2.1 Clinical trial design
COVID-19 patients suspected with GMD (i.e. accompanied with gastrointestinal symptoms or antibiotic-associated diarrhea) will be recruited for this clinical trial.
Patients with one of the following criteria will be excluded: 1) Critical type of COVID-19 according to COVID-19 diagnosis and treatment guidelines in China (7th edition);9 2) Gastrointestinal perforation; 3) Gastrointestinal obstruction; 4) Recent history of intestinal fistula; 5) Disturbance of consciousness; 6) Gastrointestinal hemorrhage; 7) Malignant tumors; and 8) Pregnant or lactating women.
In addition, the criteria for case rejection, dropout and discontinuation in this study include:
1) Patients with poor compliance and unable to perform WMT; 2) Patients who participate in other trials or could not follow the routine treatment during the present trial; 3) Patients with WMT-related adverse events that need to be treated; and 4) Patients with aggravated illness or severe complications during the trial that must be immediately managed.
Eligible patients will be randomly divided into group A, who will receive routine treatment, and group B, who will receive WMT with continuing the routine treatment. The frequency of WMT will be once a day for three consecutive days.
2.1.3 Laboratory and clinical evaluations
The laboratory and clinical indicators will be tested or recorded at admission, 1 and 2 weeks after treatment and on the day of discharge.
The laboratory indicators will include COVID-19 nucleic acid test, fasting blood glucose, muscle enzyme, D-dimer, electrolytes, myoglobin, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), procalcitonin(PCT), peripheral blood lymphocyte subsets, oxygenation index, blood gas analysis, blood routine tests, liver function indicators, renal function indicators, inflammatory cytokines such as IL-2, IL-6, tumor necrosis factor α (TNF-α), interferon-gamma (IFN-γ) and intestinal barrier function related factors, such as LPS, D-lactate and diamine oxidase (Table 1). Venous and arterial blood samples will be collected by clinical physician. Arterial blood samples will be used for the detection of blood gas analysis and oxygenation indexes with a blood gas analyzer. The venous blood samples will be used for the detection of the remaining indicators, using Sysmex 1000i blood cell analyzer, Cobas501 automatic biochemical analyzer and JYDLT intestinal barrier function analysis system, respectively.
The clinical indicators will include fever, pulmonary shadow absorption, gastrointestinal symptoms, etc. (Table 2) In addition, all known WMT-related adverse events such as abdominal pain, abdominal distention, diarrhea, etc. will be monitored. We will also grade all patients with pneumonia severity index (PSI) (Supplementary Table 1) and acute physiology and chronic health evaluation II (APACHE II) (Supplementary Table 2).
2.2 Study unit
This trial will be completed jointly by First Affiliated Hospital of Guangdong Pharmaceutical College and Guangzhou Eighth People's Hospital. First Affiliated Hospital of Guangdong Pharmaceutical College will be responsible for the preparation of stool suspension and the operation of WMT, and Guangzhou Eighth People's Hospital will be responsible for collecting all the data listed above. All the data will be analyzed mutually by the biostatisticians participated in this study.
The enrolled patients will be allocated into group A and group B by the block randomization method. Taking the admission time as the compatibility factor, two patients adjacent to the admission time will be taken as a block group. The first patient in each block group will get a random number by querying the random number table. If the random number is odd, it will be included in group A, and the remaining patient in the block group will be included in group B.
Blind methods are not considered because this trial involves invasive procedures such as endoscopic catheterization, which patients have the right to know.
2.4.1 Stool donors: The donors' age will be between 18-25 years old, and their body mass index is between 18.5-23.9. All donors need to pass the questionnaire firstly, and those who meet the requirements will receive further interview, psychological examination and physical examination. Donors with infectious diseases, digestive diseases, metabolic diseases, chronic fatigue syndrome, autoimmune diseases, allergic disease, and neuropsychiatric diseases will be excluded. All qualified donors of the above projects will receive the training required by healthy diet, and then donate stool in consultation with researchers.
2.4.2 Stool suspension: The stool samples provided by the donor will be collected, weighed, added with sterile saline according to the ratio of feces to saline 1:5, and then mixed evenly. The mixture will be filtered through the intelligent microbial separation system (GenFMTer; FMT Medical, Nanjing, China), and then five-stages of filtration will be carried out. The obtained suspension will be immediately centrifuged at a speed of 2500 rpm for three minutes, and this step will be repeated three times. The final sediment will be suspended again with sterile saline in accordance with the ratio of sediment to saline 1:1.
2.4.3 WMT preoperative preparation:
Metoclopramide will be injected intramuscularly 30 minutes before WMT, and a proton pump inhibitor will be injected intravenously one hour before WMT.
2.4.4 WMT procedure: The nasojejunal tube will be placed in the middle digestive tract. Normal saline will be used to flushed the tube first, and followed by the infusion of the stool suspension with a standard volume of 200 ml per person. After WMT, the patient will be asked to stay in the lying position for 30 minutes, with restriction of strenuous activities.
2.5 Participant rights and confidentiality
Participants have the right to withdraw from the study freely at any stage of the study without discrimination or retaliation, and to have their medical rights unaffected. All the information collected in this study belongs to the private information of patients, and none of researchers can disclose the information unless they obtain the permission from patients. Code will be used to identify the participants in all research-related documents. Only research teams and sponsors, ethics committees and regulatory agencies can access code information and other research materials, except that participants’ personal information and related information will not be disclosed to any other person. Research results may be published in relevant scientific journals, but participants’ name will not appear in any research reports or public publications.
2.6.1 Primary outcome
According to COVID-19 diagnosis and treatment guidelines in China (7th edition), COVID-19 patients can be classified as mild, common, severe and critical types.9 Mild type was defined when the clinical symptoms were mild, without any imaging change. Common type was defined when patients had clinical symptoms such as fever, respiratory tract infection, etc., with pulmonary imaging changes. Severe type was defined when patients met any of the following: 1) the respiratory rate > 30 rate/min, 2) the pulse oxygen saturation ≤ 93%, and 3) the ratio of arterial oxygen partial pressure to fractional inspired oxygen ≤ 300 mmHg. Critical type was defined when patients met any of the following: 1) respiratory failure and requiring mechanical ventilation, 2) shock, and 3) comorbid with other organ failure and requiring ICU care. In the present study, patients diagnosed with mild and common types will be defined as non-severe and those diagnosed with severe and critical types will be defined as severe.
The primary outcome of this study will be the clinical efficacy of WMT on the COVID-19 patients suspected with GMD. The clinical efficacy will be reflected by the SARS-Cov-2 infection status and the recovery of the disease, which is defined as "improvement"(from severe to non-severe). In addition, the improvement of gastrointestinal symptoms will be co-primary outcome.
2.6.2 Secondary outcomes
Several secondary outcomes are as following:
1) Advantages of WMT over routine treatment, as reflected by the recovery time, PSI grade and APACHE II score.
2) Effects of WMT on organ functions and homeostasis, as reflected by fasting blood glucose, muscle enzyme, D-dimer, electrolytes, myoglobin, liver function and renal function.
3) Effects of WMT on the inflammatory response, as reflected by blood routine tests, CRP, ESR, PCT, inflammatory cytokines such as IL-2, IL-6, TNF-α, and IFN-γ.
4) Effects of WMT on intestinal barrier function as reflected by the factors including LPS, D-lactate, and diamine oxidase.
5) Effects on cellular immune functions, as reflected by the peripheral blood lymphocyte subsets.
6) The safety of WMT, as reflected by WMT-related adverse events.
2.7 Follow-up visit
Telephone follow-up will be conducted at one week, two weeks, and six months after discharge, and the contents included the clinical indicators listed above (Table 3).
The schedule of enrollment, interventions, assessments and follow-up is shown as below. (Figure 1)
2.8 Data collection and statistical analysis
Quantitative data are expressed as the mean ± standard deviation, minimum and maximum, and qualitative data are expressed as the rate and constituent ratio. When the total variance of quantitative data is equal, student's t test will be used to determine the difference between groups. When the total variance of quantitative data is not equal, the rank sum test will be used to determine the difference. Qualitative data will be tested by Chi-square test or Fisher's exact test, where appropriate. P < 0.05 will be considered significant. All data will be analyzed by SPSS software version 19.0 (IBM, Armonk, NY, USA).