Does OCD have implications for studies
Microbiome in obsessive-compulsive disorder
Obsessive-compulsive disorder (OCD) is a highly debilitating disorder with a lifelong life. The prevalence of approximately 2% is evenly divided between men and women, starts at an early age and usually develops into a chronic course if left untreated. Many patients with obsessive-compulsive disorder after years of delay due to patient and health factors; Obsessive-compulsive disorder therefore has an impact on educational performance, work ability and interpersonal performance. In addition, it is a considerable burden and burden for family carers  and is one of the ten most severely disabled mental illnesses worldwide .
The role of the microbial community in health and disease has been neglected in human research until recently, despite the fact that human microbes make up about 1-3% of our body mass  .Good microbes play an important role in nutrient metabolism, the production of vitamins and preventing the colonization of our intestines by pathogens . Recently, intestinal microbiota has gained increasing interest in the brain due to the far-reaching connection between the gut and the gut . The bidirectional communication through neural, hormonal and immunological signal transmission, in which the central, autonomic and enteric nervous system is involved, forms the microbiome-intestinal-brain axis . Skin microbiota are very diverse and a number of factors influence bacterial composition, including gender, handedness and time since the last hand washing [7, 8]. In psychiatry, dysbioses in anorexia nervosa, major depression, have been reported. Autism and other serious psychiatric disorders [9, 10].
The relationship between the occurrence of obsessive-compulsive disorder and the infection has long been known. PANS / PANDAS are acute forms of obsessive-compulsive disorder associated with autoimmune reactions from pathogens such as streptococci [11, 12]. The link between bacterial pathogen and immunological response, This is further evidence of the importance of the microbiome-gut-brain axis in OCD. Recently there have been numerous hypotheses regarding the importance of gut microbiome and obsessive-compulsive disorder [13, 14]. Neither skin nor gut microbial composition has ever been studied in obsessive-compulsive disorder. In particular, given the central importance of washing and cleaning for obsessive-compulsive disorder, the examination of skin and intestinal microbiota in obsessive-compulsive disorder is a logical step.
Objective 1. Characterization of the composition and diversity of the intestinal microbiome in people with obsessive-compulsive disorder, obsessive-compulsive disorder with compulsive washing and healthy controls.
- We assume that OCD patients show signs of pronounced intestinal dysbiosis (imbalance) due to lower microbial diversity and specific taxonomic and gene content differences compared to healthy controls. The primary result is the α-diversity, which refers to the number of species (wealth) in one place. Second result is β-diversity, which refers to the differences in species composition between participants and taxonomic abundance of bacterial groups (at the level of strain, class, order, family and genus). These measures provide information about the individual microbial composition at each location and the homogeneity in a group of people.
- We assume that decreased α- and β-diversity in the gut microbiome correlates with the severity of obsessive-compulsive symptoms measured with the Yale-Brown Obsessive-Compulsive Scale (Y-BOCS) and Obsessive-Compulsive Inventory Revised (OCI-R). We predict that greater symptom severity is associated with less diversity. A significant negative correlation will be supporting evidence for the microbiome-gut-brain axis.
- We assume that the intestinal microbiome has a significantly greater α-diversity composition after successful psychotherapy (change in the Y-BOCS score).
Objective 2. Characterize the composition and diversity of the skin (palm and forehead) in patients with obsessive-compulsive disorder, obsessive-compulsive disorder with primary obsessional washing, and healthy controls.
- We assume that obsessive-compulsive disorder in patients with compulsive washing show signs of skin dysbiosis, characterized by lower microbial α- and β-diversity and specific taxonomic and genetic characteristics content differences.
- A disturbed microbial diversity and abundance of the skin correlate with the time spent on washing and the properties of the skin surface, documented by high-resolution photography.
- Individuals with laundry contamination who do not wash for more than an hour a day will regain normal skin flora, which is more similar to healthy controls. This would provide important information regarding the plasticity of the skin microbiome. It also improves skin health in relation to problems with eczema or other skin diseases.
Recruiting: Healthy controls are recruited through advertising or from previous studies with their consent. Male and female patients between the ages of 18 and 45 from the OCD Clinic at Huddinge Hospital with a DSM-5 diagnosis of OCD and fear of contamination are invited to participate. Age-appropriate controls will be recruited who have no personal or family history of OCD. Exclusion criteria for all groups include history of GI tract surgery (excluding appendectomy or cholecystectomy); History of inflammatory bowel disease, irritable bowel syndrome, celiac disease, or any other diagnosis that could explain chronic or recurrent bowel symptoms, use of antibiotics in the past 3 months; pro-biotic use in the past 4 weeks; Pregnancy. Intellectual Disability, Autism Spectrum Disorder, and Psychosis.
Assessment: All participants meet with a skilled psychiatrist for diagnostic assessments including an M.I.N.I. interview. Participants are asked to fill out self-assessment forms on the internet and answer questions about their dietary habits over the past three months. Weight and height for calculating the Body Mass Index (BMI) are measured. All evaluation forms, information from interviews and medical examinations are documented in their medical records. Further information (e.g. antibiotics and previous therapies) will be taken from their medical records with the consent of the participants.
The investigators chose the following battery of questionnaires to capture all relevant outcomes variables (OCD symptoms), diet, and stool characteristics and frequency.
- Yale-Brown Obsessive Compulsive Scale (Y-BOCS): A commonly used clinical, semi-structured interview for assessing the severity of OCD.
- Revised compulsory inventory (OCI-R -): An 18-point self-report questionnaire estimating the OCD symptom dimensions. The OCI-R gives a total score and 5 subscales: review, hoard, neutralize, obsess, order and wash.
- Meal Q questionnaire: A web-based questionnaire standardized in Sweden on the frequency of food is used. Validation of energy and macronutrient intake, which affects the microbial composition.
- Bristol Stool Scale: A medical diagnostic scale used to classify the shape of human feces into seven categories.
In addition, the investigators collect demographic information on all patients: age, gender, starting age, occupational status, somatic health, and psychiatric comorbidities.
Stool Sampling Procedure: Patients and controls are given clear instructions on how to collect stool samples at home. Patients collect two stool samples at the beginning of treatment and two at the end of treatment. The controls collect two samples at a time, Participants only. Participants collect the stool at home using convenient kits that are sent The stool's microbial DNA is isolated using a phenol-chloroform extraction method combined with physical destruction of bacterial cells and a DNA purification kit (QiaAmp DNAstool Minikit, Qiagen, CA) .
Skin Sampling Procedure: All participants are requested not to wash their hands prior to sampling. preferably after a two hour ERP session with a psychologist. The time since the last hand wash will later be recorded as covariate in the statistical analysis. The palm of both hands are wiped separately with cotton swabs that are moistened with a solution of 0.15 NaCl and 0.1% Tween 20. The samples are then placed in 15 ml polypropylene tubes and stored at -20 degrees Celsius for 72 Hours before DNA extraction with Mobio UltraClean Plant DNA isolation kit (Mobio Laboratories) .
Exposure and Reaction Prevention Therapy (ERP): An evidence-based psychotherapy provided by experienced psychologists, either individually, in groups, face-to-face, or through the internet. At least five exposure and reaction prevention sessions are required to be an ERP treatment.
Sample storage in the biobank: All samples are stored in the biobank of the Karolinska Institutet (KI). The application for storage of samples in the biobank is made in accordance with the rules and regulations.
Metagenomic sequencing: The researchers will use two approaches: sequencing the 16S rRNA gene and shotgun metagenomics  providing functional information about which genes are present in the microbiota of patients. This method can also provide a more detailed taxonomic resolution than 16S rRNA sequencing.
16S metagenomic sequencing technique: DNA amplification of the V1-3 regions using forward primers (8f: 5'-AGAGTTTGATCMTGGCTCAG-3 ') and reverse primers (518r: 5'-ATTACCGCGGCTGCTGG-3') by PCR.454 pyrosequencing of 16S rRNA genes is performed on a life sciences genome sequencer FLX machine (Roche, Florence, SC, USA). Sequences less than 200 base pairs (bp) or longer than 1,000 bp that contain or contain incorrect primer sequences. More than one ambiguous base is discarded. 16S rRNA sequence data is generated processed by the QIIME (Quantitative Insights Into Microbial Ecology) pipeline grouped into Operational Taxonomic Units (OTUs; similar to species level) based on their sequence similarity at a threshold of 97% using BLAST and assigned taxonomy below Use of the Greengenes The main coordinates (PC) are generated with unweighted and weighted UniFrac distances.
Metagenomic Shotgun Sequencing: Used in the CTMR under the direction of Prof. Engstrand and Dr. Scale Koistines. Sequencing is done with the Illumina HiSeq 2500 platform in high performance mode. In order to use the flow cells efficiently, about ten samples are multiplexed with two indices per lane of the flow cell.In the downstream bioinformatics analysis, the raw sequence data is quality filtered and tailored to remove bases with Phred quality values of less than 20.
Statistical analysis: The downstream bioinformatics analysis consists of: i) taxonomic analyzes composition estimation to identify which microorganisms are present in the samples resolution at the strain level [ConStrains, kraken, metaphlan2]; ii) functional annotation and profiling both with methods based on reference gene catalogs and with hidden Markov model-based genes Family approaches to determine the microbial gene content and frequency [concoct, mocat2], including determination of the antibiotic resistance potential (antibiotic resistance); and iii) compute descriptive statistics and identify groups within the data and perform statistical analysis between subgroups using additional metadata if available [hirbin, stats]. Bioinformatics analyzes are performed on Swedish national SNIC cluster computing resources (Infrastructure for Computing) at UPPMAX.Differences in alpha diversity (expressed as number of observed species and Chao-1 estimator), beta diversity (UniFrac distances) and taxa frequency of bacterial groups (on the strain, class, order, family and genus levels) will be analyzed. Because response variables are not predicted to be normally distributed, nonparametric tests are used.
Determination of sample size: In the absence of any previous obsessive-compulsive disorder studies, the researchers consulted similar studies done on patients with depression and anorexia All included between 20 and 30 patients large enough to detect significant differences in alpha and beta diversity. Since this is an initial study of obsessive-compulsive disorder and obsessive-compulsive disorder investigators are doing two samples for two cases, investigators expect this to be a reasonable sample size to indicate if there are obsessive-compulsive disorder skin and bowel dysbiosis that warrant a larger investigation .
Timetable: The ethical proposal has been approved.
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