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RESEARCH ARTICLE
Asia Pac J Clin Trials Nerv Syst Dis 2018,  3:120

Differences in intestinal microflora and metabolites between patients with schizophrenia, depression, bipolar disorder, and healthy subjects: protocol for a case-control study


1 Department of Psychiatry, Xijing Hospital, Air Force Medical University, Xi’an, Shaanxi Province, China
2 Medical Office, Chinese People’s Liberation Army General Hospital, Beijing, China

Date of Web Publication27-Aug-2018

Correspondence Address:
Hua-Ning Wang
Department of Psychiatry, Xijing Hospital, Air Force Medical University, Xi’an, Shaanxi Province
China
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Source of Support: This work was supported by the National Natural Science Foundation of China, No. 81571309; a grant from Shaanxi Provincial Key Projects in the Field of Social Development, No. 2017 ZDXM-SF-047., Conflict of Interest: None


DOI: 10.4103/2542-3932.238438

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  Abstract 

Background and objectives: The human intestine contains a large amount of commensal bacteria. Under normal conditions, the intestinal microflora is stable, forms intestinal biological barriers, and promotes the growth and development of the organism. However, changes in the external environment can lead to disturbances in intestinal micro-organisms, causing host dysfunction and resulting in various types of disease. This study will investigate the role of intestinal microbes in the development of depression, bipolar disorder, and schizophrenia.
Design: A case-control study.
Methods: We recruited 50 patients with schizophrenia, 50 with depression, 50 with bipolar disorder, 50 with bipolar depressive episode, 50 with manic or hypomanic bipolar episode, and 50 age- and sex-matched healthy individuals who received physical examinations at the Department of Psychiatry of Xijing Hospital (China).
Outcome measures: The primary outcome measure is the degree of change in fecal bacterial microflora after 3 months of pharmaceutical treatment. The secondary outcome measures are the type and content of small molecule metabolites in feces, the Hamilton Depression Scale score, the Young Mania Rating Scale score, the positive and negative syndrome scale score, and the Global Assessment of Functioning scale score before vs. after treatment.
Discussion: The results of this study will reveal changes in intestinal microflora and metabolic patterns in patients with schizophrenia, depression, and bipolar disorder. These data may lead to biomarkers for disease diagnosis and provide new directions for investigation of possible mechanisms underlying the development of mental disorders.
Ethics and dissemination: This study was approved by Medical Ethics Committee, Xijing Hospital, China (approval No. KY20172048-1). This study was disigned in May 2017, received ethical approval on September 6, 2017, and registered on October 18, 2017. Patient recuritement initiated in November 2017 and ended in February 2018. Genomics and metabolomics detection and data analysis initiated in March 2018 and will end in December 2018. Results will be disseminated through presentations at scientific meetings and/or by publication in a peer-reviewed journal. Trial data will be publicly accessible via ResMan.
Trial registration: This trial was registered with the Chinese Clinical Trial Registry (registration number: ChiCTR-ROC-17013029). Protocol version: 2.0.

Keywords: intestinal flora; metabolite; fecal bacterial DNA; schizophrenia; depression; bipolar disorder; case-control study


How to cite this article:
Chen YH, Peng ZW, Zhang X, Bai J, Yu SF, Li XS, Qiang XL, Zhou P, He H, Wang HN. Differences in intestinal microflora and metabolites between patients with schizophrenia, depression, bipolar disorder, and healthy subjects: protocol for a case-control study. Asia Pac J Clin Trials Nerv Syst Dis 2018;3:120-7

How to cite this URL:
Chen YH, Peng ZW, Zhang X, Bai J, Yu SF, Li XS, Qiang XL, Zhou P, He H, Wang HN. Differences in intestinal microflora and metabolites between patients with schizophrenia, depression, bipolar disorder, and healthy subjects: protocol for a case-control study. Asia Pac J Clin Trials Nerv Syst Dis [serial online] 2018 [cited 2021 May 12];3:120-7. Available from: https://www.actnjournal.com/text.asp?2018/3/3/120/238438


  Introduction Top


Background

There are approximately 1014–1015 bacteria in the human intestine, which is 10- to 100-fold the number of eukaryotic cells in human intestinal tissue (Savage, 1977). Commensal bacteria play a critical role in human physiological processes, such as immune system function and nutrient absorption (Macpherson and Harris, 2004). Under normal conditions, the intestinal microflora is stable, forms intestinal biological barriers, and promotes the growth and development of the organism. However, changes in the external environment changes can lead to disturbances in intestinal micro-organisms, causing host dysfunction and triggering multiple diseases, such as depression, schizophrenia, diabetes, fatty liver, and bowel stress syndrome (Li et al., 2018; Meijers et al., 2018). Previous research has indicated that intestinal microbes regulate brain function through the intestinal nervous system, endocrine system, immune system, metabolic system, neurotransmitters, neurotrophic factors, and neurotoxic products, which can affect the development and regulation of the human central nervous system. At the same time, the brain can also regulate gastrointestinal function through the immune system, endocrine system, and the nervous system (Cryan and Dinan, 2012), and brain activity has been found to affect intestinal microbial populations (Tillisch, 2014).

Jiang (2015) reported that intestinal microbiotas differ significantly between depression patients and healthy controls. They also found that compared with healthy controls, Shannon’s diversity index of intestinal microbiota was significantly greater in depression patients, who exhibited higher proportions of Bacteroidetes, Proteobacteria, and Actinobacteria, and lower proportions of Firmicutes, and that an abundance of Faecalibacterium bacteria was negatively correlated with the severity of depression. The occurrence and development of depression has been related to a reduction of beneficial bacteria and an increase in numbers of harmful bacteria in the gut (Jiang et al., 2015). A recent study reported that depression induced by intestinal dysbiosis may be related to excessive activation of the kynurenine pathway (Kennedy et al., 2017). Under the influence of inflammatory factors, corticosteroids, and stress factors, the tryptophan metabolic pathway is unbalanced, leading tryptophan to be metabolized into quinaldinic acid via the kynurenine pathway. This leads to neurotoxicity and a reduction in 5-hydroxytryptamine production, and finally depressive symptoms (Kennedy et al., 2017). This finding suggests that an imbalance in the tryptophan metabolic pathway plays an important role in the development of depression. Intestinal microbes are known to regulate a range of neurotrophic factors and protein levels, such as brain-derived neurotrophic factor, synaptophysin, and postsynaptic density protein 95 (PSD-95). These substances are associated with neuronal plasticity and neurodevelopment, and changes in their expression levels may cause abnormal psychiatric symptoms (Li et al., 2017; Francis et al., 2018). Intestinal microbes can regulate the immune inflammatory response of an organism, affecting neurodevelopment and leading to the emergence of abnormal psychiatric symptoms (Nguyen et al., 2018). Schizophrenia and the associated metabolic abnormalities that are present after treatment with antipsychotic drugs is an increasing area of research (Sugawara et al., 2018). Intestinal microbiota can regulate lipid accumulation, lipopolysaccharide content, and the production of short-chain fatty acids, while short-chain fatty acids can affect food intake, inflammation regulation, and insulin signaling (Hur and Lee, 2015). Davey et al. (2012) reported that olanzapine administration in famale rats altered the distribution of intestinal flora, and increased levels of inflammatory factors such as interleukin-8 and interleukin-1β. These findings indicate that metabolic disorders caused by antipsychotic drugs may be related to inflammatory reactions mediated by microbial changes in the gut.



A recent cross-sectional study of individuals with bipolar disorder found that women with bipolar disorder who were treated with atypical antipsychotics carried fewer intestinal flora compared with women with bipolar disorder who were not treated with atypical antipsychotics (Flowers et al., 2017). Evans et al. (2017) used a 16S ribosomal RNA gene sequencing method to compare differences in fecal microbiota between 115 patients with bipolar disorder and 64 normal controls. They found that bipolar disorder was associated with Faecalibacterium such that therapeutically increasing these levels of Faecalibacterium helped reduce the disease burden. These data suggest that certain microorganisms may have potential as effective treatments for bipolar disorder.[Table 1] contains a summary of recent studies on intestinal microorganisms in patients with psychiatric disorders.
Table 1: Representative clinical studies on intestinal microorganisms in patients with psychiatric disorders

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Features of this study

Recent developments in genomics and metabolomics, particularly regarding the structure of intestinal microflora, have enabled a deeper understanding of the metabolic characteristics and physiological functions of microflora as well as the interaction mechanism with the host (Franzosa et al., 2015). Genomics explores the activity of life at the gene level. Many cellular activities occur at the level of metabolites. For example, cell signaling, energy transmission, and cell-to-cell communication are all controlled by metabolites. Metabolomics-related techniques can measure metabolic dynamics or metabolic flux in the human intestinal ecosystem at particular time points or over durations of time. Genomics in combination with metabolomics can illuminate relationships among species and reveal corresponding interactions between microbial and metabolic pathways.

As little information is available about the characteristics of intestinal microorganisms among patients with common psychiatric disorders and the role that these microorganisms play in disease severity, there are many pertinent topics for research.

According to current international and domestic medical treatment standards, patients meeting the diagnostic criteria for schizophrenia, schizophrenia-like disorders, bipolar disorder, or major depressive disorder as per the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5) should be treated. According to international and domestic guidelines and expert consensus, the clinicians develop a treatment plan. This study will not limit the treatment of patients.

Study objective

The aim of this study is to investigate the role of intestinal microorganisms in the occurrence and development of common psychiatric disorders (depression, bipolar disorder, and schizophrenia) using a combination of techniques. Specifically, we are focused on the identification of intestinal microflora with the goal of characterizing the metabolic processes associated with specific psychiatric disorders. Characterization of the mechanisms underlying psychiatric conditions may lead to clinical diagnostic biomarkers for specific psychiatric disorders, as well as potential treatments.


  Methods/Design Top


Study design

We recruited 50 patients with schizophrenia, 50 patients with depression, 50 patients with bipolar disorder, 50 patients with bipolar depressive episode, 50 patients with manic or hypomanic bipolar episode, and 50 age- and sex-matched healthy individuals who received a physical examination at the Department of Psychiatry of Xijing Hospital (China).

Stool samples for patients in the treatment group were collected prior to treatment. The schedule for collection of stool samples was not limited for the healthy individuals in the control group. The primary outcome measure was changes in fecal bacterial microflora after 3 months of treatment relative to that before treatment. The secondary outcome measures were the types and content of small molecule metabolites in feces, and scores on the Hamilton Depression Rating Scale, Young Mania Rating Scale, positive and negative syndrome scale, and General Function Rating Scale before and after 3 months of pharmaceutical treatment.

Recruitment

Recruitment was performed using information dissemination in various WeChat groups. The distributed information served to advertise the study among patients at clinics and wards at the Department of Psychiatry of Xijing Hospital in China. After being informed about the purpose of the trial and its interventions, as well as the risks and benefits, patients who were interested in participation (or their legal guardians) contacted the project manager via telephone, email, or WeChat and provided written informed consent. Participants were assessed by a group of experts associated with the trial, and confirmed according to the following eligibility criteria.

Eligibility criteria

-Schizophrenia group

Inclusion criteria

Patients with schizophrenia who had received treatment at the Department of Psychiatry of Xijing Hospital and met all of the following inclusion criteria were considered for inclusion:

  • Met the diagnostic criteria for schizophrenia or schizophreniform disorder as per the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5; American Psychiatric Association, 2013)
  • Total positive and negative syndrome scale (PANSS; Kay et al., 1987) score of 60 or above
  • Had not eaten prebiotics, probiotics, or probiotic fermented food products within the prior 1 month
  • Had not used antibiotics within the prior 1 month
  • Aged 18–65 years, either sex
  • Provision of written informed consent


Exclusion criteria

Patients presenting with one or more of the following criteria were excluded from this study:

  • Other psychiatric disorders meeting DSM-5 diagnostic criteria
  • Serious physical diseases
  • Digestive diseases or organic diseases, such as inflammatory bowel disease, Crohn’s disease, pancreatitis, or fatty liver
  • Obesity: body mass index ≥ 28.0 kg/m2
  • Hyperglycemia: fasting blood glucose ≥ 6.1 mM or 2-hour postprandial blood glucose (2 hTC) ≥ 7.8 mM and/or receiving treatment for diabetes
  • Triglyceride (TG) ≥ 2.3 mM
  • Hypertension: systolic pressure ≥ 140 mmHg or diastolic pressure ≥ 90 mmHg
  • Severely unbalanced diet, such as high-fat diet or long-term vegetarian foods preference
  • Use of any psychotropic drug and/or antipyretic analgesics for more than 2 days within the prior 2 weeks
  • Pregnant or lactating women


Withdrawal criteria

Patients with one of the following criteria were withdrawn from this study:

  • Presence of conditions not suitable for continuation of the study, including aggravated disease condition, serious adverse events, poor compliance
  • Non-eligible fecal specimens


-Depression group

Inclusion criteria

Patients with depression who had received treatment at the Department of Psychiatry of Xijing Hospital and met all of the following inclusion criteria were considered for inclusion:

  • Met the diagnostic criteria for severe depression described in the DSM-5 (American Psychiatric Association, 2013)
  • Hamilton Depression Scale (HAMD; Hamilton, 1960) score ≥ 18
  • Had not eaten prebiotics, probiotics, or probiotic fermented food products within the prior 1 month
  • Had not used antibiotics within the prior 1 month
  • Aged 18–65 years, either sex
  • Provision of written informed consent


The exclusion criteria and withdrawal criteria for the Depression group were the same as those for the Schizophrenia group.

-Bipolar disorder group

Inclusion criteria

Patients with bipolar disorder who had received treatment at the Department of Psychiatry of Xijing Hospital and met all of the following inclusion criteria were considered for inclusion:

  • Met the diagnostic criteria for bipolar disorder described in the DSM-5 (American Psychiatric Association, 2013)
  • HAMD score ≥ 18 at the time of depressive episode
  • Young Mania Rating Scale (YMRS; Young et al., 1978) score ≥ 20 at the time of manic episode
  • Had not eaten prebiotics, probiotics, or probiotic fermented food products within the prior 1 month
  • Had not used antibiotics within the prior 1 month
  • Aged 18–65 years, either sex
  • Provision of written informed consent


The exclusion criteria and withdrawal criteria for the bipolar disorder group were the same as those for the Schizophrenia group.

-Normal control group

Inclusion criteria

Healthy people who had received physical examination at the Health Center of Xijing Hospital of China and met all of the following inclusion criteria were considered for inclusion:

  • Had not eaten prebiotics, probiotics, or probiotic fermented food products within the prior 1 month
  • Had not taken antibiotics within the prior 1 month
  • Aged 18–65 years, either sex
  • Provision of written informed consent


The exclusion criteria and withdrawal criteria for the normal control group were the same as those for the Schizophrenia group.

Blinding

The assessors responsible for evaluating outcome measures will be blinded to grouping.

Interventions

Patients with schizophrenia, depression, and bipolar manic or hypomanic episodes were routinely treated. Drugs were selected according to the clinical manifestations of the disease and patient tolerance to drugs. For example, olanzapine or risperidone was used for schizophrenia patients with mainly positive symptoms, and aripiprazole and amisulpride were used for those patients with mainly negative symptoms. Certain drugs that affect glucose and lipid metabolism, such as olanzapine and risperidone, were not used for patients who were overweight or had high blood lipids. A single antipsychotic drug was preferred for treatment of each psychiatric disease. Benzodiazepines could be taken to improve sleep.

Outcome measures

Primary outcome measure

Change in fecal bacterial microflora after 3 months of treatment relative to preoperative levels.

Fecal specimens were collected from patients with depression, bipolar disorder, and schizophrenia before and 3 months after treatment with drugs at adequate doses. The schedule for collecting fecal specimens was not limited for healthy controls. Fresh feces was quickly placed in an ice box and then transferred to a laboratory for subpackaging. All fecal specimens were processed within 30 minutes after collection and then immediately stored in a –80 °C freezer for later use. 16S ribosomal RNA gene sequencing was performed to analyze the microflora in the feces.

The genomic DNA was extracted using the QIAamp DNA Stool Mini Kit, and the extracted DNA was subjected to quality control using Qubit 2.0 and 0.8% agarose gel electrophoresis. The DNA that passed the quality check could be sequenced. Two rounds of PCR amplification were performed on the purified genomic DNA to complete the construction of the sequencing sample library. The Qubit® 2.0 Fluorometer was used to measure the concentration and the Agilent 2100 was used to measure the size of the library. After the library was constructed, it was sequenced on a sequencing machine (MiSeq, Illumin). Sequencing reagents were prepared according to the MiSeq User Guide. Flow cells were placed on the sequencer (instrument pattern: MiSeq, Illumin). Paired-end 2 × 300 nt multiplex sequencing was performed using the paired-end program. The sequencing process was controlled using the Illumina data collection software and real-time data analysis was performed.

Secondary outcome measures

- Type and content of small molecule metabolites in feces Fecal small molecule metabolites were detected via metabonomics before and 3 months after pharmaceutical treatment. After fecal specimens were slowly thawed at 4°C, 1 mL of pre-chilled methanol/acetonitrile/water solution (2:2:1, v/v) was added. The mixture was vortexed, sonicated at a low temperature, and incubated at –20°C for 1 hour to precipitate protein. After centrifugation at 13,000 r/min and 4°C for 15 minutes, the supernatant was vacuum-dried. The resulting preparation was mixed with 100 µL of acetonitrile aqueous solution (acetonitrile: water = 1:1, v/v), vortexed, and centrifuged at 14,000 × g at 4 °C for 15 minutes. The supernatant was collected for mass spectroscopy. The samples were separated using ultra high performance liquid chromatography. To avoid the influence of detection signal fluctuations, samples were continuously analyzed using a random order. One quality control sample was selected from every five experimental samples in the sample cohort to monitor and evaluate the stability of the system and the reliability of the experimental data.

- HAMD score: HAMD scores were evaluated in patients with depression and patients with bipolar depressive episode before and 3 months after treatment. Total HAMD score can effectively reflect the disease severity, i.e., a lower HAMD score indicates milder symptoms and a higher HAMD score indicates more severe symptom (Hamilton, 1960)

- YMRS score: The severity of mania in patients with bipolar manic or hypomanic episode was evaluated before and 3 months after treatment. The YMRS consists of 11 items, including elevated mood, increased motor activity-energy, sexual interest, sleep, irritability, speech (rate and amount), language-thought disorder, content, disruptive-aggressive behavior, appearance, and insight, with a maximum score of 60. Higher scores indicate more severe manic symptoms (Young et al., 1978).

- Positive and negative syndrome scale (PANSS) score: The PANSS was used to evaluate patients with depression, bipolar disorder, and schizophrenia before and 3 months after pharmaceutical treatment. The positive and negative syndrome scale is used to determine the absence or presence and severity of psychiatric symptoms. The PANSS includes the Positive Scale (n = 7 items), Negative Scale (n = 7 items), General Psychopathology scale (n = 16 items), and 3 supplementary items that evaluate the risk of attack. Higher PANSS scores indicate more severe symptoms (Kay et al., 1987).

- Global Assessment of Functioning (GAF) score: The GAF was used to evaluate patients with depression, bipolar disorder, and schizophrenia before and 3 months after treatment. The maximum GAF score is 100. Higher GAF scores indicate milder disease (American Psychiatric Association, 2013).

- Laboratory tests: We conducted routine blood tests, routine urinary tests, and routine stool tests as well as tests of liver function, kidney function, blood glucose level, blood lipids level, electrocardiograms, and abdominal B-ultrasound examinations.

Adverse events

Adverse events refer to any adverse medical events occurring during the process of a clinical trial. They include (1) all suspicious adverse events; (2) all reactions due to drug overdose, abuse, withdrawal, allergy, or toxicity; (3) unrelated diseases such as aggravation of congenital diseases; (4) trauma or accidents; and (5) abnormalities found in physical examinations that require clinical treatment or further investigation. Researchers should report all adverse events that are directly observed by physicians or reported spontaneously by participants. In addition, practitioners should inquire with patients regarding adverse events at each visit after the start of treatment. Adverse events occurring from the provision of informed consent to 1 month after the end of the trial should be recorded for each subject. Only adverse events that are clearly more serious than baseline events for each patient should be recorded. Abnormal evaluation results obtained during subject screening or baseline collection are not considered to be adverse events. The date of onset of adverse events, measures taken, and any correlations between adverse events and drug treatment should be recorded. Participants who experience adverse events should receive follow-up assessments until the disappearance of said events, or until the condition stabilizes, at the discretion of the investigator. If the participant does not return to baseline levels by the end of the trial, the investigator should continue to provide necessary treatment, reports, and records. For special circumstances, patient cases should be handled according to the opinions of the relevant management department. The adverse events should be described as mild, moderate, or severe. Investigators should assess the possible association of adverse events with treatment.

Trial procedures

Flow diagram of the case-control study is shown in [Figure 1]. Schedule of outcome measure assessment is shown in [Table 2].
Figure 1: Flow diagram of the case-control study.

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Table 2: Schedule of primary and secondary outcome measures

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Sample size

In accordance with our previous study (Flowers et al., 2017) and based on the maximum grant amount, a total of 250 participants, 50 participants per group, will be included.

Statistical analysis

Statistical analysis will be performed using SPSS 19.0 software (IBM Corp., Armonk, NY, USA).

The differences in bacterial species and operational taxonomic units between groups will be analyzed using the Mann-Whitney U test with the linear discriminant analysis effect size (LEfSe) method. Correlation analysis will be performed using the Spearman Rank correlation test. In the pretreatment of metabolomics data, outlier will be detected using robust Principal Component Analysis method. The non-conservative metabolic components will be determined by comparing intra-class and inner-class differences of variables. Data preprocessing will be performed using the method of scale equalization to eliminate scale differences. A level of P < 0.05 will be considered statistically significant.

Data collection and management

The authors PZ and HH will be responsible for participant management, including informed consent and participant’s safety. The participant’s information should be strictly kept confidential. The participants should be clearly informed of their rights and interests, for example, the right to withdraw at any time during the research without any reason and without discrimination. Prior to enrollment, participants who will be hospitalized or non-hospitalized will be inquired of their medical history, in particular history of treatment, history of drug allergy, history of severe liver, kidney or heart diseases. Physical examination and corresponding laboratory tests (routine blood, urine, and stool tests and electrocardiogram) and scale evaluation will be performed. The investigators will urge the participants to be followed up in advance. The doctors will treat the possible adverse events occurring during the study period in a careful and timely manner. During this study, normal diagnosis and treatment process will not be interfered.

Data monitoring

The quality control and supervision of the research will be performed from the aspects of laboratory index detection, implementation of relevant SOPs, researcher training, subject compliance, and research monitoring. The research and supervision of clinical trials will be completed by a monitor appointed by the sponsor. The monitor’s responsibility is to ensure that the researchers strictly follow the study protocol, relevant standard operating procedures, guiding principles, and regulatory requirements during the research process. The monitor should keep in touch with the research center throughout the research. Before the study is launched, the monitor will hold a kickoff meeting at the research center to conduct research program, standard operating procedure, and GCP trainings. During the research process, the monitor should visit the research center regularly to understand the researcher’s compliance with the research program and applicable regulations through checking the original records and case report forms, and to ensure that the study data are objective, true, and legal. At the end of the study, the monitor should verify all documents of the research center and documented.

Compensation to patients

Patients included in the clinical trial will be able to receive a certain amount of transportation subsidy. They can be also compensated in follow up-related examination and registration fees. The insurance company will bear the cost of treatment and corresponding financial compensation for the participants who have suffered damage or death related to the trial.

Ethics and dissemination

This study will be performed in accordance with the Declaration of Helsinki developed by the World Medical Association. This study was approved by Clinical Trial Ethics Committee of Xijing Hospital of China (approval No. KY20172048-1 (Additional file 1 [Additional file 1]). Prior to enrolment of each participant in this study, it is the responsibility of the investigator to provide the participant or his or her legal guardian with a complete and comprehensive introduction about the purpose, procedure, and possible benefits and risks of this study. Each participate will sign the informed consent and he or she will understand that they have the right to withdraw from the study at any time. The signed informed consent should be retained as a clinical study document for future reference. The participant’s personal privacy and data confidentiality will be protected during the study period. The manuscript was prepared in accordance with Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) statements (Additional file 2 [Additional file 2]). Results will be disseminated through presentations at scientific meetings and/or by publication in a peer-reviewed journal. Anonymized trial data will be available through the ResMan.


  Discussion Top


Previous contributions and existing problems

In recent years, several studies have examined intestinal microflora in patients with psychiatric disorders (Jiang, 2015; Evans et al., 2017; Flowers et al., 2017). For example, metabolic disorders exist in patients with schizophrenia before and after antipsychotic treatment (Sugawara et al., 2018). Olanzapine interferes with changes in intestinal microflora in mice (Davey et al., 2012). However, related studies are often directed at a single disease. Moreover, little information is available regarding the detection of intestinal microflora and metabolic changes using genomics and metabolomics methods. All clinical and other relevant data of the patients will be recorded in case report forms, which will be kept by a designated person. The hospital ethics committee will be responsible for supervising the trial process and data. Once an adverse event occurs, it should be reported to the committee immediately. The hospital ethics committee will determine whether the trial will continue. Raw data will be obtained through contacting the corresponding author.

Novelty of this study

Genomics and metabolomic techniques can be used to analyze the structure of intestinal microbiota, metabolic features, physiological functions, and interaction mechanisms with the host. However, no studies have used genomics and metabolomics methods to examine changes in intestinal microbiota in patients with common psychiatric disorders. This study is the first to investigate changes in intestinal microbiota among patients with schizophrenia, depression, and bipolar disorder using genomics and metabolomics methods.

Limitations of the study

This study only selected two time points (before and 3 months after treatment) in patients with schizophrenia, depression, and bipolar disorder. This is not sufficient to comprehensively analyze the changes and metabolic dynamics of intestinal microflora in these patients.

Significance of this study

The results of this study will indicate specific intestinal microflora and their metabolic changes in patients with schizophrenia, depression, and bipolar disorder, and may yield potential biomarkers. This will provide new approaches for investigation of the possible mechanisms underlying psychiatric disorders.


  Trial Status Top


This study was designed in May 2017. Ethical approval was achieved in September 6, 2017. The study protocol was registered with the Chinese Clinical Trial Registry (registration number: ChiCTR-ROC-17013029) on September 18th, 2017. Patient recruitment was initiated in November 2017 and ended in February, 2018. Genomics and metabolomics testing and data analysis began in March 2018 and will be ended in December 2018. Data collection and analysis is ongoing.

Additional files

Additional file 1: Hospital ethics approval in Chinese.

Additional file 2: SPIRIT checklist.

Author contributions

Patient recruitment and management and data collection: PZ and HH; outcome assessment: JB, SFY and XSL; sample collection: XLQ; laboratory tests: ZWP; data analysis: YHC; experimental management and monitoring: HNW; conception of this protocol: XZ. All authors approved the final version of this manuscript.

Conflicts of interest

All authors declare that they have no conflicts of interest.

Financial support

This work was supported by the National Natural Science Foundation of China, No. 81571309; a grant from Shaanxi Provincial Key Projects in the Field of Social Development, No. 2017ZDXM-SF-047.

Institutional review board statement

All experimental procedures were performed in strict accordance with the Declaration of Helsinki and relevant ethical requirement of Xijing Hospital, Air Force Medical University, China.

Declaration of patient consent

The authors certify that they will obtain all appropriate patient consent forms. In the form the patients will give their consent for their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Reporting statement

This manuscript was prepared in accordance with the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) guidance for protocol reporting.

Biostatistics statement

The statistical methods of this study were reviewed by the biostatisticians of Xijing Hospital, Air Force Medical University, China.

Copyright license agreement

The Copyright License Agreement has been signed by all authors before publication.

Data sharing statement

Individual participant data that underlie the results reported in this article, after deidentification (text, tables, figures, and appendices) will be available. Study protocol, informed consent, and clinical study report will be available within 6 months after completing the trial. Results will be disseminated through presentations at scientific meetings and/or by publication in a peer-reviewed journal. Anonymized trial data will be available indefinitely at www.figshare.com.

Plagiarism check

Checked twice by iThenticate.

Peer review

Externally peer reviewed.

Funding: This work was supported by the National Natural Science Foundation of China, No. 81571309; a grant from Shaanxi Provincial Key Projects in the Field of Social Development, No. 2017 ZDXM-SF-047. [22]

 
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