Inclusion and exclusion criteria for cases.

Inclusion criteria for cases:

1. Patients with a first-time diagnosis within the past year of a non-affective psychotic disorder (ICD-10: F20, F22-29)
2. Age between 18 and 50 years
3. Obtainment of written informed consent

For exclusion criteria, see Table 1.

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Table 1. Exclusion criteria.

https://doi.org/10.1371/journal.pone.0257946.t001

Healthy controls.

Inclusion criteria for healthy controls:

1. Age between 18 and 50 years
2. Obtainment of written informed consent

For exclusion criteria, see Table 1.

Recruitment and screening.

The patients will be recruited via psychiatric departments and outpatient clinics in the Capital Region of Denmark (see S1 Table for an overview of involved centers). Healthy controls will mainly be recruited via a Danish web portal used to find participants for clinical studies (www.forsøgsperson.dk).

Patients will be screened for fulfilment of inclusion and exclusion criteria through their medical journals, including lists of current and prior diagnoses, medical treatment, and blood test results. The healthy controls will be asked a series of screening questions concerning their prior and current mental and physical health, both at first contact via telephone, and on the day of inclusion (see text section 1 in S1 Methods, “Screening questions for healthy controls”). On the day of inclusion, fulfilment of criteria for all participants will be validated through initial screening prior to all other procedures.

Intervention.

If an eligible participant agrees to be included in the study, written consent will be obtained. For each participant, we aim to perform all clinical measurement and obtain all biological samples on the same day. The inclusion will start between 9 and 10 am. The approximate duration of the inclusion is 3 hours for healthy controls, and 4.5 hours for patients, the latter including a lunch break. At any time during the inclusion, participants can take breaks as needed.

All participants will undergo an interview using WHO Schedules for Clinical Assessment in Neuropsychiatry (SCAN, version 2.1) [27], covering the last 4 weeks. The following chapters were included as they are considered relevant for the included diagnoses; 4 (anxiety), 6 (depressed mood), 7 (thinking, concentration, and energy), 8 (appetite and sleep), 10 (mania), 16 (perceptual disorders), 17 (hallucinations), 18 (thought interference and replacement of will), and 19 (delusions). All interviewers are certified in performing SCAN interviews. For the patients, the interview will be used to confirm diagnoses given by the clinical doctors. Controls will be interviewed prior to the blood sampling and lumbar puncture to ensure that no psychiatric disorders are present, and the time frame for all questions will be extended to include previous possible psychiatric disorders, to ensure that no history hereof exists either.

All participants will be rated on multiple psychopathology rating scales as listed in the following: Positive and Negative Symptom Scale (PANSS) [28], Scale for Assessment of Positive/Negative Symptoms (SAPS/SANS) [29, 30], 17-item Hamilton Depression Rating Scale (HAM-D-17) [31], Montgomery-Asberg Depression Rating Scale (MADRS) [32], Hamilton Anxiety Rating Scale (HAM-A) [33], and Young Mania Rating Scale (YMRS) [34]. Functioning will be rated using Personal and Social Performance Scale (PSP) [35], and Global Assessment of Functioning (GAF) [36].

During the visit, all participants will be asked to complete a questionnaire assessing depressive symptoms, the Major Depression Inventory (MDI) [37], and a questionnaire for the assessment of quality of life and overall health, the EQ-5D-5L [38].

Cognitive testing of all participants will be done using the Brief Assessment of Cognition in Schizophrenia (BACS) [39], Montreal Cognitive Assessment (MoCA) [40], Mini-Mental State Examination (MMSE) [41], and Trail Making Test (TMT) [42] A and B. All testers are certified in using BACS. Additionally, participants are asked to spell the Danish word “klode” backwards and mention as many animals as possible in 1 minute.

All participants will undergo a thorough neurological examination including scoring on the Neurological Evaluation Scale (NES) [43] for detection of neurological soft signs, such as trouble with fine motor coordination and audiovisual integration (for a description of both, see text section 2 and 3 in S1 Methods).

During the visit, all participants will be asked to complete a Danish questionnaire on diet and exercise [44]. Additionally, the following data on possible confounders are registered; height, weight, blood pressure, pulse, smoking status, alcohol intake, use of recreational drugs, allergies, somatic illnesses, prior and concurrent psychiatric disorders, current medication, use of NSAIDs, paracetamol, and antihistamines in the prior 2 weeks, use of antibiotics in the prior 6 months, information on female participants’ menstrual cycle, time for last intake of food and years of education, as well as employment status.

Biological samples.

Time points for collection of all biological samples will be noted. To minimize diurnal variation in the biomarkers, we aim to collect all blood samples between 9.30 and 11 am, and all CSF samples between 10 and 12 am. For both blood and CSF, a sterile culture swab will be used to perform a swab sample of the skin above the point of needle entry prior to sampling. The swab sample will be stored at -80 degrees Celsius.

The procedures are described below. For further details, see text section 4 in S1 Methods on the collection of blood samples and text section 5 in S1 Methods on the collection of CSF.

Venous blood samples from all participants will be collected prior to the lumbar puncture. A maximum of 44 mL of blood will be sampled.

Lumbar puncture will be carried out according to current consensus guidelines for lumbar puncture [45]. It will be noted whether 1) the participant is placed in a lateral decubitus or sitting position, 2) a traumatic or atraumatic needle is used and 3) whether any anxiolytics (e.g. benzodiazepines) has been taken by the participant prior to the procedure. When possible, we will use lateral decubitus position and atraumatic needles. Lumbar punctures will be performed under sterile conditions, using sterile gloves, sterile covers and face masks. A maximum of 16 mL of CSF will be collected.

As part of the construction of the larger biobank, each participant is asked to provide a fecal sample, either during the visit for inclusion, or afterwards from home. Fecal samples are collected in two ways, both in a Sarstedt Faeces Container and with the OMNIgene Gut Sample Collection Kit. If done at home, the fecal samples will be sent via mail, and stored at -80 degrees Celsius when received at the research unit. Samples collected during the visit will be frozen immediately. The Bristol Stool Scale score, time and date of sampling, as well as time and date of freezing is noted.

Follow-up.

All included patients will be offered a follow-up visit, which will take place at least one year after the first visit. Controls that matched patients who agreed to a follow-up visit, will also be offered a second visit. Follow-up visits will consist of the same procedures as those mentioned above.

Initial handling and storage.

Eight mL of the collected blood and 2 mL of the collected CSF are initially analyzed. Initial blood analyses include white blood cell (WBC) count, differential WBC count, hemoglobin, platelet count, HbA1c, glucose, hs-CRP, albumin, and IgG. Initial CSF analyses include WBC count, differential WBC count, erythrocyte count, protein, glucose, albumin, and IgG. Analyses of cells in CSF and blood are done using Sysmex XN9000, with distribution of cells in the CSF analyzed using CellaVision DM 96. HbA1c is measured using TOSOH G8, albumin and glucose using an ABL800 FLEX radiometer and the rest of the initial CSF and blood analyses are performed on Cobas 8000 module c502 (Roche). We aim to perform all initial CSF analyses and freeze all the remaining CSF no later than 1 hour after sampling. All laboratory personnel will be blinded to all clinical information.

A total of 36 mL of blood and 14 mL of CSF are processed for storage for later analyses, both as droplets on PKU cards, and as small aliquots frozen at -80°C. For details on handling and storage, see text section 6 in S1 Methods on handling of samples.

Analyses of stored samples.

The initial analyses of the biobank samples stored in small aliquots will be conducted when the first 100 patients with psychotic disorders and 100 healthy controls have been included.

Cytokines and chemokines will be analyzed using the Mesoscale V-PLEX Neuroinflammation Panel 1 Human Kit, by Statens Serum Institut, according to the manufacturer’s instructions.

Antineuronal autoantibodies will be analyzed at Odense University Hospital, with the Euroimmun Mosaic 1 kit (“Autoimmune encephalitis”) combined with the Euroimmun Gad65 Elisa kit, both used according to the manufacturer’s instructions.

All analyses will be performed in a blinded fashion, with no laboratory staff having access to clinical information on participants. Analyses will be performed according to a statistically designed experimental schedule based on a randomized block design or similar, to minimize the risk of confounding by time of analysis, batch, and other factors.

Biomarkers.

Table 2 gives an overview of the below mentioned outcomes.

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Table 2. Outcomes.

https://doi.org/10.1371/journal.pone.0257946.t002

The four co-primary outcomes for our initial screening analyses of CSF and blood will be CSF leukocytes, blood/CSF albumin ratio, CSF total protein and IgG index. For cytokine and chemokine CSF levels, IL-6 and IL-8 are the two co-primary outcomes. For antineuronal autoantibodies the two co-primary outcomes will be presence of any one of seven antineuronal autoantibodies (CASPR2-Ab, LGI1-Ab, NMDAR1-Ab, GABA-B receptor 1, Glutamate receptor 1, Glutamate receptor 2, GAD65) in 1) CSF and 2) in either blood or CSF.

As, secondary outcomes, more exploratory analyses will be performed on broader panels of cytokines, specific antineuronal autoantibodies, and other neurological/inflammatory markers. Possible associations between all biomarker outcomes and several clinical measurements will additionally be analyzed.

In our primary publication we will furthermore include reports of adverse events to the lumbar puncture procedure.

Measures of safety.

In order to assess the risk of adverse events associated with lumbar puncture, participants are contacted by telephone the day after the intervention. Adverse event that could be due to the procedures are noted, with specific focus on possible post lumbar headache and infections.

Registry data.

Data in the biobank will eventually be linked to the nationwide Danish registers to obtain information on, amongst others, prior infections treated in the primary care sector, comorbidities, treatment outcome (e.g. psychiatric readmissions and change in medication), and later psychiatric diagnoses.

Case assessment and definition.

Previous studies have found an increased peripheral inflammatory response in patients with psychotic disorders, particularly during the first psychotic episode [11, 18]. In order to investigate if also neuroinflammation is present in these patients, we only include patients with recent debut of the psychotic illness. Thus, the case group is also more homogenous making the patients more comparable.

To ensure an accurate case definition, we will independently, through our comprehensive SCAN-interview, validate the patients’ diagnoses given by the clinicians. WHO’s SCAN-interview is a broadly tested and widely used instrument in assessment of psychopathology [27]. In our interview we mainly focus on anxiety, depressive, manic and psychotic symptoms. This raises the possibility of not diagnosing other concurrent psychiatric disorders, such as ADHD or eating disorders, but we chose to do so to limit the time needed for the interview and make it more bearable even to the most severely ill patients. Furthermore, with the chosen chapters of the SCAN-interview we are certain that we can reliably validate the diagnoses within the psychotic spectrum.

Our follow up visit and the possibility of linking data to Danish registers, further makes it possible for us to, over time, validate the diagnoses given.

Control assessment and definition.

An important strength of our study is our healthy control group, as only few previous studies investigating immunological alterations in the CSF of patients with psychotic disorders have included a group of healthy controls. We thoroughly assess possible psychopathology in our healthy controls before including them in our study and interview them using the SCAN-interview just as we do with our cases. This allows us to exclude any subjects with current or previous psychiatric disorders. Additionally, many of the symptoms occurring in our patients might also to some degree be present in otherwise completely healthy controls (such as increased/decreased appetite for a period of time, issues with sleep, or some level of concentration difficulties). With our thorough SCAN-interview and multiple rating scales, we can adequately evaluate the severity of any possible symptoms. Recruiting healthy volunteers from the website forsøgsperson.dk allows us to include individuals from the same broad community as the cases.

Risk of bias.

When including a vulnerable group of patients, a risk of selection bias is difficult to avoid. The most severely ill patients might be less likely to agree to participate, particularly since the intervention is somewhat comprehensive. To overcome this to the greatest possible extent, we have tried to keep our intervention as brief as possible, whilst including all relevant variables, and have managed to limit the duration of the intervention to a single day. When recruiting patients, we will strongly emphasize the possibility of multiple breaks, and that the patients may leave at any time, should they feel the need to stop the intervention. Additionally, we arrange and pay for transport via taxi to and from the inclusion, and for those who are admitted to hospital wards, we offer to perform the intervention at their ward. Another possibility of selection bias stems from our strict inclusion and exclusion criteria. However, the nature of the study, investigating multiple biomarkers that broadly represent many aspects of the immune system, establishes the need for stringent exclusion of other factors with impact hereon.

Some confounders such as smoking, alcohol use, and eating habits, rely on self-reported data, raising the issue of possible recall bias, and limits the validity of some of these data.

Unexpected findings.

Some of the clinical examinations and biological samples will result in unexpected findings regarding the health status of patients or healthy controls. If a consent to be contacted regarding such findings has been given, the participant will be contacted and referred to further relevant investigations (e.g., at a neurological department). If the findings are found to be severe or life threatening, the participant will be contacted regardless of consent.