abs in serum. The finding of anti-neuronal Abs in serum from healthy individuals is in line with the largest previous study of IgG type anti-neuronal Abs among healthy individuals to date (N = 1 703), revealing prevalences of IgG NMDAR, CASPR2 and GAD65 Abs to be approximately 1.2%, 0.3% and 0.3%, respectively (
      • Dahm L.
      • Ott C.
      • Steiner J.
      • Stepniak B.
      • Teegen B.
      • Saschenbrecker S.
      • et al.
      Seroprevalence of autoantibodies against brain antigens in health and disease.
      ). Thus, the prevalence of anti-neuronal Abs in serum are approximately the same in healthy individuals as in patients, questioning the relevance of serum IgG anti-neuronal Abs, when there is no clinical suspicion of AE. This has previously been addressed by others (
      • de Witte L.D.
      • Hoffmann C.
      • van Mierlo H.C.
      • Titulaer M.J.
      • Kahn R.S.
      • Martinez-Martinez P.
      Absence of N-Methyl-D-Aspartate Receptor IgG Autoantibodies in Schizophrenia: The Importance of Cross-Validation Studies.
      ), and anti-neuronal Abs as minimum need to reach the brain to be pathogenic (
      • Diamond B.
      • Huerta P.T.
      • Mina-Osorio P.
      • Kowal C.
      • Volpe B.T.
      Losing your nerves? Maybe it’s the antibodies.
      ); thus, mainly the prevalence of anti-neuronal Abs in the CSF is of clinical relevance. Thus, based on the findings from our study, we do not recommend routine CSF and serum investigation for AE among patients with isolated depression of moderate severity. However, AE should still be considered for patients with signs of possible AE or atypical presentation of depression.
      We estimated the specific CSF prevalence of anti-neuronal Abs from healthy controls to be 0.0% defined by three consistently positive results CBAs (borderline positives not included). However, in the first CBA run, the CSF prevalence was 0.9% in the healthy group. Only two previous studies investigated CSF anti-neuronal Abs among 40 (
      • Bien C.G.
      • Rohleder C.
      • Mueller J.K.
      • Bien C.I.
      • Koethe D.
      • Leweke F.M.
      Neural Autoantibodies in Cerebrospinal Fluid and Serum in Clinical High Risk for Psychosis, First-Episode Psychosis, and Healthy Volunteers.
      ) and 48 (
      • Theorell J.
      • Ramberger M.
      • Harrison R.
      • Mgbachi V.
      • Jacobson L.
      • Waters P.
      • et al.
      Screening for pathogenic neuronal autoantibodies in serum and CSF of patients with first-episode psychosis.
      ) healthy individuals respectively and found no anti-neuronal Abs in CSF from the healthy controls. Thus—to our knowledge—CSF anti-neuronal Abs among healthy individuals have not previously been reported in the peer-reviewed scientific literature. However, according to the manufacturer’s instruction, in one of their cohorts one (1.7%) of 60 healthy individuals had CSF NMDAR Abs (

      Euroimmune (2018): IIFT: Neurology Mosaics Instructions for the indirect immunofluorescence test. p 9.

      ). When followed up 6 months after inclusion, the healthy control in our cohort was still borderline positive for NMDAR Abs in CSF and weakly seropositive. Nevertheless, the healthy control was continuously healthy at re-examination and therefore the CSF was sent for re-test in the reference laboratory and the results here from were negative. As CSF analysis is considered the most important test in evaluation of AE (
      • Abboud H.
      • Probasco J.C.
      • Irani S.
      • Ances B.
      • Benavides D.R.
      • Bradshaw M.
      • et al.
      Autoimmune encephalitis: proposed best practice recommendations for diagnosis and acute management.
      ), the finding of false positive NMDAR Abs in CSF from a healthy individual highlights the unconditional need for a clinical context when interpreting Ab results, as also addressed by others (
      • Bastiaansen A.E.M.
      • de Bruijn M.A.A.M.
      • Schuller S.L.
      • Martinez-Hernandez E.
      • Brenner J.
      • Paunovic M.
      • et al.
      Anti-NMDAR Encephalitis in the Netherlands, Focusing on Late-Onset Patients and Antibody Test Accuracy.
      ). Furthermore, our results emphasize the importance of a healthy control group when investigating the contribution of anti-neuronal Abs in diseases.
      This study was strengthened by a large sample size, including a large control group and a well-characterized population. It is an additional strength that positive or borderline positive results by first CBA were re-run twice by CBA and once by TBA. However, the generalizability of the study was limited by design, as patients, who met most of the criteria for possible AE (
      • Graus F.
      • Titulaer M.J.
      • Balu R.
      • Benseler S.
      • Bien C.G.
      • Cellucci T.
      • et al.
      A clinical approach to diagnosis of autoimmune encephalitis.
      ), were not included. Likewise, the age criterium for participation (18-50 years) is a limitation, since e.g. anti-LGI1 encephalitis is more prevalent above 50 years of age (
      • Uy C.E.
      • Binks S.
      • Irani S.R.
      Autoimmune encephalitis: clinical spectrum and management.
      ) and as the prevalence of anti-neuronal Abs in general increases with age (
      • Ehrenreich H.
      Autoantibodies against the N-Methyl-d-Aspartate Receptor Subunit NR1: Untangling Apparent Inconsistencies for Clinical Practice.
      ). Furthermore, anti-NMDAR encephalitis has been reported among individuals younger than 18 years of age (
      • Uy C.E.
      • Binks S.
      • Irani S.R.
      Autoimmune encephalitis: clinical spectrum and management.
      ) and our findings do not apply to a pediatric population. Many other anti-neuronal Abs have been identified (
      • Prüss H.
      Autoantibodies in neurological disease.
      ), including paraneoplastic Abs and novel Abs like neurexin 3α that has also been associated with depression at case report level (

      Hansen N, Lange C, Maass F, Hassoun L, Bouter C, Stöcker W, et al. (2021, May): Mild Amnestic Cognitive Impairment and Depressive Symptoms in Autoimmune Encephalitis Associated with Serum Anti-Neurexin-3α Autoantibodies. Brain Sciences, vol. 11. https://doi.org/10.3390/brainsci11060673

      ). Thus, the results of our study are limited by the Ab panel chosen. Additionally, it might be a limitation that we used fixed CBA instead of live CBA, as live CBA has been shown to reveal more serum IgG Abs against NMDAR among patients with psychosis as compared to fixed CBA (
      • Jézéquel J.
      • Rogemond V.
      • Pollak T.
      • Lepleux M.
      • Jacobson L.
      • Gréa H.
      • et al.
      Cell- and Single Molecule-Based Methods to Detect Anti-N-Methyl-D-Aspartate Receptor Autoantibodies in Patients With First-Episode Psychosis From the OPTiMiSE Project.
      ). It is also a potential limitation that TBA was only used confirmatory and not consistently as this might have revealed reactivity not found by CBA (
      • Endres D.
      • von Zedtwitz K.
      • Matteit I.
      • Bünger I.
      • Foverskov-Rasmussen H.
      • Runge K.
      • et al.
      Spectrum of Novel Anti-Central Nervous System Autoantibodies in the Cerebrospinal Fluid of 119 Patients With Schizophreniform and Affective Disorders.
      ).
      Despite our study revealing a low prevalence of the seven most commonly occurring anti-neuronal Abs among patients with isolated depression, depression is a highly heterogenous disorder (
      • Malhi G.S.
      • Mann J.J.
      Depression.
      ) estimated to affect more than 260 million people worldwide (

      Global, regional, and national incidence, prevalence, and years lived with disability for 354 diseases and injuries for 195 countries and territories, 1990-2017: a systematic analysis for the Global Burden of Disease Study 2017. (2018): Lancet (London, England) 392: 1789–1858.

      ). Thus, we cannot rule out that specific subgroups of patients with symptoms mimicking depression can have anti-neuronal Abs contributing to the symptomatology. However, in individuals with isolated depression symptoms, we conclude that anti-neuronal Abs are very rare in the CSF and should mainly be investigated in individuals with specific symptoms of probable AE or CSF alterations. Commercial fixed CBAs are more sensitive than TBAs using monkey cerebellum, and borderline or weak Abs can only rarely be confirmed by a positive reaction on the tissue. Thus, when designing future studies, the methods (e.g. fixed CBA, live CBA, TBA, a combination of modalities or even other modalities (
      • Jézéquel J.
      • Johansson E.M.
      • Dupuis J.P.
      • Rogemond V.
      • Gréa H.
      • Kellermayer B.
      • et al.
      Dynamic disorganization of synaptic NMDA receptors triggered by autoantibodies from psychotic patients.
      )) and management of reproducibility with re-testing of positive results should be considered.

      Conclusion

      This is the first large, systematic, prospective case-control study to estimate the prevalence of anti-neuronal Abs in CSF and serum from patients with isolated depression compared to healthy controls. Anti-neuronal Abs were not present in CSF from this group of mainly outpatients with recent onset depression of moderate severity and without signs of AE. Thus, much larger sample sizes will be needed to estimate the prevalence and other modalities for anti-neuronal Ab evaluation could be considered. The study emphasizes the importance of a healthy control group and highlights the need of a clinical evaluation when interpreting—especially weak—Ab results. The results supported the hypothesis that anti-neuronal Abs are rare among patients with depression without symptoms of potential AE. Therefore, we do not recommend evaluating CSF or serum anti-neuronal Abs as part of the routine examination of patients with isolated unipolar depression of moderate severity in the clinical setting, unless there is an abnormal CSF test or a clinical suspicion of AE.

      ACKNOWLEDGEMENTS

      The present study was funded by an unrestricted grant from The Lundbeck Foundation (grant number R268-2016-3925). The funders had no role in the acquisition of the data, interpretation of the results or the decision to publish the findings.
      DISCLOSURES
      The authors report no biomedical financial interests or potential conflicts of interest.
      DATA AVAILABILITY STATEMENT
      The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy and ethical restrictions.

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