Patent Publication Number: US-2022226305-A1

Title: Method and combination for treating viral infection and long hauler syndrome

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation-in-part of U.S. patent application Ser. No. 17/406,774, titled “Methods of Preventing and Treating COVID-19 Infection with Probiotics,” filed Aug. 19, 2021, which is a continuation-in-part of U.S. patent application Ser. No. 17/200,585, titled “Methods of Preventing and Treating COVID-19 Infection with Probiotics,” filed Mar. 12, 2021, which claims priority to U.S. patent application Ser. No. 17/026,051, now U.S. Pat. No. 11,253,534, titled “Method of Preventing and Treating COVID-19 Infection, filed Sep. 18, 2020, which claims priority to U.S. Provisional Patent Application No. 62/993,345, titled “Method of Treating and Preventing COVID-19 Infection,” filed Mar. 23, 2020, U.S. Provisional Patent Application No. 63/022,371, titled “Method of Treating and Preventing COVID-19 Infection,” filed May 8, 2020, U.S. Provisional Patent Application No. 63/002,494, titled “Method of Using Vitamin C, Vitamin D, Zinc, and Optionally Hydroxychloroquine, to Prevent COVID-19 Infection,” filed Mar. 31, 2020, U.S. Provisional Patent Application No. 63/022,368, titled “Method of Using Vitamin C, Vitamin D, Zinc, and Optionally Hydroxychloroquine, to Prevent COVID-19 Infection,” filed May 8, 2020, and U.S. Provisional Patent Application No. 63/001,161, titled “Method of Using Aerosolized Hydroxychloroquine, Vitamin C, and Zinc to Treat Covid-19 Infection,” filed Mar. 27, 2020, and is continuation-in-part of U.S. patent application Ser. No. 17/350,520, titled “Method of Preventing and Treating COVID-19 Infection,” filed Jun. 17, 2021, which is a continuation application of U.S. patent application Ser. No. 17/126,715, now U.S. Pat. No. 11,166,971, titled “Method of Preventing and Treating COVID-19 Infection,” filed Dec. 18, 2020, which is a divisional application of U.S. patent application Ser. No. 17/026,051, titled “Method of Preventing and Treating COVID-19 Infection, filed Sep. 18, 2020, which claims priority to U.S. Provisional Patent Application No. 62/993,345, titled “Method of Treating and Preventing COVID-19 Infection,” filed Mar. 23, 2020, U.S. Provisional Patent Application No. 63/022,371, titled “Method of Treating and Preventing COVID-19 Infection,” filed May 8, 2020, U.S. Provisional Patent Application No. 63/002,494, titled “Method of Using Vitamin C, Vitamin D, Zinc, and Optionally Hydroxychloroquine, to Prevent COVID-19 Infection,” filed Mar. 31, 2020, U.S. Provisional Patent Application No. 63/022,368, titled “Method of Using Vitamin C, Vitamin D, Zinc, and Optionally Hydroxychloroquine, to Prevent COVID-19 Infection,” filed May 8, 2020, and U.S. Provisional Patent Application No. 63/001,161, titled “Method of Using Aerosolized Hydroxychloroquine, Vitamin C, and Zinc to Treat Covid-19 Infection,” filed Mar. 27, 2020, the contents of which are incorporated by reference in their entirety. 
    
    
     BACKGROUND 
     Viral infections are a constant and recurring issue for human health. With the emergence of COVID-19, viral infections have become a focus of the health industry. 
     It is known that single anti-viral agents work poorly when used alone in other chronic viral infections such as Hepatitis C or HIV infection. Therefore, the greater the number of anti-viral agents used in combination, the greater the cure rate. 
     Thus, there is a significant unmet need for treating viral infections and long hauler syndrome. 
     SUMMARY 
     The present invention addresses this need. The invention herein is directed to my method of treating viral infection and long hauler syndrome. The method of treatment comprises the steps of: providing an individual having a viral infection; and administering at least eight antimicrobials to the individual. 
     The antimicrobials comprise: administering hydroxychloroquine in a daily dosage range of 20 mg to 2,000 mg; administering azithromycin or doxycycline in a daily dosage range of 250 mg to 500 mg; administering ivermectin in a daily dosage range of 3 mg to 200 mg; administering vitamin C in a daily dosage range of 1,000 mg to 20,000 mg; administering vitamin D in a daily dosage range of 1,000 IU to 100,000 IU; administering zinc in a daily dosage range of 25 mg to 100 mg; and administering bifidobacterial probiotics in a daily dosage range of 1 billion to 100,000 billion bifidobacterial. 
     Optionally, the method can further comprise administering famotidine in a daily dosage range of 20 mg to 80 mg; administering oseltamivir phosphate in a daily dosage range of 30 mg to 75 mg; administering aspirin in a daily dosage range of 81 mg to 500 mg; administering cyproheptadine in a daily dosage range of 4 mg to 8 mg; administering a budesonide inhaler in daily dosage range of one to two puffs; and administering an iodine nasal spray in a daily dosage range of one spray in one nostril to two sprays in both nostrils. 
     Optionally, 400 mg of hydroxychloroquine can be administered, 250 mg of azithromycin or doxycycline can be administered, 36 mg of ivermectin can be administered, and 8 mg of cyproheptadine can be administered. 
     The method can be administered for at least one day, at least two days, or fourteen days. 
     The method can further comprise monitoring the individual&#39;s condition over a pre-determined period of time to determine whether the individual no longer has the viral infection. 
     The probiotic can be administered anally, orally, or topically, and if the probiotic is administered anally, it can be in the form of a fecal transplant, colonoscopy, suppository, enema, or topical application. 
     A combination of two or more antivirals for treating a viral infection, the combination comprising at least two of the following: hydroxychloroquine, azithromycin or doxycycline, ivermectin, oseltamivir phosphate, famotidine, vitamin c, vitamin d, zinc, aspirin, bifidobacterial probiotics, cyproheptadine, budesonide inhaler, and iodine nasal spray. 
     Preferably, the combination comprises at least hydroxychloroquine, azithromycin or doxycycline, ivermectin, oseltamivir phosphate, vitamin c, vitamin d, zinc, and bifidobacterial probiotics. 
     Optionally, combination comprises: 20 mg to 2,000 mg of hydroxychloroquine, 250 mg to 500 mg of azithromycin or doxycycline, 3 mg to 200 mg of ivermectin, 30 mg to 75 mg of oseltamivir phosphate, 1,000 mg to 20,000 mg of vitamin C, 1,000 IU to 100,000 IU of vitamin D, 25 mg to 100 mg of zinc, and 1 billion to 100,000 billion bifidobacterial in the form of a probiotic. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       Further advantages of the present invention may become apparent to those skilled in the art with the benefit of the following detailed description of the preferred embodiments and upon reference to the accompanying drawings in which: 
         FIG. 1  is a flow chart depicting the steps of a method of preventing infection of an individual with COVID-19; 
         FIG. 2  is a flow chart depicting the steps of a first method of treating an individual infected with COVID-19; 
         FIG. 3A  a graphical representation of whole genome alignment of SARS-CoV-2 in patient 1 of Example 6; 
         FIG. 3B  a graphical representation of whole genome alignment of SARS-CoV-2 in patient 3 of Example 6; 
         FIG. 3C  a graphical representation of whole genome alignment of SARS-CoV-2 in patient 4 of Example 6; 
         FIG. 3D  a graphical representation of whole genome alignment of SARS-CoV-2 in patient 6 of Example 6; 
         FIG. 3E  a graphical representation of whole genome alignment of SARS-CoV-2 in patient 8 of Example 6; 
         FIG. 3F  a graphical representation of whole genome alignment of SARS-CoV-2 in patient 10 of Example 6; 
         FIG. 3G  a graphical representation of whole genome alignment of SARS-CoV-2 in patient 11 of Example 6; 
         FIG. 3H  a graphical representation of whole genome alignment of SARS-CoV-2 in patient 12 of Example 6; 
         FIG. 4A  is a graph showing the Alpha diversity of SARS-CoV-2 positive patients and exposed healthy controls using a Shannon index [P=0.026]; 
         FIG. 4B  is a graph showing the Alpha diversity index of SARS-CoV-2 positive patients and exposed healthy controls using a Simpson index [P=0.043]; 
         FIG. 5  is a graph showing the relative abundance of Bifidobacteria for each subject, grouped by COVID-19 severity; 
         FIG. 6  is a diagram showing a proposed mechanism for cytokine storm and immune hyper-response in SARS-CoV-2 positive patients; and 
         FIG. 7  is a flow chart depicting the steps of a second method of treating an individual with a viral infection. 
     
    
    
     DETAILED DESCRIPTION 
     As used herein, the following terms and variations thereof have the meanings given below, unless a different meaning is clearly intended by the context in which such term is used. 
     The terms “a,” “an,” and “the” and similar referents used herein are to be construed to cover both the singular and the plural unless their usage in context indicates otherwise. 
     As used in this disclosure, the term “comprise” and variations of the term, such as “comprising” and “comprises,” are not intended to exclude other additives, components, integers ingredients or steps. 
     Referring now to the drawings, wherein like reference numerals designate identical or corresponding features throughout the several views. Further, described herein are certain non-limiting embodiments of my pipeline filter assembly for pool filtering and maintenance. 
     The following discussion describes in detail multiple embodiments of the invention with several variations of those embodiments. This discussion should not be construed, however, as limiting the invention to those particular embodiments. Practitioners skilled in the art will recognize numerous other embodiments as well. 
     In a first embodiment, the present invention is directed to a method of preventing COVID-19 infection in an individual. The method involves administration of chloroquine or hydroxychloroquine, Vitamin C, Vitamin D, and Zinc. Both methods are discussed in greater detail below. 
     Referring now to  FIG. 1 , there is shown the method of prevention. The method of prevention comprises administering four different antimicrobials. The four antimicrobials comprise: chloroquine or hydroxychloroquine, vitamin C, vitamin D, and zinc. 
     Day 1 the individual takes the following, ideal, regimen outlined in Table 1: 
     
       
         
           
               
               
               
               
             
               
                   
                 TABLE 1 
               
               
                   
                   
               
               
                   
                 Drug 
                 AM Dose 
                 PM Dose 
               
               
                   
                   
               
             
            
               
                   
                 Hydroxychloroquine 
                  200 mg 
                 200 mg 
               
               
                   
                 Vitamin C 
                 3000 mg 
                 — 
               
               
                   
                 Vitamin D 
                 3000 mg 
                 — 
               
               
                   
                 Zinc 
                  50 mg 
                 — 
               
               
                   
                   
               
            
           
         
       
     
     Optionally, the method of prevention can comprise administering on day 2: 3,000 mg of vitamin C; 3,000 mg of vitamin D; and 50 mg of zinc. 
     Hydroxychloroquine is administered only on day 1. The half-life of hydroxychloroquine is up to 32 days, thus treatment with this drug for one day should be sufficient. However, should the need to prevent the infection or disease last longer than 32 days, repeat dosing can be considered. Accordingly, if necessary, the cycle of day 1 followed by day 2 can be repeated weekly, 2 nd -weekly, every 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks. 
     Vitamin C is administered at 3,000 mg per day ongoing. This 3,000 mg can be broken up into two 1500 mg doses, one taken in the morning and one taken at night. 
     Vitamin D is administered at 3,000 mg per day ongoing. This 3,000 mg can be broken up into two 1500 mg doses, one taken in the morning and one taken at night. 
     Zinc is administered at 50 mg per day ongoing. This 50 mg can be broken up into two 25 mg doses, one taken in the morning and one taken at night. 
     Chloroquine or hydroxychloroquine can be administered in a daily dosage range of 20 mg to 2,000 mg. The above amounts recited in the tables are not limiting. 
     Vitamin C can be administered in a daily dosage range of 250 mg to 10,000 mg. The above amounts recited in the tables are not limiting. 
     Vitamin D can be administered in a daily dosage rage of 1,000 mg to 100,000 mg. The above amounts recited in the tables are not limiting. 
     Zinc can be administered in a daily dosage of 5 mg to 100 mg. The amount of Zinc can be reduced to 25 mg per day if gastrointestinal upset occurs. 
     Zinc, Vitamin C and D help in numerous aspects of viral protection through cellular metabolism, including catalytic activity of enzymes, and play roles in immune function, protein synthesis. 
     The chloroquine or hydroxychloroquine can come in various forms: as a pill, liquid solution, lozenges, topical treatment such as a cream or oil, or any other means of delivery. Hydroxychloroquine prevents cytokine release, and cytokine release is what causes anaphylactic flush. Optionally, the hydroxychloroquine is sprayed directly on the users tongue. 
     In the method of prevention described above, all, or any combination of, the four antimicrobials disclosed above can be administered in the form of a single small atomizer. The patient sprays the atomizer towards the back of their throat. The spray is administered at least once a day, but preferably twice a day when coughing starts. Use of the atomizer continues as directed by the supervising physician. 
     In a second embodiment, the present invention is directed to a method of treating an individual with infection or disease with five different antimicrobials. The five antimicrobials comprise: hydroxychloroquine, azithromycin, vitamin C, vitamin D and zinc. Referring now to  FIG. 2 , there is shown the method of treatment. 
     Day 1 following positive test (isolation), the individual takes the following regimen outlined in Table 2: 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 2 
               
               
                   
                   
               
               
                   
                 Drug 
                   
                 AM Dose 
                 PM Dose 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 Hydroxychloroquine 
                 200 
                 mg 
                 200 mg 
               
               
                   
                 Azithromycin 
                 500 
                 mg 
                 — 
               
               
                   
                 Vitamin C 
                 3000 
                 mg 
                 — 
               
               
                   
                 Vitamin D 
                 3000 
                 mg 
                 — 
               
               
                   
                 Zinc 
                 50 
                 mg 
                 — 
               
               
                   
                   
               
            
           
         
       
     
     On Day 2-Day 5 the individual takes the following regimen outlined in Table 3: 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 3 
               
               
                   
                   
               
               
                   
                 Drug 
                   
                 AM Dose 
                 PM Dose 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 Hydroxychloroquine 
                 200 
                 mg 
                 200 mg 
               
               
                   
                 Azithromycin 
                 250 
                 mg 
                 — 
               
               
                   
                 Vitamin C 
                 3000 
                 mg 
                 — 
               
               
                   
                 Vitamin D 
                 3000 
                 mg 
                 — 
               
               
                   
                 Zinc 
                 50 
                 mg 
                 — 
               
               
                   
                   
               
            
           
         
       
     
     On Day 6-Day 10, the individual takes the following regimen outlined in Table 4: 
     
       
         
           
               
               
               
               
             
               
                   
                 TABLE 4 
               
               
                   
                   
               
               
                   
                 Drug 
                 AM Dose 
                 PM Dose 
               
               
                   
                   
               
             
            
               
                   
                 Hydroxychloroquine 
                  200 mg 
                 200 mg 
               
               
                   
                 Vitamin C 
                 3000 mg 
                 — 
               
               
                   
                 Vitamin D 
                 3000 mg 
                 — 
               
               
                   
                 Zinc 
                  50 mg 
                 — 
               
               
                   
                   
               
            
           
         
       
     
     Hydroxychloroquine is administered daily, at 200 mg twice daily for days 1-10. 
     Azithromycin is administered daily, at 500 mg on day 1, and 250 mg days 2-5. Optionally, azithromycin can be administered at 250 mg per day for 5-10 days with a loading dose of 500 mg×day 1 or simply 250 mg on day 1. Azithromycin may be substituted with doxycycline at a daily dosage range of 25 mg to 800 mg, for those unable to take azithromycin. 
     Vitamin C is administered at 3,000 mg per day ongoing. This 3,000 mg can be broken up into two 1500 mg doses, one taken in the morning and one taken at night. 
     Vitamin D is administered at 3,000 mg per day ongoing. This 3,000 mg can be broken up into two 1500 mg doses, one taken in the morning and one taken at night. 
     Zinc is administered at 50 mg per day ongoing. This 50 mg can be broken up into two 25 mg doses, one taken in the morning and one taken at night. 
     Chloroquine or hydroxychloroquine can be administered in a daily dosage range of 20 mg to 2,000 mg. The above amounts recited in the tables are not limiting. 
     Vitamin C can be administered in a daily dosage range of 250 mg to 10,000 mg. The above amounts recited in the tables are not limiting. 
     Vitamin D can be administered in a daily dosage rage of 1,000 mg to 100,000 mg. The above amounts recited in the tables are not limiting. 
     Zinc can be administered in a daily dosage of 5 mg to 100 mg. The amount of Zinc can be reduced to 25 mg per day if gastrointestinal upset occurs. 
     Concurrently with the above treatment, the individual is self-quarantined per CDC recommendations. 
     Optionally, for the method of treatment noted above, as the dosages of the hydroxychloroquine, vitamin C, vitamin D and zinc remain the same throughout treatment, all four of those antimicrobials can be administered in the form of a single small atomizer. The patient sprays the atomizer towards the back of their throat. The spray is administered at least once a day, but preferably twice a day when coughing starts. Use of the atomizer continues as directed by the supervising physician. 
     In an additional embodiment, the present invention is directed to a method of treatment of viral infections and long hauler syndrome. Long hauler syndrome is when a individual has had a viral infection, has not fully recovered, and continues to have symptoms over a long period of time, such as weeks or months, or they continue to relapse with symptoms. 
     The method comprises administering one or more of the following compounds/components/antivirals for at least one (1) day, and up to fourteen (14) days: hydroxychloroquine, azithromycin or doxycycline, ivermectin, oseltamivir phosphate (Tamiflu™), famotidine (Pepcid™), vitamin c, vitamin d, zinc, aspirin, bifidobacterial probiotics, cyproheptadine, budesonide inhaler, and iodine nasal spray. 
     Preferably, at least hydroxychloroquine, azithromycin or doxycycline, ivermectin, oseltamivir phosphate (Tamiflu™), vitamin c, vitamin d, zinc, and bifidobacterial probiotics are administered. 
     Hydroxychloroquine or chloroquine can be administered as follows: orally, daily in a dosage range of 20 mg to 2,000 mg (including any amounts or ranges in between). Preferably, 200 mg is administered twice daily for 1 to 14 days. 
     Azithromycin or doxycycline can be administered as follows: administered daily, at 500 mg on day 1, and 250 mg days 2-14. Azithromycin may be substituted with doxycycline at a daily dosage range of 25 mg to 800 mg (including any amount or range in between), for those unable to take azithromycin. 
     Ivermectin can be administered as follows: 3 mg to 200 mg (including any amount or range in between), orally, daily, for 1 to 14 days; preferably, 36 mg, orally, twice a day, for 1 to 14 days. 
     Oseltamivir phosphate can be administered twice a day orally in the form of 30 mg, 45 mg, or 75 mg capsules or as an oral suspension made from 360 mg oseltamivir base supplied as powder (constituted to a final concentration of 6 mg/mL), for 1 to 14 days. Additional dosage options are as follows:
         Adults and adolescents (13 years and older): 75 mg twice daily for 5 days   Pediatric patients 1 to 12 years of age: Based on weight twice daily for 5 days   Pediatric patients 2 weeks to less than 1 year of age: 3 mg/kg twice daily for 5 days   Renally impaired adult patients (creatinine clearance &gt;30-60 mL/min): Reduce to 30 mg twice daily for 5 days   Renally impaired adult patients (creatinine clearance &gt;10-30 mL/min): Reduce to 30 mg once daily for 5 days   ESRD patients on hemodialysis: Reduce to 30 mg immediately and then 30 mg after every hemodialysis cycle. Treatment duration not to exceed 5 days   ESRD patients on CAPD: Reduce to a single 30 mg dose immediately       

     Famotidine can be administered as follows: 20 mg to 80 mg (including any amounts or ranges in between), orally, three times a day, for 1 to 14 days. 
     Vitamin C can be administered as follows: 1000 mg to 20,000 mg (including any amounts or ranges in between), orally, daily for 1 to 14 days. 
     Vitamin D can be administered as follows: 1,000 IU to 100,000 IU (including any amounts or ranges in between), orally, daily for 1 to 14 days. 
     Zinc can be administered as follows: any form or type of zinc: 50 mg to 100 mg (including any amounts or ranges in between), orally, daily for 1 to 14 days 
     Aspirin can be administered as follows: 81 mg to 325 mg to 500 mg (including any amounts or ranges in between) orally, daily, for 1 to 14 days. 
     Bifidobacteria probiotics can be administered as follows: one or more probiotics containing 1 billion to 100,000 billion bifidobacteria (including but not limited to Bifidobacteriales, Bifidobacteriacae) administered orally in form of a pill, lozenge, liquid tincture, spray, drink, chewable tablet, or food stuff such as yogurt, or rectally, in the form of a fecal transplant, suppository, or cream, daily, for 1 to 14 days. In all instances, amount of probiotic, and the amount or dosage of the bacteria contained within the probiotic can vary from patient to patient, depending on the microbiome needs. The probiotics can be administered for as many days as the supervising physician deems necessary. 
     Cyproheptadine can be administered as follows: 4 mg to 8 mg (including any amount or range in between) orally, daily, for 1 to 14 days; preferably, 4 mg, orally, twice a day, for 1 to 14 days. 
     Budesonide inhaler can be administered as follows: For inhalation dosage form (powder inhaler): Adults and children 6 years of age and older—At first, one or two puffs two times a day. For inhalation dosage form (suspension in a nebulizer): Children 12 months to 8 years of age—0.5 to 1 milligram (mg) in a nebulizer once a day, or divided and given twice a day. Each container of liquid has one dose and a new container is used for each dose. Regardless of form, the budesonide is administered daily, for 1 to 14 days. 
     Iodine nasal spray can be administered as follows: daily, one or more squirts in one or both nostrils, for 1 to 14 days. 
     For all protocols provided in this application, vitamin C dosage can range from 250 mg to 10,000 mg per day (including any amounts or ranges in between), vitamin D dosage can range from 1000 IU (mg) to 100,000 IU (mg) per day (including any amounts or ranges in between), zinc (which can be any form or type of zinc) dosage can range from 5 mg to 100 mg per day (including any amounts or ranges in between), and hydroxychloroquine/chloroquine dosage can range from 50 mg to 2,000 mg per day (including any amounts or ranges in between) for a treatment period of 1 to 14 days treatment (including any range in between). Optionally, hydroxychloroquine can be administered once as single dose. For all protocols provided in this application, when a dosage range is provided, any dosage amount that is included in that range can be administered. Accordingly, the invention is not limited to the dosage ranges disclosed and includes all dosage amounts contained in those ranges. 
     Optionally, the above protocols can include selenium, copper and other vitamins that are deemed acceptable supplements for vitamin C, vitamin D or zinc or to counteract the negative depletion of certain vitamins, which is why copper or selenium are typically used. 
     The above protocols can be used to treat all viruses, including but not limited to, covid-19, other flu and various respiratory viruses, including more benign coronaviruses and rhinoviruses, Ebola, 
     The above protocols can also be used to treat Autism, Parkinson&#39;s, Alzheimer&#39;s and other neurological diseases. 
     In an additional embodiment, the present invention is directed to the combination of one or more antivirals for treating a viral infection. The combination can comprise one or more of the following: hydroxychloroquine, azithromycin or doxycycline, ivermectin, oseltamivir phosphate (Tamiflu™), famotidine (Pepcid™), vitamin c, vitamin d, zinc, aspirin, bifidobacterial probiotics, cyproheptadine, budesonide inhaler, and iodine nasal spray, in the amounts/ranges noted above. 
     EXAMPLES 
     Example 1: Hydroxychloroquine, Vitamin C, Vitamin D, and Zinc for Prevention of COVID-19 Infection 
     Objective: To determine whether treatment with hydroxychloroquine, vitamin C, vitamin D, and zinc in combination will prevent infection with COVID-19 and to assess the safety and tolerability of hydroxychloroquine, vitamin C, vitamin D, and zinc in healthy, high-risk individuals without hypertension, and no evidence of COVID-19 infection. 
     Procedure: Day 1: Prescription of study drugs and home health monitoring equipment. The study drugs are hydroxychloroquine 200 mg twice a day for 1 day only, vitamin C 3000 mg per day ongoing, vitamin D 3000 mg per day ongoing, and zinc 50 mg per day ongoing per day. The home health monitoring equipment is an electrocardiogram (EKG) which synchs up with a smartphone 
     Days 2-7: Patient collects the EKG once during this week using the home health equipment 
     Weeks 1-23: Patient provides an assessment of any COVID-19 symptoms and continues to collect EKG weekly throughout the remainder of the trial 
     Week 24: Patient undergoes confirmatory COVID-19 testing which consists of nasopharyngeal (NP) and oropharyngeal (OP) swabs collected according to CDC (Center for Disease Control) protocol. The swabs consist of synthetic fiber swabs with plastic shafts. NP swabs are collected by insertion of a swab into the nostril parallel to the palate. The swab is left in place a few seconds to allow it to absorb secretions. OP swabs are inserted into the oropharynx parallel to the palate, avoiding the tongue. The swab is left in place a few seconds to allow it to absorb secretions. The swabs are then immediately placed in sterile tubes with 2-3 mL of viral transport media. The tubes are placed in biohazard bags then boxes and couriered to the local Public Health Lab. 
     Table 5 provides a schedule of events for Example 1. 
     
       
         
           
               
               
               
               
               
             
               
                 TABLE 5 
               
               
                   
               
               
                   
                 Screening 
                   
                 Weeks 
                 Week 
               
               
                 Assessment 
                 (Day 1) 
                 Day 3 
                 1-23 
                 24 
               
               
                   
               
             
            
               
                 Informed consent and 
                 X 
                   
                   
                   
               
               
                 demographics 
               
               
                 Review of prior and 
                 X 
               
               
                 concomitant medications 
               
               
                 EKG at home 
                   
                 X 
                 X 
               
               
                 Prescription of 
                 X 
               
               
                 Hydroxychloroquine, 
               
               
                 vitamin C, vitamin D, 
               
               
                 and zinc 
               
               
                 Provision of home health 
                 X 
               
               
                 EKG and pulse oximeter 
               
               
                 Update list of prior and 
                 X 
                   
                 X 
                 X 
               
               
                 concomitant medications 
               
               
                 Ask about any adverse 
                   
                 X 
                 X 
                 X 
               
               
                 events and any serious 
               
               
                 adverse events 
               
               
                 Evaluation of COVID-19 
                   
                   
                 X 
                 X 
               
               
                 symptoms 
               
               
                 COVID-19 testing 
                   
                   
                   
                 X 
               
               
                 Blood draw for future 
                   
                   
                   
                 X 
               
               
                 testing 
               
               
                 Vitals in-clinic 
                   
                   
                   
                 X 
               
               
                 Physical exam 
                   
                   
                   
                 X 
               
               
                   
               
            
           
         
       
     
     Regarding Table 5: Vitals at home to include EKG and O2Sat, future testing will require separate informed consent and could possibly include antibody or cytokine testing, and vitals in-clinic to include height, weight, blood pressure (following 5 minutes sitting) pulse, respiratory rate, temperature, and oxygen saturation. 
     Example 2: Randomized, Double-Blind, Placebo-Controlled Phase IIA Study of Hydroxychloroquine, Vitamin C, Vitamin D, and Zinc for the Prevention of COVID-19 Infection 
     Objectives: Prevention of COVID-19, Lack of COVID-19 symptoms, and assessment of safety and tolerability 
     Procedure: Screening Period (Days −5 to −1): Prescription of home health monitoring equipment that includes a thermometer, a pregnancy test if applicable, and a daily diary. There are two groups being studied: Arm 1 and Arm 2. 
     Arm 1 is prescribed the following antimicrobials: hydroxychloroquine 200 mg twice a day for 1 day only, vitamin C 3000 IU per day for 12 weeks, vitamin D 3000 IU per day for 12 weeks, and zinc 50 mg per day for 12 weeks. The zinc can be reduced to 25 mg if GI upset occurs. 
     Arm 2 is prescribed the following antimicrobials: Placebo twice a day for 1 day only, vitamin C 3000 IU per day for 12 weeks, vitamin D 3000 IU per day for 12 weeks, and zinc 50 mg per day for 12 weeks. The zinc can be reduced to 25 mg if GI upset occurs. 
     Day 1: Patient is called to teach them how to use the diary in the EDC, discuss the medication regimen, and answer any questions they may have. Patient takes pregnancy test if applicable, collects a temperature reading, completes their diary, and takes the prescribed treatment regimen. Table 6 outlines the prescribed treatment regimen for Day 1. 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 6 
               
               
                   
                   
               
               
                   
                 Drug 
                   
                 AM Dose 
                 PM Dose 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 Hydroxychloroquine (or placebo) 
                 200 
                 mg 
                 200 mg 
               
               
                   
                 Vitamin C 
                 3000 
                 mg 
                 — 
               
               
                   
                 Vitamin D 
                 3000 
                 IU 
                 — 
               
               
                   
                 Zinc 
                 50 
                 mg 
                 — 
               
               
                   
                   
               
            
           
         
       
     
     Day 2: Patient collects a temperature reading, completes their diary, and is called on the phone for assessment of any adverse events or serious adverse events, assessment of any COVID-19 symptoms, the list of prior and concomitant medications is updated, any questions the patient has are answered, and the patient takes their prescribed treatment regimen. Table 7 outlines the prescribed treatment regimen for Day 2. 
     
       
         
           
               
               
               
               
             
               
                   
                 TABLE 7 
               
               
                   
                   
               
               
                   
                 Drug 
                 AM Dose 
                 PM Dose 
               
               
                   
                   
               
             
            
               
                   
                 Vitamin C 
                 3000 mg 
                 — 
               
               
                   
                 Vitamin D 
                 3000 IU  
                 — 
               
               
                   
                 Zinc 
                  50 mg 
                 — 
               
               
                   
                   
               
            
           
         
       
     
     Days 3-10: Patient collects a temperature reading, completes their diary, and takes their prescribed treatment regimen. Table 8 outlines the prescribed treatment regimen for Days 3-10. 
     
       
         
           
               
               
               
               
             
               
                   
                 TABLE 8 
               
               
                   
                   
               
               
                   
                 Drug 
                 AM Dose 
                 PM Dose 
               
               
                   
                   
               
             
            
               
                   
                 Vitamin C 
                 3000 mg 
                 — 
               
               
                   
                 Vitamin D 
                 3000 IU  
                 — 
               
               
                   
                 Zinc 
                  50 mg 
                 — 
               
               
                   
                   
               
            
           
         
       
     
     Day 14: The patient is called for instruction on how to collect a nasal swab and package for shipping, assessment COVID-19 symptoms, updating their list of prior and concomitant medications, and answering any questions they may have. 
     Weeks 3-11: The patient is called weekly for assessment of any adverse events or serious adverse events, assessment of any COVID-19 symptoms, updating their list of prior and concomitant medications, and answering any questions they may have. The patient takes weekly temperature readings, completes their diary, and takes the prescribed treatment regimen. During Week 4 the patient is reminded to collect a nasal swab. The prescribed treatment regimen for Weeks 3-11 is shown in Table 9. 
     
       
         
           
               
               
               
               
             
               
                   
                 TABLE 9 
               
               
                   
                   
               
               
                   
                 Drug 
                 AM Dose 
                 PM Dose 
               
               
                   
                   
               
             
            
               
                   
                 Vitamin C 
                 3000 mg 
                 — 
               
               
                   
                 Vitamin D 
                 3000 IU  
                 — 
               
               
                   
                 Zinc 
                  50 mg 
                 — 
               
               
                   
                   
               
            
           
         
       
     
     Week 12: The patient presents to the clinic for evaluation that includes assessment for adverse events and serious adverse events, updating their list of prior and concomitant medications, a physical exam, nasal swab collection and COVID-19 sample collection. 
     Samples for COVID-19 testing are collected using synthetic swabs with plastic shafts. Nasal swabs are collected and immediately placed into a sterile vial with 2-3 mL of viral transport media. The vials are placed into biohazard bags, boxed up, the box sterilized, and picked up for shipment to the central laboratory. Samples are tested by RT-PCR. 
     Table 10 presents the schedule of events for Example 2. 
     
       
         
           
               
               
               
               
               
               
               
               
               
               
             
               
                 TABLE 10 
               
               
                   
               
               
                   
                   
                   
                   
                 Day 
                   
                 Weeks 
                   
                   
                   
               
               
                 Assessment 
                 Screening 
                 Day 1 
                 Day 2 
                 3-10 
                 Day 14 
                 3-12 
                 Month 1 
                 Month 2 
                 Month 3 
               
               
                   
               
             
            
               
                 Informed Consent 
                 X 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 &amp; Demographics 
               
               
                 Initial Review of 
                 X 
               
               
                 Prior and 
               
               
                 Concomitant 
               
               
                 Medications 
               
               
                 Medical Records 
                 X 
               
               
                 review, including 
               
               
                 baseline EKG 
               
               
                 Randomization 
                 X 
               
               
                 Prescription of 
                 X 
               
               
                 Hydroxychloroquine 
               
               
                 or placebo, vitamin C, 
               
               
                 vitamin D, and zinc 
               
               
                 Provision of daily 
                 X 
               
               
                 diary, thermometer 
               
               
                 and pregnancy test 
               
               
                 Pregnancy Test if 
                   
                 X 
               
               
                 applicable 
               
               
                 Temperature at home 
                   
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                 Complete daily diary 
                   
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                 Hydroxychloroquine 
                   
                 X 
               
               
                 200 mg 
               
               
                 Vitamin C 3000 mg 
                   
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                 Vitamin D 3000 IU 
                   
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                 Zinc 50 mg a   
                   
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                 Phone/video call 
                 X 
                 X 
                 X 
                   
                 X 
                 X 
                 X 
                 X 
               
               
                 to patient b   
               
               
                 Update list of prior 
                   
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                 and concomitant 
               
               
                 medications 
               
               
                 Ask about AE and 
                   
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                 SAE 
               
               
                 Assessment of 
                   
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                 COV1D-19 symptoms 
               
               
                 Swabs for RT-PCR 
                   
                   
                   
                   
                 X 
                   
                 X 
                   
                 X 
               
               
                 Vitals in-clinic c   
                   
                   
                   
                   
                   
                   
                   
                   
                 X 
               
               
                 Physical Exam 
                   
                   
                   
                   
                   
                   
                   
                   
                 X 
               
               
                   
               
            
           
         
       
     
     Regarding Table 10: The Zinc may be reduced to 25 mg if GI upset occurs, phone/video calls to the patient occur weekly during Weeks 2-11, and vitals in-clinic include height, weight, blood pressure (following 5 minutes sitting) pulse, respiratory rate, temperature, and oxygen saturation. 
     Statistical Analysis: The treated patients in this study are compared to the placebo group. Measurements include PCR test results, presence or absence of symptoms, and symptom severity. 
     The change of these measurements from the end to the baseline (post-pre) are used as the primary outcome, for example, μe=μe1−μe0, where μe1 and μe0 are the outcome of patients from the treatment group at the end and at baseline, respectively. 
     H 0 :Δ≤δ 0  against H α :δ&gt;δ 0  where δ0 is clinically meaningful threshold to measure the disease symptoms. In this study, that meaningful threshold is calculated as mean change in clinical symptoms as recorded in the diary, from baseline through week 12. Each category in the diary is assigned a number, 0 for None, 1 for Mild, 2 for Moderate and 3 for severe. Each category is analyzed independently and as a group. 
     Categorical variables are summarized by presenting the number (n) and percent (%) of subjects in each category. All Statistical tests for the analysis are performed using the p&lt;0.05 level of significance. All confidence intervals are one-sided. 
     Since these are healthcare workers who are exposed to COVID-19 at every shift, efficacy is determined by RT-PCR testing, as well as the presence or absence of symptoms as recorded in the patient diary via EDC. 
     Example 3: Use of Hydroxychloroquine, Azithromycin, Vitamin C, Vitamin D, and Zinc to Treat COVID-19 Infection 
     Objectives: test the efficacy of hydroxychloroquine, azithromycin, vitamin C, vitamin D, and zinc in the treatment of patients with COVID-19 infection and to assess the safety and tolerability of this treatment in patients with COVID-19 infection. 
     Procedure: First, the patient is determined to have COVID-19. 
     Day 1 following positive test (isolation): The patient takes prescribed regimen outlined in Table 11. 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 11 
               
               
                   
                   
               
               
                   
                 Drug 
                   
                 AM Dose 
                 PM Dose 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 Hydroxychloroquine 
                 200 
                 mg 
                 200 mg 
               
               
                   
                 Azithromycin 
                 500 
                 mg 
                 — 
               
               
                   
                 Vitamin C 
                 3000 
                 mg 
                 — 
               
               
                   
                 Vitamin D 
                 3000 
                 mg 
                 — 
               
               
                   
                 Zinc 
                 50 
                 mg 
                 — 
               
               
                   
                   
               
            
           
         
       
     
     Day 2-Day 5: The patient takes the prescribed regimen outlined in Table 12. 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 12 
               
               
                   
                   
               
               
                   
                 Drug 
                   
                 AM Dose 
                 PM Dose 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 Hydroxychloroquine 
                 200 
                 mg 
                 200 mg 
               
               
                   
                 Azithromycin 
                 250 
                 mg 
                 — 
               
               
                   
                 Vitamin C 
                 3000 
                 mg 
                 — 
               
               
                   
                 Vitamin D 
                 3000 
                 mg 
                 — 
               
               
                   
                 Zinc 
                 50 
                 mg 
                 — 
               
               
                   
                   
               
            
           
         
       
     
     Day 6-Day 10: The patient takes the prescribed regimen outlined in Table 13. 
     
       
         
           
               
               
               
               
             
               
                   
                 TABLE 13 
               
               
                   
                   
               
               
                   
                 Drug 
                 AM Dose 
                 PM Dose 
               
               
                   
                   
               
             
            
               
                   
                 Hydroxychloroquine 
                  200 mg 
                 200 mg 
               
               
                   
                 Vitamin C 
                 3000 mg 
                 — 
               
               
                   
                 Vitamin D 
                 3000 mg 
                 — 
               
               
                   
                 Zinc 
                  50 mg 
                 — 
               
               
                   
                   
               
            
           
         
       
     
     Follow-Up Period: Month 1 (outpatient only AFTER negative test): vital signs are taken that include blood pressure, heart rate, respiratory rate, oxygen saturation, temperature), the patient is assessed for adverse events and serious adverse events, and swabs for PCR are taken. 
     Month 3 (outpatient): vital signs are taken that include blood pressure, heart rate, respiratory rate, oxygen saturation, temperature), the patient is assessed for adverse events and serious adverse events, and swabs for PCR are taken. 
     Month 6 (outpatient) vital signs are taken that include blood pressure, heart rate, respiratory rate, oxygen saturation, temperature), the patient is assessed for adverse events and serious adverse events, and swabs for PCR are taken. 
     Month 9 (outpatient) vital signs are taken that include blood pressure, heart rate, respiratory rate, oxygen saturation, temperature), the patient is assessed for adverse events and serious adverse events, and swabs for PCR are taken. 
     Month 12 (outpatient) vital signs are taken that include blood pressure, heart rate, respiratory rate, oxygen saturation, temperature), the patient is assessed for adverse events and serious adverse events, and swabs for PCR are taken. 
     COVID-19 sample collection procedure is as follows: nasopharyngeal (NP) and oropharyngeal (OP) swabs are collected according to CDC protocol. The swabs comprise synthetic fiber swabs with plastic shafts. NP swabs are collected by insertion of a swab into the nostril parallel to the palate. The swab is left in place a few seconds to allow it to absorb secretions. OP swabs are inserted into the oropharynx parallel to the palate, avoiding the tongue. The swab is left in place a few seconds to allow it to absorb secretions. NP and OP swabs are immediately placed in sterile tubes with 2-3 mL of viral transport media. The tubes are placed in biohazard bags then boxes and couriered to the local Public Health Lab. Table 14 outlines the schedule of events for Example 3. 
     
       
         
           
               
               
               
               
               
               
               
               
             
               
                 TABLE 14 
               
               
                   
               
               
                   
                 Diagnosis and Rx 
                   
                 Month 1 
                 Month 3 
                 Month 6 
                 Month 9 
                 Month 12 
               
               
                 Assessment 
                 (Day 1) 
                 Day 1→ 7 
                 (±4 d) 
                 (±4 d) 
                 (±4 d) 
                 (±4 d) 
                 (±4 d) 
               
               
                   
               
             
            
               
                 Informed Consent &amp; Demographics 
                 X 
                   
                   
                   
                   
                   
                   
               
               
                 Confirmation of Positive PCR 
                 X 
               
               
                 for COVID-19 
               
               
                 Review of Medical Records 
                 X 
               
               
                 Vitals a   
                   
                   
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                 Prescription of Antimicrobials b   
                 X 
               
               
                 Ask about AE and SAE 
                   
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                   
               
            
           
         
       
     
     Regarding Table 14: Vitals to include height (only at first visit), weight, blood pressure (following 5 minutes sitting) pulse, respiratory rate, temperature, and oxygen saturation. Dosage to be given as in section 8, below 
     Table 15 provides a summary of the antimicrobial dosage of Example 3. 
     
       
         
           
               
               
               
               
             
               
                 TABLE 15 
               
               
                   
               
               
                   
                   
                   
                 Fre- 
               
               
                 Treatment Method 
                 Medication 
                 Dose 
                 quency 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Quintuple Therapy 
                 Hydroxychloroquine 
                 200 
                 mg 
                 BID 
               
            
           
           
               
               
               
               
            
               
                   
                 Azithromycin 
                 500 mg\Day 
                 Daily 
               
               
                   
                   
                 1→250 mg Day 2-5 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Vitamin C 
                 3000 
                 mg 
                 Daily 
               
               
                   
                 Vitamin D 
                 5000 
                 mg 
                 Daily 
               
               
                   
                 Zinc 
                 50 
                 mg 
                 Daily 
               
               
                   
                   
               
            
           
         
       
     
     Example 4: Randomized, Double-Blind, Placebo-Controlled Phase IIA Study of Hydroxychloroquine, Azithromycin, Vitamin C, Vitamin D, and Zinc to Treat COVID-19 Infection 
     Objectives: Test the efficacy of hydroxychloroquine, azithromycin, vitamin C, vitamin D, and zinc in the treatment of patients with COVID-19 infection and to assess the safety and tolerability of this treatment in patients with COVID-19 infection. 
     Procedure: First, the patient&#39;s diagnosis of COVID-19 infection is confirmed. 
     Screening Period (Days −3 to −1): Prescription of home health monitoring equipment that includes a thermometer, a pulse oximeter, a pregnancy test if applicable, and a daily diary. There are two groups being studied: Arm 1 and Arm 2. 
     Arm 1 is prescribed the following antimicrobials: Hydroxychloroquine 200 MG BID for 10 days; Azithromycin 500 mg on day 1, 250 mg day 2-5; Vitamin C 3000 mg for 10 days, then 1500 mg for 20 days; Vitamin D 3000 IU for 10 days, then 1500 IU for 20 days; and Zinc 50 mg for 10 days, then 25 mg for 20 days. 
     Arm 2 is prescribed the following antimicrobials: Placebo for Hydroxychloroquine BID for 10 days; Placebo for Azithromycin to be taken 2 the on Day 1, then 1 on Days 2-5; Vitamin C 3000 mg for 10 days, then 1500 mg for 20 days; Vitamin D 3000 IU for 10 days, then 1500 IU for 20 days; and Zinc 50 mg for 10 days, then 25 mg for 20 days. Table 16 outlines the prescribed antimicrobials discussed above. 
     
       
         
           
               
               
               
               
             
               
                 TABLE 16 
               
               
                   
               
               
                   
                   
                   
                 Fre- 
               
               
                 Treatment Method 
                 Medication 
                 Dose 
                 quency 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Quintuple Therapy 
                 Hydroxychloroquine 
                 200 
                 mg 
                 BID 
               
            
           
           
               
               
               
               
            
               
                   
                 Azithromycin 
                 500 mg\Day 
                 Daily 
               
               
                   
                   
                 1→250 mg Day 2-5 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Vitamin C 
                 3000 
                 mg 
                 Daily 
               
               
                   
                 Vitamin D 
                 5000 
                 mg 
                 Daily 
               
               
                   
                 Zinc 
                 50 
                 mg 
                 Daily 
               
            
           
           
               
               
               
               
            
               
                 Placebo 
                 Placebo 
                 1 tablet 
                 BID 
               
               
                   
                 Placebo 
                 2 tablets day 1, then 
                 Daily 
               
               
                   
                   
                 1 tablet day 2-5 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Vitamin C 
                 3000 
                 mg 
                 Daily 
               
               
                   
                 Vitamin D 
                 3000 
                 IU 
                 Daily 
               
               
                   
                 Zinc 
                 50 
                 mg 
                 Daily 
               
               
                   
                   
               
            
           
         
       
     
     Treatment Period: Day 1 following positive test (isolation), the patient is video called to ensure they have all study materials and the following is discussed: Use of home health equipment, Subject will take baseline measurements at this time and record it in the diary, Diary and how to transmit its contents, Medication dosing, Subject will take pregnancy test if applicable, Subject will use provided equipment to measure vital signs such as EKG, Oxygen Saturation, and Temperature. The patient takes prescribed regimen outlined in Table 17. 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 17 
               
               
                   
                   
               
               
                   
                 Drag 
                   
                 AM Dose 
                 PM Dose 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 Hydroxychloroquine (or Placebo) 
                 200 
                 mg 
                 200 mg 
               
               
                   
                 Azithromycin (or Placebo) 
                 500 
                 mg 
                 — 
               
               
                   
                 Vitamin C 
                 3000 
                 mg 
                 — 
               
               
                   
                 Vitamin D 
                 3000 
                 IU 
                 — 
               
               
                   
                 Zinc 
                 50 
                 mg 
                 — 
               
               
                   
                   
               
            
           
         
       
     
     Day 2: The patient completes the AM and PM diary entries, the patient uses the provided equipment to measure vital signs such as EKG, Oxygen saturation, and temperature, and the patient takes the prescribed regimen outlined in Table 18. 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 18 
               
               
                   
                   
               
               
                   
                 Drug 
                   
                 AM Dose 
                 PM Dose 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 Hydroxychloroquine (or Placebo) 
                 200 
                 mg 
                 200 mg 
               
               
                   
                 Azithromycin (or Placebo) 
                 250 
                 mg 
                 — 
               
               
                   
                 Vitamin C 
                 3000 
                 mg 
                 — 
               
               
                   
                 Vitamin D 
                 3000 
                 IU 
                 — 
               
               
                   
                 Zinc 
                 50 
                 mg 
                 — 
               
               
                   
                   
               
            
           
         
       
     
     Day 3: The patient is called and asked if they are experiencing any difficulties with swab collection, whether there have been any adverse events and/or serious adverse events, the list of prior and concomitant medications is updated, the patient is asked about symptom resolution or progression, the patient is instructed on how to collect nasal swabs, and the patient then collects the first nasal swab. The patient completes their AM and PM diary entries, and collects their vital signs such as EKG, oxygen saturation and temperature. The patient takes the prescribed regimen outlined in Table 19. 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 19 
               
               
                   
                   
               
               
                   
                 Drug 
                   
                 AM Dose 
                 PM Dose 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 Hydroxychloroquine (or Placebo) 
                 200 
                 mg 
                 200 mg 
               
               
                   
                 Azithromycin (or Placebo) 
                 250 
                 mg 
                 — 
               
               
                   
                 Vitamin C 
                 3000 
                 mg 
                 — 
               
               
                   
                 Vitamin D 
                 3000 
                 IU 
                 — 
               
               
                   
                 Zinc 
                 50 
                 mg 
                 — 
               
               
                   
                   
               
            
           
         
       
     
     Day 4: The patient completes their AM and PM diary entries, collects their vital signs, and takes the prescribed regimen outlined in Table 20. 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 20 
               
               
                   
                   
               
               
                   
                 Drug 
                   
                 AM Dose 
                 PM Dose 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 Hydroxychloroquine (or Placebo) 
                 200 
                 mg 
                 200 mg 
               
               
                   
                 Azithromycin (or Placebo) 
                 250 
                 mg 
                 — 
               
               
                   
                 Vitamin C 
                 3000 
                 mg 
                 — 
               
               
                   
                 Vitamin D 
                 3000 
                 IU 
                 — 
               
               
                   
                 Zinc 
                 50 
                 mg 
                 — 
               
               
                   
                   
               
            
           
         
       
     
     Day 5: The patient completes their AM and PM diary entries, collects their vital signs, collects a nasal swab, and takes the prescribed regimen outlined in Table 21. 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 21 
               
               
                   
                   
               
               
                   
                 Drug 
                   
                 AM Dose 
                 PM Dose 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 Hydroxychloroquine (or Placebo) 
                 200 
                 mg 
                 200 mg 
               
               
                   
                 Azithromycin (or Placebo) 
                 250 
                 mg 
                 — 
               
               
                   
                 Vitamin C 
                 3000 
                 mg 
                 — 
               
               
                   
                 Vitamin D 
                 3000 
                 IU 
                 — 
               
               
                   
                 Zinc 
                 50 
                 mg 
                 — 
               
               
                   
                   
               
            
           
         
       
     
     Day 6: The patient completes their AM and PM diary entries, collects their vital signs, and takes the prescribed regimen outlined in Table 22. 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 22 
               
               
                   
                   
               
               
                   
                 Drug 
                   
                 AM Dose 
                 PM Dose 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 Hydroxychloroquine (or Placebo) 
                 200 
                 mg 
                 200 mg 
               
               
                   
                 Vitamin C 
                 3000 
                 mg 
                 — 
               
               
                   
                 Vitamin D 
                 3000 
                 IU 
                 — 
               
               
                   
                 Zinc 
                 50 
                 mg 
                 — 
               
               
                   
                   
               
            
           
         
       
     
     Day 7: The patient is called and asked whether there have been any adverse events and/or serious adverse events, the list of prior and concomitant medications is updated, the patient is asked about symptom resolution or progression, and the patient then collects a nasal swab. The patient also completes their AM and PM diary entries and collects their vital signs. The patient takes the prescribed regimen outlined in Table 23. 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 23 
               
               
                   
                   
               
               
                   
                 Drug 
                   
                 AM Dose 
                 PM Dose 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 Hydroxychloroquine (or Placebo) 
                 200 
                 mg 
                 200 mg 
               
               
                   
                 Vitamin C 
                 3000 
                 mg 
                 — 
               
               
                   
                 Vitamin D 
                 3000 
                 IU 
                 — 
               
               
                   
                 Zinc 
                 50 
                 mg 
                 — 
               
               
                   
                   
               
            
           
         
       
     
     Day 8: The patient completes their AM and PM diary entries, collects their vital signs, and takes the prescribed regimen outlined in Table 24. 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 24 
               
               
                   
                   
               
               
                   
                 Drug 
                   
                 AM Dose 
                 PM Dose 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 Hydroxychloroquine (or Placebo) 
                 200 
                 mg 
                 200 mg 
               
               
                   
                 Vitamin C 
                 3000 
                 mg 
                 — 
               
               
                   
                 Vitamin D 
                 3000 
                 IU 
                 — 
               
               
                   
                 Zinc 
                 50 
                 mg 
                 — 
               
               
                   
                   
               
            
           
         
       
     
     Day 9: The patient completes their AM and PM diary entries, collects their vital signs, and takes the prescribed regimen outlined in Table 25. 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 25 
               
               
                   
                   
               
               
                   
                 Drug 
                   
                 AM Dose 
                 PM Dose 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 Hydroxychloroquine (or Placebo) 
                 200 
                 mg 
                 200 mg 
               
               
                   
                 Vitamin C 
                 3000 
                 mg 
                 — 
               
               
                   
                 Vitamin D 
                 3000 
                 IU 
                 — 
               
               
                   
                 Zinc 
                 50 
                 mg 
                 — 
               
               
                   
                   
               
            
           
         
       
     
     Day 10: The patient is called reminded to decrease dosage of vitamins C, vitamin D, and zinc tomorrow. The patient is asked whether there have been any adverse events and/or serious adverse events, the list of prior and concomitant medications is updated, and the patient is asked about symptom resolution or progression. The patient also completes their AM and PM diary entries and collects their vital signs. The patient takes the prescribed regimen outlined in Table 26. 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 26 
               
               
                   
                   
               
               
                   
                 Drug 
                   
                 AM Dose 
                 PM Dose 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 Hydroxychloroquine (or Placebo) 
                 200 
                 mg 
                 200 mg 
               
               
                   
                 Vitamin C 
                 3000 
                 mg 
                 — 
               
               
                   
                 Vitamin D 
                 3000 
                 IU 
                 — 
               
               
                   
                 Zinc 
                 50 
                 mg 
                 — 
               
               
                   
                   
               
            
           
         
       
     
     Day 11 to Day 13: The patient completes their AM and PM diary entries and takes the prescribed regimen outlined in Table 27. 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 27 
               
               
                   
                   
               
               
                   
                 Drug 
                   
                 AM Dose 
                 PM Dose 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 Vitamin C 
                 1500 
                 mg 
                 — 
               
               
                   
                 Vitamin D 
                 1500 
                 IU 
                 — 
               
               
                   
                 Zinc 
                 25 
                 mg 
                 — 
               
               
                   
                   
               
            
           
         
       
     
     Day 14: The patient is called and asked whether there have been any adverse events and/or serious adverse events, the patient is asked about symptom resolution or progression, and the patient then collects a nasal swab. The patient also completes their AM and PM diary entries and collects their vital signs. The patient takes the prescribed regimen outlined in Table 28. 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 28 
               
               
                   
                   
               
               
                   
                 Drug 
                   
                 AM Dose 
                 PM Dose 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 Vitamin C 
                 1500 
                 mg 
                 — 
               
               
                   
                 Vitamin D 
                 1500 
                 IU 
                 — 
               
               
                   
                 Zinc 
                 25 
                 mg 
                 — 
               
               
                   
                   
               
            
           
         
       
     
     Day 15 to Day 30: The patient completes their AM and PM diary entries and takes the prescribed regimen outlined in Table 29. 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 29 
               
               
                   
                   
               
               
                   
                 Drug 
                   
                 AM Dose 
                 PM Dose 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 Vitamin C 
                 1500 
                 mg 
                 — 
               
               
                   
                 Vitamin D 
                 1500 
                 IU 
                 — 
               
               
                   
                 Zinc 
                 25 
                 mg 
                 — 
               
               
                   
                   
               
            
           
         
       
     
     Follow-Up Period: Month 1 (outpatient only AFTER negative test): Vital signs are taken (BP, HR, RR, oxygen saturation, temperature), the patient is assessed for adverse events and/or serious adverse events, and EKG is administered as well as physical exam and a blood draw for CBC/Complete metabolic panel/C-Reactive Protein. The list of prior and concomitant medications is updated and a nasal swab is collected. 
     Month 2 (outpatient): Vital signs are collected (BP, HR, RR, oxygen saturation, temperature) the patient is assessed for adverse events and/or serious adverse events, and EKG is administered as well as physical exam and a blood draw for CBC/Complete metabolic panel/C-Reactive Protein. The list of prior and concomitant medications is updated. 
     Month 3 (outpatient): Vital signs are taken (BP, HR, RR, oxygen saturation, temperature), the patient is assessed for adverse events and/or serious adverse events, and EKG is administered as well as physical exam and a blood draw for CBC/Complete metabolic panel/C-Reactive Protein. The list of prior and concomitant medications is updated and a nasal swab is collected. 
     COVID-19 Sample Collection Procedure is the same as that outlined above in prior Examples. Table 30 outlines the schedule of events for Example 4. 
     
       
         
           
               
               
               
               
               
               
               
               
               
               
               
               
             
               
                 TABLE 30 
               
               
                   
               
             
            
               
                   
                 Screening 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 (Da −3 to 
               
               
                 Assessment 
                 Day −1) 
                 Day 1 
                 Day 2 
                 Day 3 
                 Day 4 
                 Day 5 
                 Day 6 
                 Day 7 
                 Day 8 
                 Day 9 
                 Day 10 
               
               
                   
               
               
                 Informed Consent &amp; 
                 X 
               
               
                 Demographics 
               
               
                 Make list of prior 
                 X 
                   
                   
                 x 
                   
                   
                   
                 x 
                   
                   
                 x 
               
               
                 and concomitant 
               
               
                 medications 
               
               
                 Confirmation of 
                 X 
               
               
                 Positive PCR for 
               
               
                 COVID-19 
               
               
                 Review of Medical 
                 X 
               
               
                 Records 
               
               
                 Prescription of 
                 X 
               
               
                 Antimicrobials a   
               
               
                 Provide home health 
                 X 
               
               
                 equipment b   
               
               
                 Pregnancy test c   
                   
                 X 
               
               
                 Call Subject at home d   
                   
                 X 
                   
                 X 
                   
                   
                   
                 X 
                   
                   
                 X 
               
               
                 Vitals at home e   
                   
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                 Subject will complete 
                   
                 X 
                 x 
                 x 
                 x 
                 x 
                 x 
                 x 
                 x 
                 x 
                 x 
               
               
                 AM/PM diary 
               
               
                 Hydroxychloroquine 
                   
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                 200 mg BID (or 
               
               
                 placebo 
               
               
                 Azithromycin 500 mg 
                   
                 X 
               
               
                 (2 tablets) once 
               
               
                 daily (or placebo) 
               
               
                 Azithromycin 250 mg 
                   
                   
                 X 
                 X 
                 X 
                 X 
               
               
                 (1 tablet) once 
               
               
                 daily (or placebo) 
               
               
                 Vitamin C 3000 mg 
                   
                   
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                 (4 capsules) once 
               
               
                 daily 
               
               
                 Vitamin D 3000 IU 
                   
                   
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                 (4 capsules) once 
               
               
                 daily 
               
               
                 Zinc 50 mg 
                   
                   
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                 (4 capsules) once 
               
               
                 daily 
               
               
                 Vitamin C 1500 mg 
               
               
                 (2 capsules) once 
               
               
                 daily 
               
               
                 Vitamin D 1500 IU 
               
               
                 (2 capsules) once 
               
               
                 daily 
               
               
                 Zinc 25 mg 
               
               
                 (2 capsules) once 
               
               
                 daily 
               
               
                 Ask about AE and 
                   
                   
                   
                 X 
                   
                   
                   
                 X 
                   
                   
                 X 
               
               
                 SAE 
               
               
                 Vitals in-clinic f   
               
               
                 Physical exam 
               
               
                 EKG in-clinic 
               
               
                 Swabs for RT-PCR 
                   
                   
                   
                 X 
                   
                 X 
                   
                 X 
               
               
                 Bloodwork g   
               
               
                 Review list of 
                   
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                 prior and 
               
               
                 concomitant 
               
               
                 medications 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 Assessment 
                 Day 11 
                 Day 12 
                 Day 13 
                 Day 14 
                 Days 15-30 
                 Month 1 
                 Month 2 
                 Month 3 
               
               
                   
                   
               
               
                   
                 Informed Consent &amp; 
               
               
                   
                 Demographics 
               
               
                   
                 Make list of prior 
                   
                   
                   
                 x 
                   
                 X 
                 X 
                 X 
               
               
                   
                 and concomitant 
               
               
                   
                 medications 
               
               
                   
                 Confirmation of 
               
               
                   
                 Positive PCR for 
               
               
                   
                 COVID-19 
               
               
                   
                 Review of Medical 
               
               
                   
                 Records 
               
               
                   
                 Prescription of 
               
               
                   
                 Antimicrobials a   
               
               
                   
                 Provide home health 
               
               
                   
                 equipment b   
               
               
                   
                 Pregnancy test c   
               
               
                   
                 Call Subject at home d   
                   
                   
                   
                 X 
               
               
                   
                 Vitals at home e   
               
               
                   
                 Subject will complete 
                 x 
                 x 
                 x 
                 x 
                 x 
               
               
                   
                 AM/PM diary 
               
               
                   
                 Hydroxychloroquine 
               
               
                   
                 200 mg BID (or 
               
               
                   
                 placebo 
               
               
                   
                 Azithromycin 500 mg 
               
               
                   
                 (2 tablets) once 
               
               
                   
                 daily (or placebo) 
               
               
                   
                 Azithromycin 250 mg 
               
               
                   
                 (1 tablet) once 
               
               
                   
                 daily (or placebo) 
               
               
                   
                 Vitamin C 3000 mg 
               
               
                   
                 (4 capsules) once 
               
               
                   
                 daily 
               
               
                   
                 Vitamin D 3000 IU 
               
               
                   
                 (4 capsules) once 
               
               
                   
                 daily 
               
               
                   
                 Zinc 50 mg 
               
               
                   
                 (4 capsules) once 
               
               
                   
                 daily 
               
               
                   
                 Vitamin C 1500 mg 
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                   
                 (2 capsules) once 
               
               
                   
                 daily 
               
               
                   
                 Vitamin D 1500 IU 
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                   
                 (2 capsules) once 
               
               
                   
                 daily 
               
               
                   
                 Zinc 25 mg 
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                   
                 (2 capsules) once 
               
               
                   
                 daily 
               
               
                   
                 Ask about AE and 
                   
                   
                   
                 X 
                   
                 X 
                 X 
                 X 
               
               
                   
                 SAE 
               
               
                   
                 Vitals in-clinic f   
                   
                   
                   
                   
                   
                 X 
                 X 
                 X 
               
               
                   
                 Physical exam 
                   
                   
                   
                   
                   
                 X 
                 X 
                 X 
               
               
                   
                 EKG in-clinic 
                   
                   
                   
                   
                   
                 X 
                 X 
                 X 
               
               
                   
                 Swabs for RT-PCR 
                   
                   
                   
                 X 
                   
                 X 
                   
                 X 
               
               
                   
                 Bloodwork g   
                   
                   
                   
                   
                   
                 X 
                 X 
                 X 
               
               
                   
                 Review list of 
                 X 
                 X 
                 X 
                 x 
                 X 
                 X 
                 X 
                 x 
               
               
                   
                 prior and 
               
               
                   
                 concomitant 
               
               
                   
                 medications 
               
               
                   
                   
               
            
           
         
       
     
     Regarding Table 30: The antimicrobials include hydroxychloroquine 200 mg tablets (#20), azithromycin 250 mg tablets (#6), vitamin C 750 mg capsules (#80), vitamin D 750 IU capsules (#80), zinc 12.5 mg capsules (#80); the home health equipment includes an EKG (worn continuously), pulse oximeter, and thermometer; the pregnancy test is administered if the patient is a woman of childbearing potential; the patients are called at home to remind them to collect swabs for RT-PCT, ask about AE/SAE, ask about symptoms, and answer any questions; the vitals taken at home include an EKG, oxygen saturation, and temperature; the vitals in-clinic include height (only at first visit), weight, blood pressure (following 5 minutes sitting) pulse, respiratory rate, temperature, and oxygen saturation; and the bloodwork includes CBC, Complete Metabolic Panel, and CRP (details in section 9 Sample Collection). 
     Statistical Analysis: The treated patients in this study are compared to the placebo group. Measurements include PCR test results, presence or absence of symptoms, and symptom severity. PCR results will be compared between the groups as positive or negative 
     In this study, the meaningful threshold is calculated as mean change in clinical symptoms as recorded in the diary, from Day 1 through week 12. Each category in the diary is assigned a number, 0 for None, 1 for Mild, 2 for Moderate and 3 for severe. Each category is analyzed independently and as a group. Ultimately, efficacy is determined based upon reduction and/or progression of symptomatic days, reduction of symptom severity, as well as analysis of the subject&#39;s RT-PCR testing per protocol. These data are compared to an existing database of de-identified Subject data. 
     The change of these measurements from the end to the baseline (post-pre) are used as the primary outcome, for example, μe=μ e1 −μ e0 , where μ e1  and μ e0  are the outcome of Subjects from the treatment group at the end and at baseline, respectively. 
     Categorical variables are summarized by presenting the number (n) and percent (%) of subjects in each category. All Statistical tests for the analysis are performed using the p&lt;0.05 level of significance. All confidence intervals will be one-sided 
     Sample size was calculated as follows: 
     
       
         
           
             n 
             = 
             
               
                 log 
                 ⁢ 
                 
                     
                 
                 ⁢ 
                 B 
               
               
                 log 
                 ⁢ 
                 
                     
                 
                 ⁢ 
                 ρ 
               
             
           
         
       
     
     Where β9 =the probability of a Type II error 
     p=the proportion of the population NOT affected 
     The proportion of the population affected by COVID-19 is 0.005 percent, thus 0.995 percent aren&#39;t affected 
     The probability of a type II error is 0.05 
     Thus: 
     
       
         
           
             n 
             = 
             
               
                 log 
                 ⁢ 
                 
                     
                 
                 ⁢ 
                 0.050 
               
               
                 log 
                 ⁢ 
                 
                     
                 
                 ⁢ 
                 
                   0 
                   . 
                   9 
                 
                 ⁢ 
                 95 
               
             
           
         
       
       
         
           
             n 
             = 
             
               5 
               ⁢ 
               9 
               ⁢ 
               
                 7 
                 . 
                 6 
               
               ⁢ 
               4 
               ⁢ 
               7 
             
           
         
       
     
     A sample size of 600 was used. 
     Example 5: Successful Treatment of COVID-19 Infected Outpatients and Prophylaxis of Immediate Associates 
     Objective: to successfully treat COVID-19 infected outpatients and prophylaxis of immediate associates. 
     Procedure: Prospective COVID-19 infected individuals were diagnosed using a Pangea DNA/RNA Shield™ Collection Tube to obtain a nasopharyngeal swab and PCR +ve patients were entered into the study. They were immediately commenced on a 10 day course of Hydroxychloroquine (200 mg, twice a day, for 10 days), Azithromycin extended release (500 mg on day 1, then 250 mg a day for days 9-10), zinc (50 mg a day for days 1-10), Vitamin D (3000 IU a day for days 1-10) and Vitamin C (3000 mg a day for days 1-10). Some individuals lived alone, otherwise immediate partners and family deemed to be most exposed were given a prophylactic which comprised hydroxychloroquine 200 mg twice a day on day 1 only with Zinc, Vitamin C and Vitamin D for given at the same doses as above for days 1-10. 
     Results: In 11 families a total of 21 family members were identified to be PCR COVID-19 positive index cases and were treated with the above treatment protocol while 22 exposed associates with negative PCR were given the above prophylaxis protocol. This is shown below in Table 31. All 21 index cases were cured of COVID-19 infection as judged by the repeat swab PCR on day 10 and accompanying symptom resolution. None of the 22 highly exposed associates developed COVID-19 infection when retested on day 10 (day 14 in Family 10) in spite of close co-habitation with the infected index cases. TABLE 31: Results from families received 10-day course of daily HCQ (200 mg bd), AZ extended release (500 mg day 1, then 250 mg), zinc (50 mg), Vitamin D (3000 IU) and Vitamin C (3000 mg) 
     
       
         
           
               
               
               
               
               
               
             
               
                 TABLE 31 
               
               
                   
               
               
                   
                   
                   
                   
                 After treating 
                   
               
               
                   
                   
                   
                   
                 with HAZDPAC 
                 After treating 
               
               
                   
                   
                 Age (years)- 
                   
                 Cured/PCR test 
                 ZINCD + H 
               
               
                   
                 Patient/s 
                 sex of the 
                 Comorbidities of the 
                 positive/negative/ 
                 family member 
               
               
                 Family 
                 treated 
                 patient 
                 patients 
                 other symptoms 
                 prophylaxed 
               
               
                   
               
             
            
               
                 #1 
                 1 
                 23 - male 
                 Asthma 
                 Negative PCR - 
                 Parents 
               
               
                   
                   
                   
                   
                 day 10 
                 overweight, 
               
               
                   
                   
                   
                   
                   
                 diabetes, father 
               
               
                   
                   
                   
                   
                   
                 with heart 
               
               
                   
                   
                   
                   
                   
                 disease never got 
               
               
                   
                   
                   
                   
                   
                 the disease 
               
               
                 #2 
                 3 
                 60- male 
                 Asthma 
                 All 3 patients 
                 Mother didn&#39;t 
               
               
                   
                   
                 (father) 
                   
                 cured 
                 get the virus 
               
               
                   
                   
                 18- male 
               
               
                   
                   
                 (son) 
               
               
                   
                   
                 16- female 
               
               
                   
                   
                 (daughter) 
               
               
                 #3 
                 1 
                 40-feniale 
                 Avoided intubation 
                 Sever multiple 
                 Husband did not 
               
               
                   
                   
                   
                 by leaving hospital 
                 symptoms 
                 turn positive 
               
               
                   
                   
                   
                   
                 resolved at home 
               
               
                 #4 
                 2 
                 78- male 
                 BCG + COPD, 
                 Both cured 
                 Daughter, son- 
               
               
                   
                   
                   
                 diabetes, heart 
                   
                 in-law and 2 
               
               
                   
                   
                   
                 disease (heart 
                   
                 grandkids didn&#39;t 
               
               
                   
                   
                   
                 surgery month prior 
                   
                 catch the virus 
               
               
                   
                   
                 77- female 
                 Pacemaker, BCG 
               
               
                 #5 
                 3 
                 56-male 
                 Unable to eat or 
                 Started treatment 
                 Wife never got 
               
               
                   
                   
                   
                 drink 
                 on day 10, Sever 
                 the disease 
               
               
                   
                   
                   
                   
                 symptoms 
               
               
                   
                   
                   
                   
                 resolved 
               
               
                   
                   
                 27- male 
                 Asthma 
                 Cured 
               
               
                   
                   
                 24- female 
               
               
                 #6 
                 1 
                 56- female 
                   
                 Admitted to 
                 Husband never 
               
               
                   
                   
                   
                   
                 hospital -sent 
                 got the disease 
               
               
                   
                   
                   
                   
                 home, as two 
               
               
                   
                   
                   
                   
                 kids were sick 
               
               
                   
                   
                   
                   
                 with fever. All 
               
               
                   
                   
                   
                   
                 recovered with 
               
               
                   
                   
                   
                   
                 vitamins 
               
               
                 #7 
                 1 
                 44-female 
                   
                 Cured, 
                 Husband and 4 
               
               
                   
                   
                   
                   
                 autoimmune 
                 kids never got 
               
               
                   
                   
                   
                   
                 issues started 
                 the disease 
               
               
                 #8 
                 3 
                 52- female 
                 Lupus 
                 Cured 
                 Boyfriend (54 y) 
               
               
                   
                   
                 53- male 
                   
                 Cured 
                 with Diabetes, 
               
               
                   
                   
                 19- male 
                 Severe asthma 
                 Cured 
                 son (18) and 
               
               
                   
                   
                   
                   
                   
                 daughter (16) 
               
               
                   
                   
                   
                   
                   
                 never got the 
               
               
                   
                   
                   
                   
                   
                 disease 
               
               
                 #9 
                 2 
                 45-female 
                   
                 Both cured from 
                 Husband and 
               
               
                   
                   
                 16-male 
                   
                 severe symptoms 
                 daughter never 
               
               
                   
                   
                   
                   
                 of cough and 
                 got the disease 
               
               
                   
                   
                   
                   
                 fever 
               
               
                 #10  
                 2 
                 44- male 
                   
                 Cured from loss 
                 Mother (50 y) 
               
               
                   
                   
                   
                   
                 of smell, fever, 
                 never got the 
               
               
                   
                   
                   
                   
                 cough 
                 disease 
               
               
                   
                   
                 25- female 
                   
                 Cured 
               
               
                   
                   
                 21-female 
                   
                 Partially treated 
               
               
                   
                   
                   
                   
                 with HAZDPAC - 
               
               
                   
                   
                   
                   
                 slowly turned −ve 
               
               
                 #11  
                 1 
                 33-female 
                 No 
                 ICU nurse 
                 Boyfriend never 
               
               
                   
                   
                   
                   
                 recovered 
                 got the disease 
               
               
                   
               
            
           
         
       
     
     Apart from the families a further 11 single infected individuals found to be swab PCR positive were treated with the above treatment protocol. This is shown below in Table 32. All were also successfully cured of the infection. 
     
       
         
           
               
               
               
               
             
               
                 TABLE 32 
               
               
                   
               
               
                   
                   
                   
                 After 
               
               
                   
                 Age (years)- 
                   
                 treating with 
               
               
                   
                 sex of the 
                 Comorbidities/symptoms 
                 HAZDPAC 
               
               
                 Patient 
                 patient 
                 of the patients 
                 Cured/not 
               
               
                   
               
             
            
               
                 #1 
                 44-female 
                 Asthma 
                 Cured 
               
               
                   
                 (no BCG in 
               
               
                   
                 childhood) 
               
               
                 #2 
                 81- male 
                 Diarrhea 
                 Cured 
               
               
                 #3 
                 52-female 
                 Fever 
                 Cured 
               
               
                 #4 
                 66-male 
                 Valve surgery 
                 Cured 
               
               
                 #5 
                 29-female 
                 Asthma 
                 Cured 
               
               
                   
                 (no BCG in 
               
               
                   
                 childhood) 
               
               
                 #6 
                 50-female 
                 Auto immune thyroiditis, sever 
                 Cured 
               
               
                   
                   
                 cough and fever 
               
               
                 #7 
                 43-male 
                 Cough, desaturation of oxygen 
                 Cured 
               
               
                   
                 53-male 
                 Diarrhea, cough, fever, 
                 Cured 
               
               
                   
                   
                 desaturation of oxygen 
               
               
                 #9 
                 44- female 
                 Autoimmune history, fever, 
                 Cured 
               
               
                   
                   
                 increase heart rate 
               
               
                 #10  
                 44- female 
                 Pneumothorax discharged from 
                 Cured 
               
               
                   
                   
                 ICU with COVID-19 
               
               
                 #11  
                 43- male 
                 Fever +ve Covid-19 PCR 
                 Cured 
               
               
                   
               
            
           
         
       
     
     A further 9 individuals recently closely exposed to Covid-19 infected persons were given the prophylaxis protocol outlined above. The prophylactic worked very well with no exposed person acquiring the infection. This is shown in Table 33. 
     
       
         
           
               
               
               
             
               
                 TABLE 33 
               
               
                   
               
               
                 Individual 
                 Age (years) &amp; details of the individual 
                 After treating with ZINCD + H 
               
               
                   
               
             
            
               
                 #1 
                 Mother of 16 year old child who had 
                 Prophylaxed and never got the 
               
               
                   
                 COVID-19 
                 disease 
               
               
                 #2 
                 24- female, ICU nurse - multiple 
                 Prophylaxed and never got the 
               
               
                   
                 exposures 
                 disease 
               
               
                 #3 
                 47- male, cardiologist exposed to +ve 
                 Prophylaxed and never got the 
               
               
                   
                 patients 
                 disease 
               
               
                 #4 
                 70- male medical director of a hospital 
                 Prophylaxed and never got the 
               
               
                   
                 (exposed to numerous doctors with 
                 disease 
               
               
                   
                 COVID-19) 
               
               
                 #5 
                 55- male anaesthesiologist (intubates 
                 Prophylaxed and never got the 
               
               
                   
                 COVID-19 patients) 
                 disease 
               
               
                 #6 
                 55- ICU nurse (worked on COVID-19 
                 Prophylaxed and never got the 
               
               
                   
                 floor) 
                 disease 
               
               
                 #7 
                 40- ICU nurse many Covid-19 patients 
                 Prophylaxed and never got the 
               
               
                   
                   
                 disease 
               
               
                 #8 
                 53-Doctor with pancreatitis 
                 Prophylaxed and never got the 
               
               
                   
                   
                 disease 
               
               
                 #9 
                 28-Paramedic - healthy 
                 Prophylaxed and never got the 
               
               
                   
                   
                 disease 
               
               
                   
               
            
           
         
       
     
     Discussion: It was demonstrated that a 10 day combination of hydroxychloroquine, azithromycin (for 5 days only), zinc with vitamin D and vitamin C, can result in uniform cure of COVID-19 infection when used in an outpatient population. The prophylaxis treatment noted above for those closely exposed to proven, infected patients can completely prevent spread of COVID-19. This combination of test-and-treat permits abolishing of new outbreaks of infection such as a ‘next wave’—by avoiding quarantine to treat the infected and give prophylactics to surrounding staff and family. 
     In conclusion, this is an effective anti-Covid-19 therapy as well as an effective prophylactic combination capable of arresting the spread of coronavirus infection throughout the community. This is achieved by treating the index case and the surrounding associates of the patient as early as possible after infection is identified and then treating the people they live with and close associates. 
     Example 6: Presence of the SARS-CoV-2 by NGS of Fecal Samples 
     Objective: In view of the large percentage of SARS-CoV-2 detectible by RT-PCR in stools of infected patients, the objective was to identify the presence of the SARS-CoV-2 by NGS of fecal samples from symptomatic study participants positive for SARS-CoV-2 by nasopharyngeal sample RT-PCR, in addition to asymptomatic individuals (with or without prior nasopharyngeal sample RT-PCR). The objective was also to execute whole genome analysis to characterize SARS-CoV-2 mutational variations to identify potentially significant nucleotide changes. 
     Procedure: Study participants (n=14) underwent testing for SARS-CoV-2 from fecal samples by whole genome enrichment NGS. Following fecal collection (Zymo Research Shield Fecal Collection Tubes), RNA was extracted (Qiagen Allprep Power Viral Kit), reverse transcribed (New England Biolabs NEBNext 1st and 2nd Strand Synthesis Modules), library prepped (Illumina Nextera Flex for Enrichment), enriched (Ilumina Respiratory Virus Oligo Panel), and sequenced on Illumina&#39;s NextSeq 550 System. Sequences were then mapped to the SARS-CoV-2 Wuhan-Hu-1 (MN90847.3) complete genome utilizing One Codex&#39;s SARS-CoV-2 bioinformatics analysis pipeline. SARS-CoV-2 positive samples were further analyzed for mutational variants that differed from the reference genome. Of the 14 study participants, 12 also had their nasopharyngeal swabs tested for SARS-CoV-2 by RT-PCR. 
     Results: The results from patients that had their stool samples tested by whole genome enrichment NGS, and their nasopharyngeal swabs tested by RT-PCR for the presence of SARS-CoV-2 were evaluated. Of the 14 study participants, ten were symptomatic and tested positive for SARS-CoV-2 by RT-PCR, two asymptomatic individuals tested negative, and two other asymptomatic individuals did not undergo RT-PCR testing (Table 34). Patients 5 and 7, which tested positive by RT-PCR from nasopharyngeal swabs, were treated with the protocol from Example 5 above (Hydroxychloroquine, Azithromycin, vitamin C, vitamin D, and zinc for 10 days prior to fecal collection). Similarly, after positive nasopharyngeal swab, patient 13 was treated with vitamin C, vitamin D, and zinc for 10 days (the same protocol as noted above in Example 5) before fecal collection. The concordance of SARS-CoV-2 detection by enrichment NGS from stools among positive non-treated patients tested by RT-PCR nasopharyngeal analysis was 100% (7/7). Patient 8, who did not undergo nasopharyngeal analysis, tested positive for SARS-CoV-2 by NGS. The three patients (5, 7, 13) that received treatment prior to providing fecal samples, all tested negative by NGS. Asymptomatic patients 2 and 9, who tested negative by nasopharyngeal swab, were also negative by NGS, as was asymptomatic patient 14. Table 34 outlines the symptoms and SARS-CoV-2 testing results. 
     
       
         
           
               
               
               
               
               
               
             
               
                 TABLE 34 
               
               
                   
               
               
                   
                   
                 Nasopharyngeal 
                   
                 Fecal 
                 Patient 
               
               
                 Sample ID 
                 Symptoms 
                 Swab (RT-PCR) 
                 Treated 
                 (NGS) 
                 Location 
               
               
                   
               
             
            
               
                 Patient 1 
                 febrile, diarrhea, anosmia, O2 sat. &lt;90% 
                 + 
                 no 
                 + 
                 PA 
               
               
                 Patient 3 
                 febrile, diarrhea, O2 sat. &lt;90% 
                 + 
                 no 
                 + 
                 CA 
               
               
                 Patient 4 
                 febrile, diarrhea, anosmia, O2 sat. &lt;90% 
                 + 
                 no 
                 + 
                 AZ 
               
               
                 Patient 6 
                 febrile, cough, anosmia 
                 + 
                 no 
                 + 
                 AZ 
               
               
                 Patient 8 
                 none 
                 n/a 
                 no 
                 + 
                 CA 
               
               
                 Patient 10 
                 febrile, cough, headache 
                 + 
                 no 
                 + 
                 GA 
               
               
                 Patient 11 
                 febrile, cough, headache 
                 + 
                 no 
                 + 
                 GA 
               
               
                 Patient 12 
                 febrile, cough 
                 + 
                 no 
                 + 
                 GA 
               
               
                 Patient 5 
                 febrile, cough 
                 + 
                 yes 
                 − 
                 CA 
               
               
                 Patient 7 
                 febrile, cough 
                 + 
                 yes 
                 − 
                 GA 
               
               
                 Patient 13 
                 febrile, cough 
                 + 
                 yes 
                 − 
                 GA 
               
               
                 Patient 2 
                 none 
                 − 
                 no 
                 − 
                 CA 
               
               
                 Patient 9 
                 none 
                 − 
                 no 
                 − 
                 CA 
               
               
                 Patient 14 
                 none 
                 n/a 
                 no 
                 − 
                 CA 
               
               
                   
               
            
           
         
       
     
     All fecal samples analyzed by enrichment NGS from positive patients by RT-PCR, achieved 100% genome coverage of SARS-CoV-2 except for patient 3 which had 45%, and patient 10 which had 93% coverage. Table 35 outlines the enrichment NGS metrics. 
     
       
         
           
               
               
               
               
               
             
               
                 TABLE 35 
               
               
                   
               
               
                   
                 Genome 
                 # Variants 
                 Mapped 
                   
               
               
                 Sample ID 
                 Coverage 
                 (over 10x) 
                 Reads 
                 Mean Depth 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Patient 1 
                 100% 
                 11 
                 465645 
                 1129.8x 
               
               
                 Patient 3 
                  45% 
                 11 
                 5984 
                 31.7x 
               
               
                 Patient 4 
                 100% 
                 9 
                 131582 
                 318.6x 
               
               
                 Patient 6 
                 100% 
                 10 
                 793603 
                 1924.6x 
               
               
                 Patient 8 
                 100% 
                 10 
                 496852 
                 1206.7 
               
               
                 Patient 10 
                  93% 
                 9 
                 5929 
                 15.6x 
               
               
                 Patient 11 
                 100% 
                 10 
                 1270734 
                 3075.3x 
               
               
                 Patient 12 
                 100% 
                 10 
                 38256 
                 92.7x 
               
               
                   
               
            
           
         
       
     
     The total number of SARS-CoV-2 mapped reads for patients 1, 3, 4, 6, 8, 10, 11, and 12 were 465645, 5984, 131582, 793603, 496852, 5929, 1270734, and 38256 respectively. The mean read depths of SARS-CoV-2 for patients 1, 3, 4, 6, 8, 10, 11, and 12 were 1129.8x, 31.7x, 318.6x, 1924.6x, 1206.7x, 15.5x, 3075.3x, and 92.7x, and respectively. The read depths at specific coordinates along the SARS-CoV-2 genome for each patient are captured in  FIGS. 3A-3H . Whole genome alignment of SARS-CoV-2 in patients 1, 3, 4, 6, 8, 10, 11, and 12 (respectively) as identified by One Codex&#39;s SARS-CoV-2 analysis pipeline. The x-axis depicts the genomic coordinates as aligned to the MN908947.3 reference genome, and the y-axis represents the read depth at specific loci. 
     Following alignment and mapping of SARS-CoV-2, patient genomes were compared to the Wuhan-Hu-1 (MN90847.3) SARS-CoV-2 reference genome via One Codex&#39;s bioinformatics pipeline to identify mutational variations. This analysis identified nucleotide variants at positions nt241 (C→T) and nt23403 (A→G) across all positive patients, and variants at positions nt3037 (C→T) and nt25563 (G→T) in seven of the eight patients (Table 3). Interestingly, patients 8, 11, and 12 harbored the same set of variants, as did patients 4 and 6 (who were kindred). Unique variants not identified in any of the other individuals were detected in patients 1, 3, 6, and 10, with patient 3 harboring the most distinct SARS-CoV-2 genome with eight unique variants, followed by patient 1 with seven. Collectively, there were thirty-three different mutations among the patients in which SARS-CoV-2 was detected by whole genome enrichment NGS. Table 36 outlines the SARS-CoV-2 genomic positions, variant changes, and frequencies across the positive patient cohort. 
     
       
         
           
               
               
               
               
               
               
               
               
               
               
               
             
               
                 TABLE 36 
               
               
                   
               
               
                 Region 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 (ORF) 
                 Po 
                 Varian 
                 Patien 
                 Patien 
                 Patien 
                 Patien 
                 Patien 
                 Patient 
                 Patient 
                 Patient 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 5′-UTR 
                 24 
                 C → T 
                 100% 
                 100% 
                 100% 
                 100% 
                 100% 
                 100% 
                 100% 
                 100% 
               
               
                 1a 
                 83 
                 T → C 
                 x 
                 x 
                 x 
                 x 
                 100% 
                 x 
                 100% 
                 100% 
               
               
                 1a 
                 10 
                 C → T 
                 x 
                 x 
                 100% 
                 100% 
                  99% 
                 100% 
                 100% 
                 100% 
               
               
                 1a 
                 17 
                 C → T 
                 x 
                 x 
                 100% 
                 100% 
                 x 
                 x 
                 x 
                 x 
               
               
                 1a 
                 19 
                 C → T 
                 x 
                 x 
                 x 
                  87% 
                 x 
                 x 
                 x 
                 x 
               
               
                 1a 
                 30 
                 C → T 
                 100% 
                 x 
                 100% 
                 100% 
                 100% 
                 100% 
                 100% 
                 100% 
               
               
                 1a 
                 30 
                 C → T 
                 x 
                  89% 
                 x 
                 x 
                 x 
                 x 
                 x 
                 x 
               
               
                 1a 
                 48 
                 G → T 
                  75% 
                 x 
                 x 
                 x 
                 x 
                 x 
                 x 
                 x 
               
               
                 1a 
                 67 
                 C → T 
                  93% 
                 x 
                 x 
                 x 
                 x 
                 x 
                 x 
                 x 
               
               
                 1a 
                 81 
                 G → T 
                 x 
                 100% 
                 x 
                 x 
                 x 
                 x 
                 x 
                 x 
               
               
                 1a 
                 94 
                 T → C 
                 x 
                 x 
                 x 
                 x 
                 x 
                  64% 
                 x 
                 x 
               
               
                 1a 
                 94 
                 T → A 
                 x 
                 x 
                 x 
                 x 
                 x 
                  64% 
                 x 
                 x 
               
               
                 1a 
                 10 
                 G → T 
                 x 
                 x 
                 100% 
                 100% 
                 x 
                 x 
                 x 
                 x 
               
               
                 1a 
                 11 
                 G → A 
                 x 
                 x 
                 100% 
                 100% 
                 x 
                 x 
                 x 
                 x 
               
               
                 1b 
                 14 
                 C → T 
                 100% 
                 x 
                 100% 
                 100% 
                 100% 
                 x 
                 100% 
                 100% 
               
               
                 1b 
                 14 
                 C → T 
                 x 
                 100% 
                 x 
                 x 
                 x 
                 x 
                 x 
                 x 
               
               
                 1b 
                 16 
                 C → T 
                 x 
                 x 
                 x 
                 x 
                 100% 
                 x 
                 100% 
                 100% 
               
               
                 1b 
                 16 
                 C → T 
                 100% 
                 x 
                 x 
                 x 
                 x 
                 x 
                 x 
                 x 
               
               
                 1b 
                 18 
                 C → A 
                 x 
                 x 
                 x 
                 x 
                 x 
                  83% 
                 x 
                 x 
               
               
                 1b 
                 19 
                 T → G 
                 x 
                 100% 
                 x 
                 x 
                 x 
                 x 
                 x 
                 x 
               
               
                 Spike 
                 21 
                 T → G 
                 x 
                  83% 
                 x 
                 x 
                 x 
                 x 
                 x 
                 x 
               
               
                 Spike 
                 23 
                 G → A 
                 x 
                  75% 
                 x 
                 x 
                 x 
                 x 
                 x 
                 x 
               
               
                 Spike 
                 23 
                 A → G 
                 100% 
                 100% 
                 100% 
                 100% 
                 100% 
                 100% 
                 100% 
                 100% 
               
               
                 Spike 
                 23 
                 C → T 
                  82% 
                 x 
                 x 
                 x 
                 x 
                 x 
                 x 
                 x 
               
               
                 3a 
                 25 
                 G → T 
                 x 
                 100% 
                 100% 
                 100% 
                 100% 
                 100% 
                 100% 
                 100% 
               
               
                 3a 
                 25 
                 C → A 
                 x 
                 x 
                 x 
                 x 
                 100% 
                 x 
                 100% 
                 100% 
               
               
                 8 
                 27 
                 C → T 
                 x 
                 x 
                 x 
                 x 
                 100% 
                 x 
                 100% 
                 100% 
               
               
                 Nucleopr 
                 28 
                 G → A 
                 100% 
                 x 
                 x 
                 x 
                 x 
                 x 
                 x 
                 x 
               
               
                 Nucleopr 
                 28 
                 G → A 
                 100% 
                 x 
                 x 
                 x 
                 x 
                 x 
                 x 
                 x 
               
               
                 Nucleopr 
                 28 
                 G → C 
                 100% 
                 x 
                 x 
                 x 
                 x 
                 x 
                 x 
                 x 
               
               
                 Nucleopr 
                 28 
                 C → T 
                 x 
                 100% 
                 x 
                 x 
                 x 
                 x 
                 x 
                 x 
               
               
                 Nucleopr 
                 29 
                 A → T 
                 x 
                 100% 
                 x 
                 x 
                 x 
                 x 
                 x 
                 x 
               
               
                 Nucleopr 
                 29 
                 C → G 
                 x 
                 x 
                 x 
                 x 
                 x 
                  85% 
                 x 
                 x 
               
               
                   
               
            
           
         
       
     
     Discussion: Although previous studies have identified SARS-CoV-2 in fecal collections by RT-PCR, this study was able to report whole genome sequencing (WGS) of SARS-CoV-2 from stool samples. SARS-CoV-2 was identified in patients that tested positive by nasopharyngeal swab RT-PCR analysis and unique genomes in 62.5% of the NGS positive patients was observed. The overall homology among the genomes was high (99.97%), with variations identified in the ORF regions 1a, 1b, S, 3a, 8, and N. Of particular interest, was the adenine to guanine change in the S protein at position nt23403 which converts aspartic acid to glycine (D→G). 
     Conclusion: Next generation sequencing identified the SARS-CoV-2 whole genome sequence in 100% of patients with positive nasopharyngeal RT-PCR and did not detect it in treated patients, or those with negative rt-PCR. These results highlight the importance of metagenomic analysis of the SARS-CoV-2 viral genome. 
     Example 7: Randomized, Double-Blind, Placebo-Controlled Phase IIA Study of Hydroxychloroquine, Vitamin C, Vitamin D, and Zinc for the Prevention of COVID-19 Infection 
     Objectives: Prevention of COVID-19, Lack of COVID-19 symptoms, and assessment of safety and tolerability 
     Procedure: Screening Period (Days −7 to −1): Prescription of home health monitoring equipment that includes a thermometer, a pregnancy test if applicable, and a daily diary. There are two groups being studied: Arm 1 and Arm 2. 
     Arm 1 is prescribed the following antimicrobials: hydroxychloroquine 200 mg twice a day for 1 day only, vitamin C 3000 IU per day for 12 weeks, vitamin D 3000 IU per day for 12 weeks, and zinc 50 mg per day for 12 weeks. The zinc can be reduced to 25 mg if GI upset occurs. The hydroxychloroquine is to be taken first thing in the morning as soon as subject has eaten and again right before bed, and must be separated from vitamin dose by at least 2 hours. 
     Arm 2 is prescribed the following antimicrobials: Placebo twice a day for 1 day only, vitamin C 3000 IU per day for 12 weeks, vitamin D 3000 IU per day for 12 weeks, and zinc 50 mg per day for 12 weeks. The zinc can be reduced to 25 mg if GI upset occurs. 
     Day 1: Patient is called to teach them how to use the diary in the EDC, discuss the medication regimen, and answer any questions they may have. Patient takes pregnancy test if applicable, collects a temperature reading, completes their diary, and takes the prescribed treatment regimen. Table 37 outlines the prescribed treatment regimen for Day 1. 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 37 
               
               
                   
                   
               
               
                   
                 Drug 
                   
                 AM Dose 
                 PM Dose 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Hydroxychloroquine (or placebo) 
                 200 
                 mg 
                 200 
                 mg 
               
               
                   
                 Vitamin C 
                 1500 
                 mg 
                 1500 
                 mg 
               
               
                   
                 Vitamin D 
                 1500 
                 IU 
                 1500 
                 IU 
               
               
                   
                 Zinc 
                 25 
                 mg 
                 25 
                 mg 
               
               
                   
                   
               
            
           
         
       
     
     Day 2: Patient collects a temperature reading, completes their diary, and is called on the phone for assessment of any adverse events or serious adverse events, assessment of any COVID-19 symptoms, the list of prior and concomitant medications is updated, any questions the patient has are answered, and the patient takes their prescribed treatment regimen. Table 38 outlines the prescribed treatment regimen for Day 2. 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 38 
               
               
                   
                   
               
               
                   
                 Drug 
                   
                 AM Dose 
                 PM Dose 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Vitamin C 
                 1500 
                 mg 
                 1500 
                 mg 
               
               
                   
                 Vitamin D 
                 1500 
                 IU 
                 1500 
                 IU 
               
               
                   
                 Zinc 
                 25 
                 mg 
                 25 
                 mg 
               
               
                   
                   
               
            
           
         
       
     
     Days 3-10: Patient collects a temperature reading, completes their diary, and takes their prescribed treatment regimen. Table 8 outlines the prescribed treatment regimen for Days 3-10. 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 8 
               
               
                   
                   
               
               
                   
                 Drug 
                   
                 AM Dose 
                 PM Dose 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Vitamin C 
                 1500 
                 mg 
                 1500 
                 mg 
               
               
                   
                 Vitamin D 
                 1500 
                 IU 
                 1500 
                 IU 
               
               
                   
                 Zinc 
                 25 
                 mg 
                 25 
                 mg 
               
               
                   
                   
               
            
           
         
       
     
     Day 14: The patient is called for instruction on how to collect a nasal swab and package for shipping, assessment COVID-19 symptoms, updating their list of prior and concomitant medications, and answering any questions they may have. 
     Week 3: The patient is called for assessment of any adverse events or serious adverse events, assessment of any COVID-19 symptoms, updating their list of prior and concomitant medications, and answering any questions they may have. The patient takes a temperature reading, completes their diary, and takes the prescribed treatment regimen. 
     Table 39 outlines the treatment regime for week 3. 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 39 
               
               
                   
                   
               
               
                   
                 Drug 
                   
                 AM Dose 
                 PM Dose 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Vitamin C 
                 1500 
                 mg 
                 1500 
                 mg 
               
               
                   
                 Vitamin D 
                 1500 
                 IU 
                 1500 
                 IU 
               
               
                   
                 Zinc 
                 25 
                 mg 
                 25 
                 mg 
               
               
                   
                   
               
            
           
         
       
     
     Week 4: The patient is called for assessment of any adverse events or serious adverse events, assessment of any COVID-19 symptoms, updating their list of prior and concomitant medications, and answering any questions they may have. The patient takes a temperature reading, completes their diary, and takes the prescribed treatment regimen. The patient also collects a nasal swab. 
     Table 40 outlines the treatment regime for week 4. 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 40 
               
               
                   
                   
               
               
                   
                 Drug 
                   
                 AM Dose 
                 PM Dose 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Vitamin C 
                 1500 
                 mg 
                 1500 
                 mg 
               
               
                   
                 Vitamin D 
                 1500 
                 IU 
                 1500 
                 IU 
               
               
                   
                 Zinc 
                 25 
                 mg 
                 25 
                 mg 
               
               
                   
                   
               
            
           
         
       
     
     Weeks 5-11: The patient is called weekly for assessment of any adverse events or serious adverse events, assessment of any COVID-19 symptoms, updating their list of prior and concomitant medications, and answering any questions they may have. The patient takes a weekly temperature reading, completes their diary, and takes the prescribed treatment regimen. 
     Table 41 outlines the treatment regime for weeks 5-11. 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 41 
               
               
                   
                   
               
               
                   
                 Drug 
                   
                 AM Dose 
                 PM Dose 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Vitamin C 
                 1500 
                 mg 
                 1500 
                 mg 
               
               
                   
                 Vitamin D 
                 1500 
                 IU 
                 1500 
                 IU 
               
               
                   
                 Zinc 
                 25 
                 mg 
                 25 
                 mg 
               
               
                   
                   
               
            
           
         
       
     
     Week 12: The patient presents to the clinic (or video conference) for evaluation that includes assessment for adverse events and serious adverse events, updating their list of prior and concomitant medications, a physical exam, nasal swab collection and COVID-19 sample collection. 
     Samples for COVID-19 testing are collected using synthetic swabs with plastic shafts. Nasal swabs are collected and immediately placed into a sterile vial with 2-3 mL of viral transport media. The vials are placed into biohazard bags, boxed up, the box sterilized, and picked up for shipment to the central laboratory. Samples are tested by RT-PCR. 
     Table 42 presents the schedule of events for Example 7. 
     
       
         
           
               
               
               
               
               
               
               
               
               
               
             
               
                 TABLE 42 
               
               
                   
               
             
            
               
                   
                 Screening 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 (Day −7 
               
               
                 Assessment 
                 to Day 1) 
                 Day 1 
                 Day 2 
                 Day 3-10 
                 Day 14 
                 Week 3 
                 Week 4/Month 1 
                 Week 5 
                 Week 6 
               
               
                   
               
               
                 Informed Consent &amp; 
                 X 
               
               
                 Demographics 
               
               
                 Initial Review of 
                 X 
               
               
                 Prior and Concomitant 
               
               
                 Medications 
               
               
                 Medical Records 
                 X 
               
               
                 review, including 
               
               
                 baseline EKG 
               
               
                 Randomization 
                 X 
               
               
                 Prescription of 
                 X 
               
               
                 Hydroxychloroquine 
               
               
                 or placebo, vitamin C, 
               
               
                 vitamin D, and zinc 
               
               
                 Provision of daily 
                 X 
               
               
                 diary, thermometer 
               
               
                 and pregnancy test 
               
               
                 Pregnancy Test if 
                   
                 X 
               
               
                 applicable 
               
               
                 Temperature at home 
                   
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                 Complete daily diary 
                   
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                 Hydroxychloroquine 
                   
                 X 
               
               
                 200 mg 
               
               
                 Vitamin C 3000 mg 
                   
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                 Vitamin D 3000 IU 
                   
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                 Zinc 50 mg a   
                   
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                 Phone/video call 
                 X 
                 X 
                 X 
                   
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                 to patient b   
               
               
                 Update list of prior 
                   
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                 and concomitant 
               
               
                 medications 
               
               
                 Ask about AE and SAE 
                   
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                 Assessment of 
                   
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                 COVID-19 symptoms 
               
               
                 Swabs for RT-PCR 
                   
                 X 
                   
                   
                 X 
                   
                 X 
               
               
                 Vitals in-clinic c   
               
               
                 Physical Exam 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                 Assessment 
                 Week 7 
                 Week 8/Month 2 
                 Week 9 
                 Week 10 
                 Week 11 
                 Week 12/Month 3 
               
               
                   
                   
               
               
                   
                 Informed Consent &amp; 
               
               
                   
                 Demographics 
               
               
                   
                 Initial Review of 
               
               
                   
                 Prior and Concomitant 
               
               
                   
                 Medications 
               
               
                   
                 Medical Records 
               
               
                   
                 review, including 
               
               
                   
                 baseline EKG 
               
               
                   
                 Randomization 
               
               
                   
                 Prescription of 
               
               
                   
                 Hydroxychloroquine 
               
               
                   
                 or placebo, vitamin C, 
               
               
                   
                 vitamin D, and zinc 
               
               
                   
                 Provision of daily 
               
               
                   
                 diary, thermometer 
               
               
                   
                 and pregnancy test 
               
               
                   
                 Pregnancy Test if 
               
               
                   
                 applicable 
               
               
                   
                 Temperature at home 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                   
                 Complete daily diary 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                   
                 Hydroxychloroquine 
               
               
                   
                 200 mg 
               
               
                   
                 Vitamin C 3000 mg 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                   
                 Vitamin D 3000 IU 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                   
                 Zinc 50 mg a   
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                   
                 Phone/video call 
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                   
                 to patient b   
               
               
                   
                 Update list of prior 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                   
                 and concomitant 
               
               
                   
                 medications 
               
               
                   
                 Ask about AE and SAE 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                   
                 Assessment of 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                   
                 COVID-19 symptoms 
               
               
                   
                 Swabs for RT-PCR 
                   
                   
                   
                   
                   
                 X 
               
               
                   
                 Vitals in-clinic c   
                   
                   
                   
                   
                   
                 X 
               
               
                   
                 Physical Exam 
                   
                   
                   
                   
                   
                 X 
               
               
                   
                   
               
            
           
         
       
     
     Regarding Table 42: The Zinc may be reduced to 25 mg if GI upset occurs, phone/video calls to the patient occur weekly during Weeks 2-11, and vitals in-clinic include height, weight, blood pressure (following 5 minutes sitting) pulse, respiratory rate, temperature, and oxygen saturation. 
     Statistical Analysis: The treated patients in this study are compared to the placebo group. Measurements include PCR test results, presence or absence of symptoms, and symptom severity. 
     The change of these measurements from the end to the baseline (post-pre) are used as the primary outcome, for example, μe=μe1−μe0, where μe1 and μe0 are the outcome of patients from the treatment group at the end and at baseline, respectively. 
     H 0 :Δ≤δ 0  against H α :Δ&gt;δ 0  where δ0 is a clinically meaningful threshold to measure the disease symptoms. In this study, that meaningful threshold is calculated as mean change in clinical symptoms as recorded in the diary, from baseline through week 12. Each category in the diary is assigned a number, 0 for None, 1 for Mild, 2 for Moderate and 3 for severe. Each category is analyzed independently and as a group. 
     Categorical variables are summarized by presenting the number (n) and percent (%) of subjects in each category. All Statistical tests for the analysis are performed using the p&lt;0.05 level of significance. All confidence intervals are one-sided. 
     Since these are healthcare workers who are exposed to COVID-19 at every shift, efficacy is determined by RT-PCR testing, as well as the presence or absence of symptoms as recorded in the patient diary via EDC. 
     Example 8: Study of Microbiome of Patients with COVID Versus Patients without COVID 
     36 Covid-19 patients were studied in a cross-sectional study with 14 healthy controls (HC) to identify microbiome diversity by whole genome enrichment NGS. Patient records were included and compared any significant changes on species, genus, family, order, class, and phylum levels of the Covid-19 positive and HC population. 
     Deep shotgun microbiome sequencing analysis was performed on fecal samples from the 36 Covid-19 positive patients by whole genome enrichment NGS. The observed versus expected rates were primarily reported. 
     All individuals aged 3 years and older were eligible for inclusion. For each patient, a so-called period of eligibility for study inclusion was defined, which commenced on the latest of the study start date; A patient&#39;s period of eligibility ended on the earliest of registration termination; the end of data collection from their practice; or death. 
     Data Analysis 
     The differential taxa was conducted between the Gastrointestinal Microbiome of Covid-19 positive and HC relative abundance utilizing One Codex&#39;s bioinformatics analysis pipeline. For evaluating any statistical significance of the patient&#39;s data at each Classification at phylum, class, order, family, and genus levels, a Fisher&#39;s exact test was conducted between the two variables. Statistical analysis was conducted using chi-squared statistics by R version 3.6.1 (2019 Jul. 5). In the statistical analysis, p-value, Confidence interval and Odds ratio were considered for all comparisons. 
     Results 
     The study population included 50 patients (36 Covid-19 positives and 14 HC). For the HC population, data from patients after December 2019 were excluded from the study to avoid overlap between any Covid-19 possible positive patients. 
     The results from the 50 total patients that had their stool samples tested by whole genome enrichment NGS was evaluated. Detailed demographic and summary data, clinical characteristics including total numbers of diagnoses and events were analyzed for the study cohorts and are included in Table 43. 
     
       
         
           
               
               
               
               
               
               
               
               
             
               
                 TABLE 43 
               
               
                   
               
               
                   
                   
                   
                 Significant 
                   
                   
                 Stool 
                 Sx 
               
               
                   
                   
                   
                 Past Medical 
                 PCR 
                 Covid -19 
                 Collection 
                 Severity 
               
               
                 No 
                 Age 
                 Sex 
                 History 
                 Date 
                 PCR Results 
                 Date 
                 at BL 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 1 
                 20 
                 Female 
                 Hypothyroid, 
                 Jul. 10, 2020 
                 Positive 
                 Jun. 24, 2020 
                 Severe 
               
               
                   
                   
                   
                 Hashimoto, LCH 
               
               
                 2 
                 21 
                 Female 
                 Nothing to 
                 No info 
                 Positive 
                 Apr. 5, 2020 
                 Moderate 
               
               
                   
                   
                   
                 report 
               
               
                 3 
                 56 
                 Male 
                 Nothing to 
                 No info 
                 Positive 
                 Apr. 5, 2020 
                 No info 
               
               
                   
                   
                   
                 report 
               
               
                 4 
                 25 
                 Female 
                 Asthma, Reflux, 
                 No info 
                 Positive 
                 Mar. 29, 2020 
                 Mild 
               
               
                   
                   
                   
                 GI issue 
               
               
                 5 
                 25 
                 Female 
                 No info 
                 No info 
                 Positive 
                 No info 
                 Moderate 
               
               
                 6 
                 44 
                 Female 
                 Food 
                 Apr. 8, 2020 
                 Positive 
                 Apr. 13, 2020 
                 Severe 
               
               
                   
                   
                   
                 sensitivities, gut 
               
               
                   
                   
                   
                 issues 
               
               
                 7 
                   
                 No 
                 No info 
                 No info 
                 Positive 
                 No info 
                 No info 
               
               
                   
                   
                 info 
               
               
                 8 
                 53 
                 Male 
                 No info 
                 No info 
                 Positive 
                 No info 
                 Severe 
               
               
                 9 
                 23 
                 Male 
                 No info 
                 No info 
                 Positive 
                 No info 
                 No info 
               
               
                 10 
                 19 
                 Male 
                 Nothing to 
                 Apr. 22, 2020 
                 Positive 
                 May 6, 2020 
                 No info 
               
               
                   
                   
                   
                 report 
               
               
                 11 
                 35 
                 Female 
                 Seizure 
                 No info 
                 Positive 
                 Jun. 23, 2020 
                 No info 
               
               
                 12 
                 32 
                 Male 
                 Nothing to 
                   
                 Positive 
                 Jun. 26, 2020 
                 Mild 
               
               
                   
                   
                   
                 report 
               
               
                 13 
                 63 
                 Female 
                 No info 
                 No info 
                 Positive 
                 Jul. 20, 2020 
                 No info 
               
               
                 14 
                 No 
                 Male 
                 No info 
                 No info 
                 Positive 
                 Jun. 28, 2020 
                 No info 
               
               
                   
                 info 
               
               
                 15 
                 61 
                 Male 
                 Nothing to 
                 No info 
                 Positive 
                 No info 
                 No info 
               
               
                   
                   
                   
                 report 
               
               
                 16 
                  50+ 
                 Male 
                 No info 
                 No info 
                 Positive 
                 No info 
                 No info 
               
               
                 17 
                 48 
                 Female 
                 No info 
                 No info 
                 Positive 
                 No info 
                 Severe 
               
               
                 18 
                 No 
                 Female 
                 No info 
                 No info 
                 Positive 
                 Jul. 27, 2020 
                 No info 
               
               
                   
                 info 
               
               
                 19 
                 70 
                 Male 
                 No info 
                 No info 
                 Positive 
                 Aug. 5, 2020 
                 Severe 
               
               
                 20 
                 56 
                 Male 
                 Nothing to 
                 No info 
                 Positive 
                 Aug. 10, 2020 
                 Severe 
               
               
                   
                   
                   
                 report 
               
               
                 21 
                  50+ 
                 Male 
                 No info 
                 No info 
                 Positive 
                 No info 
                 Moderate 
               
               
                 22 
                 59 
                 Female 
                 No info 
                 No info 
                 Positive 
                 Sep. 4, 2020 
                 Severe 
               
               
                 23 
                 35 
                 Female 
                 No info 
                 No info 
                 Positive 
                 No info 
                 Moderate 
               
               
                 24 
                 71 
                 Male 
                 No info 
                 No info 
                 Positive 
                 No info 
                 No info 
               
               
                 25 
                 58 
                 Female 
                 No info 
                 No info 
                 Positive 
                 No info 
                 Moderate 
               
               
                 26 
                 61 
                 Female 
                 No info 
                 No info 
                 Positive 
                 No info 
                 No info 
               
               
                 27 
                 55 
                 Male 
                 Nothing to 
                   
                 Positive 
                 Oct. 21, 2020 
                 Severe 
               
               
                   
                   
                   
                 report 
               
               
                 28 
                 66 
                 Female 
                 No info 
                 No info 
                 Positive 
                 No info 
                 Moderate 
               
               
                 29 
                 No 
                 No 
                 No info 
                 No info 
                 Positive 
                 No info 
                 No info 
               
               
                   
                 info 
                 info 
               
               
                 30 
                 66 
                 Female 
                 No info 
                 No info 
                 Positive 
                 No info 
                 Severe 
               
               
                 31 
                 No 
                 No 
                 No info 
                 No info 
                 Positive 
                 No info 
                 No info 
               
               
                   
                 info 
                 info 
               
               
                 32 
                 61 
                 Female 
                 No info 
                 No info 
                 Positive 
                 No info 
                 Severe 
               
               
                 33 
                 69 
                 Male 
                 No info 
                 No info 
                 Positive 
                 No info 
                 No info 
               
               
                 34 
                 No 
                 No 
                 No info 
                 No info 
                 Positive 
                 No info 
                 No info 
               
               
                   
                 info 
                 info 
               
               
                 35 
                 40 
                 Female 
                 No info 
                 No info 
                 Positive 
                 No info 
                 Moderate 
               
               
                 36 
                 No 
                 No 
                 No info 
                 No info 
                 Positive 
                 No info 
                 No info 
               
               
                   
                 info 
                 info 
               
               
                 37 
                  6 
                 Male 
                 Nothing to 
                 Not 
                 Not applicable- 
                 No info 
                 Not 
               
               
                   
                   
                   
                 report 
                 applicable 
                 Negative 
                   
                 applicable- 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Negative 
               
               
                 38 
                  6 
                 Female 
                 Nothing to 
                 Not 
                 Not applicable- 
                 No info 
                 Not 
               
               
                   
                   
                   
                 report 
                 applicable 
                 Negative 
                   
                 applicable- 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Negative 
               
               
                 39 
                  8 
                 Female 
                 Nothing to 
                 Not 
                 Not applicable- 
                 No info 
                 Not 
               
               
                   
                   
                   
                 report 
                 applicable 
                 Negative 
                   
                 applicable- 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Negative 
               
               
                 40 
                  3 
                 Female 
                 Nothing to 
                 Not 
                 Not applicable- 
                 No info 
                 Not 
               
               
                   
                   
                   
                 report 
                 applicable 
                 Negative 
                   
                 applicable- 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Negative 
               
               
                 41 
                 11 
                 Male 
                 Nothing to 
                 Not 
                 Not applicable- 
                 No info 
                 Not 
               
               
                   
                   
                   
                 report 
                 applicable 
                 Negative 
                   
                 applicable- 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Negative 
               
               
                 42 
                  3 
                 Male 
                 Nothing to 
                 Not 
                 Not applicable- 
                 No info 
                 Not 
               
               
                   
                   
                   
                 report 
                 applicable 
                 Negative 
                   
                 applicable- 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Negative 
               
               
                 43 
                  4 
                 Female 
                 Nothing to 
                 Not 
                 Not applicable- 
                 No info 
                 Not 
               
               
                   
                   
                   
                 report 
                 applicable 
                 Negative 
                   
                 applicable- 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Negative 
               
               
                 44 
                  7 
                 Female 
                 Nothing to 
                 Not 
                 Not applicable- 
                 No info 
                 Not 
               
               
                   
                   
                   
                 report 
                 applicable 
                 Negative 
                   
                 applicable- 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Negative 
               
               
                 45 
                 16 
                 Female 
                 Nothing to 
                 Not 
                 Not applicable- 
                 No info 
                 Not 
               
               
                   
                   
                   
                 report 
                 applicable 
                 Negative 
                   
                 applicable- 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Negative 
               
               
                 46 
                  9 
                 Male 
                 Nothing to 
                 Not 
                 Not applicable- 
                 No info 
                 Not 
               
               
                   
                   
                   
                 report 
                 applicable 
                 Negative 
                   
                 applicable- 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Negative 
               
               
                 47 
                 10 
                 Female 
                 Nothing to 
                 Not 
                 Not applicable- 
                 No info 
                 Not 
               
               
                   
                   
                   
                 report 
                 applicable 
                 Negative 
                   
                 applicable- 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Negative 
               
               
                 48 
                  7 
                 Female 
                 Nothing to 
                 Not 
                 Not applicable- 
                 No info 
                 Not 
               
               
                   
                   
                   
                 report 
                 applicable 
                 Negative 
                   
                 applicable- 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Negative 
               
               
                 49 
                 55 
                 Female 
                 Nothing to 
                 Not 
                 Not applicable- 
                 No info 
                 Not 
               
               
                   
                   
                   
                 report 
                 applicable 
                 Negative 
                   
                 applicable- 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Negative 
               
               
                 50 
                 17 
                 Female 
                 Nothing to 
                 Not 
                 Not applicable- 
                 No info 
                 Not 
               
               
                   
                   
                   
                 report 
                 applicable 
                 Negative 
                   
                 applicable- 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Negative 
               
               
                   
               
            
           
         
       
     
     Tables 44-49 demonstrate the association between relative abundance of the gastrointestinal microbiome at Phylum, Class, Order, Family and Species level of Covid-19 positive (Covid+) vs. healthy control (Covid−) patients. A 95% Confidence interval (CI) and odds ratio (OR) were used in addition to the p-value for better comparison. 
     
       
         
           
               
               
               
             
               
                   
                 TABLE 44 
               
             
            
               
                   
                   
               
               
                   
                   
                 Fisher exact test value 
               
               
                   
                 Statistical 
                 with 95% Confidence 
               
            
           
           
               
               
               
               
               
            
               
                 Classification 
                 Covid + Patients 
                 Covid − Patients 
                 Significance 
                 Intervals(CI) and odds 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Phylum 
                 Presence 
                 Absence 
                 Presence 
                 Absence 
                 (P &lt; 0.05) 
                 ratio(OR) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Actinobacteria 
                 31 
                 5 
                 14 
                 0 
                 Yes* 
                 P &lt; 0.00001(CI; 
               
               
                   
                   
                   
                   
                   
                   
                 0.00-0.3, OR = 0) 
               
               
                 Firmicutes 
                 36 
                 0 
                 14 
                 0 
                 No** 
                 p-value 1 
               
               
                 Proteobacteria 
                 33 
                 3 
                 14 
                 0 
                 Yes  
                 P value 0.003(CI; 
               
               
                   
                   
                   
                   
                   
                   
                 0.00-0.48, OR = 0) 
               
               
                 Bacteroidetes 
                 36 
                 0 
                 14 
                 0 
                 No  
                 p-value 1 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
             
               
                   
                 TABLE 45 
               
             
            
               
                   
                   
               
               
                   
                   
                 Fisher exact test value 
               
               
                   
                 Statistical 
                 with 95% Confidence 
               
            
           
           
               
               
               
               
               
            
               
                 Classification 
                 Covid + Patients 
                 Covid − Patients 
                 Significance 
                 Intervals(CI) and odds 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Class 
                 Presence 
                 Absence 
                 Presence 
                 Absence 
                 (P &lt; 0.05) 
                 ratio(OR) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Actinobacteria 
                 24 
                 12 
                 14 
                 0 
                 Yes 
                 P &lt; 0.00001(CI; 
               
               
                   
                   
                   
                   
                   
                   
                 0.00-0.07, OR = 0) 
               
               
                 Clostridia 
                 36 
                 0 
                 14 
                 0 
                 No 
                 p-value 1 
               
               
                 Negativicutes 
                 30 
                 6 
                 14 
                 0 
                 Yes 
                 P &lt; 0.00001(CI; 
               
               
                   
                   
                   
                   
                   
                   
                 0.00-0.21, OR = 0) 
               
               
                 Betaproteo- 
                 21 
                 15 
                 12 
                 2 
                 Yes 
                 P &lt; 0.00001(CI; 
               
               
                 bacteria 
                   
                   
                   
                   
                   
                 0.10-0.46, OR = 0.22) 
               
               
                 Bacteroidia 
                 36 
                 0 
                 14 
                 0 
                 No 
                 p-value 1 
               
               
                 Gammaproteo- 
                 21 
                 15 
                 9 
                 5 
                 No 
                 p-value 0.46(CI; 
               
               
                 bacteria 
                   
                   
                   
                   
                   
                 0.4-1.4, OR = 0.77) 
               
               
                 Erysipelotrichia 
                 33 
                 3 
                 11 
                 3 
                 Yes 
                 p-value 0.009(CI; 
               
               
                   
                   
                   
                   
                   
                   
                 01.27-8.77, OR = 3.19) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
             
               
                   
                 TABLE 46 
               
             
            
               
                   
                   
               
               
                   
                   
                 Fisher exact test value 
               
               
                   
                 Statistical 
                 with 95% Confidence 
               
            
           
           
               
               
               
               
               
            
               
                 Classification 
                 Covid + Patients 
                 Covid − Patients 
                 Significance 
                 Intervals(CI) and odds 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Order 
                 Presence 
                 Absence 
                 Presence 
                 Absence 
                 (P &lt; 0.05) 
                 ratio(OR) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Bifidobacteriales 
                 20 
                 16 
                 14 
                 0 
                 Yes 
                 P &lt; 0.00001(CI; 
               
               
                   
                   
                   
                   
                   
                   
                 0.00-0.05, OR = 0) 
               
               
                 Clostridiales 
                 36 
                 0 
                 14 
                 0 
                 No 
                 p-value 1 
               
               
                 Veillonellales 
                 14 
                 22 
                 11 
                 3 
                 Yes 
                 P &lt; 0.00001(CI; 
               
               
                   
                   
                   
                   
                   
                   
                 0.08-0.30, OR = 0.16) 
               
               
                 Burkholderiales 
                 21 
                 15 
                 12 
                 2 
                 Yes 
                 P &lt; 0.00001(CI; 
               
               
                   
                   
                   
                   
                   
                   
                 0.11-0.50, OR = 0.24) 
               
               
                 Bacteroidales 
                 36 
                 0 
                 14 
                 0 
                 No 
                 p-value 1 
               
               
                 Erysipelotrichales 
                 33 
                 3 
                 14 
                 0 
                 Yes 
                 0.003(CI; 0.00-0.48, 
               
               
                   
                   
                   
                   
                   
                   
                 OR = 0) 
               
               
                 Enterobacterales 
                 21 
                 15 
                 6 
                 8 
                 Yes 
                 p-value 0.03(CI; 
               
               
                   
                   
                   
                   
                   
                   
                 01.04-3.47, OR = 3.90) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
             
               
                   
                 TABLE 47 
               
             
            
               
                   
                   
               
               
                   
                   
                 Fisher exact test value 
               
               
                   
                 Statistical 
                 with 95% Confidence 
               
            
           
           
               
               
               
               
               
            
               
                 Classification 
                 Covid + Patients 
                 Covid − Patients 
                 Significance 
                 Intervals(CI) and odds 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Family 
                 Presence 
                 Absence 
                 Presence 
                 Absence 
                 (P &lt; 0.05) 
                 ratio(OR) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Bifidobacteriacae 
                 20 
                 16 
                 14 
                 0 
                 Yes 
                 P &lt; 0.00001(CI; 
               
               
                   
                   
                   
                   
                   
                   
                 0.00-0.05, OR = 0) 
               
               
                 Clostridiacae 
                 35 
                 1 
                 14 
                 0 
                 No 
                 p-value 0.24(CI; 
               
               
                   
                   
                   
                   
                   
                   
                 0.0-2.4, OR = 0) 
               
               
                 Veilloneacellae 
                 15 
                 22 
                 11 
                 3 
                 Yes 
                 P &lt; 0.00001(CI; 
               
               
                   
                   
                   
                   
                   
                   
                 0.05-0.25, OR = 0.12) 
               
               
                 Ruminococcacea 
                 36 
                 0 
                 14 
                 0 
                 No 
                 p-value 1 
               
               
                 Sutterellacae 
                 19 
                 17 
                 11 
                 3 
                 Yes 
                 p-value 0.0003(CI; 
               
               
                   
                   
                   
                   
                   
                   
                 0.16-20.61, OR = 0.32) 
               
               
                 Faecalibacterium 
                 31 
                 5 
                 14 
                 0 
                 Yes 
                 p-value 0.00007(CI; 
               
               
                   
                   
                   
                   
                   
                   
                 0.00-0.27, OR = 0) 
               
               
                 Prevotellaceae 
                 17 
                 19 
                 10 
                 4 
                 Yes 
                 p-value 0.0009(CI; 
               
               
                   
                   
                   
                   
                   
                   
                 0.19-0.67, OR = 0.36) 
               
               
                 Erysipelotrichaceae 
                 33 
                 3 
                 14 
                 0 
                 Yes 
                 p-value 0.006(CI; 
               
               
                   
                   
                   
                   
                   
                   
                 0.00-0.57, OR = 0) 
               
               
                 Entero-bacteriaceae 
                 21 
                 15 
                 8 
                 6 
                 No 
                 p-value 1 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
             
               
                   
                 TABLE 48 
               
             
            
               
                   
                   
               
               
                   
                 Statistical 
                   
               
            
           
           
               
               
               
               
               
            
               
                 Classification 
                 Covid + Patients 
                 Covid − Patients 
                 Significance 
                 Fisher exact test 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Genus 
                 Presence 
                 Absence 
                 Presence 
                 Absence 
                 (P &lt; 0.05) 
                 value 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 
                   Bifidobacterium 
                 
                 20 
                 16 
                 14 
                 0 
                 Yes 
                 P &lt; 0.00001(CI; 
               
               
                   
                   
                   
                   
                   
                   
                 0.00-0.05, OR = 0) 
               
               
                 
                   Clostridium 
                 
                 33 
                 3 
                 14 
                 0 
                 Yes 
                 p-value 0.006(CI: 
               
               
                   
                   
                   
                   
                   
                   
                 0.00-0.57, OR = 0) 
               
               
                 
                   Veillonella 
                 
                 14 
                 22 
                 11 
                 3 
                 Yes 
                 P &lt; 0.00001(CI: 
               
               
                   
                   
                   
                   
                   
                   
                 0.08-0.30 0R = 0.16) 
               
               
                 
                   Ruminococcus 
                 
                 30 
                 6 
                 14 
                 0 
                 Yes 
                 p-value 0.00007(CI; 
               
               
                   
                   
                   
                   
                   
                   
                 0.00-0.21, OR = 0) 
               
               
                 
                   Sutterella 
                 
                 7 
                 29 
                 12 
                 2 
                 Yes 
                 P &lt; 0.00001(CI; 
               
               
                   
                   
                   
                   
                   
                   
                 0.01-0.08 OR = 0.03) 
               
               
                 
                   Faecalibacterium 
                 
                 31 
                 5 
                 14 
                 0 
                 Yes 
                 p-value 0.00007(CI; 
               
               
                   
                   
                   
                   
                   
                   
                 0.00-0.21, OR = 0) 
               
               
                 
                   Prevotella 
                 
                 10 
                 26 
                 3 
                 11 
                 No 
                 p-value 0.40(CI; 
               
               
                   
                   
                   
                   
                   
                   
                 0.68-2.83, OR = 1.38) 
               
               
                 
                   Erysiplatoclostridium 
                 
                 26 
                 10 
                 12 
                 2 
                 Yes 
                 p-value 0.03(CI; 
               
               
                   
                   
                   
                   
                   
                   
                 0.20-0.96, OR = 0.45) 
               
               
                 
                   Escherichia 
                 
                 21 
                 15 
                 7 
                 7 
                 No 
                 p-value 0.32(CI; 
               
               
                   
                   
                   
                   
                   
                   
                 0.76-2.5, OR = 1.37) 
               
               
                 
                   Klebsiella 
                 
                 3 
                 33 
                 1 
                 13 
                 No 
                 p-value 1 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
             
               
                   
                 TABLE 49 
               
             
            
               
                   
                   
               
               
                   
                 Statistical 
                   
               
            
           
           
               
               
               
               
               
            
               
                 Classification 
                 Covid + Patients 
                 Covid − Patients 
                 Significance 
                 Fisher exact test 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Species 
                 Presence 
                 Absence 
                 Presence 
                 Absence 
                 (P &lt; 0.05) 
                 value 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 
                   Bifidobacterium 
                 
                 20 
                 16 
                 14 
                 0 
                 Yes 
                 P &lt; 0.00001(CI; 
               
               
                   
                   
                   
                   
                   
                   
                 0.00-0.05, OR = 0) 
               
               
                 
                   Clostridium 
                 
                 33 
                 3 
                 14 
                 0 
                 Yes 
                 p-value 0.006(CI; 
               
               
                   
                   
                   
                   
                   
                   
                 0.00-0.57. OR = 0) 
               
               
                 
                   Veillonella 
                 
                 14 
                 22 
                 11 
                 3 
                 Yes 
                 P &lt; 0.00001(CI; 
               
               
                   
                   
                   
                   
                   
                   
                 0.08-0.30 0R = 0.16) 
               
               
                 
                   Ruminococcus 
                 
                 30 
                 6 
                 14 
                 0 
                 Yes 
                 p-value 0.00007(CI; 
               
               
                   
                   
                   
                   
                   
                   
                 0.00-0.21, OR = 0) 
               
               
                 
                   Sutterella 
                 
                 7 
                 29 
                 12 
                 2 
                 Yes 
                 P &lt; 0.00001(CI; 
               
               
                   
                   
                   
                   
                   
                   
                 0.01-0.08 OR = 0.03) 
               
               
                 
                   Prevotella 
                 
                 10 
                 26 
                 3 
                 11 
                 No 
                 p-value 0.40(CI; 
               
               
                   
                   
                   
                   
                   
                   
                 0.68-2.83, OR = 1.38) 
               
               
                 
                   Erysiplatoclostridium 
                 
                 26 
                 10 
                 12 
                 2 
                 Yes 
                 p-value 0.03(CI; 
               
               
                   
                   
                   
                   
                   
                   
                 0.20-0.96, OR = 0.45) 
               
               
                 
                   Escherichia coli 
                 
                 21 
                 15 
                 7 
                 7 
                 No 
                 p-value 0.32(CI; 
               
               
                   
                   
                   
                   
                   
                   
                 0.76-2.5, OR = 1.37) 
               
               
                 
                   Klebsiella 
                 
                 3 
                 33 
                 1 
                 13 
                 No 
                 p-value 1 
               
               
                   
               
            
           
         
       
     
     According to Tables 44-49, in the 50 patients tested in this study, the relative abundance of Actinobacteria P&lt;0.00001 (CI; 0.00-0.3, OR=0) and Proteobacteria bacteria phyla p-value 0.003 (CI; 0.00-0.48, OR=0) were significantly less than HC. Although there was not any significant dysbiosis in both Bacteroidetes and Firmicutes phyla. At the class level, there was a significant reduction in Actinobacteria P&lt;0.00001 (CI; 0.00-0.07, OR=0), Negativicutes P&lt;0.00001 (CI; 0.00-0.21, OR=0), Betaproteobacteria P&lt;0.00001 (CI; 0.10-0.46, OR=0.22) and Erysipelotrichia p-value 0.009 (CI; 01.27-8.77, OR=3.19). 
     At the order level, there were significant reductions in more bacteria, including, Bifidobacteriales P&lt;0.00001 (CI; 0.00-0.05, OR=0), Veillonellales P&lt;0.00001 (CI; 0.08-0.30, OR=0.16), Burkholderiales P&lt;0.00001 (CI; 0.11-0.50, OR=0.24), and Erysipelotrichales 0.003 (CI; 0.00-0.48, OR=0) and significant increase in Enterobacterales p-value 0.03 (CI; 01.04-3.47, OR=3.90) compare to the HC. 
     At the family levels, Bifidobacteriacae P&lt;0.00001 (CI; 0.00-0.05, OR=0), Veilloneacellae P&lt;0.00001 (CI; 0.05-0.25, OR=0.12), Sutterellacae p-value 0.0003 (CI; 0.16-20.61, OR=0.32),  Faecalibacterium  p-value 0.00007 (CI; 0.00-0.27, OR=0), Prevotellaceae p-value 0.0009 (CI; 0.19- 0.67, OR=0.36) and Erysipelotrichaceae p-value 0.006 (CI; 0.00-0.57, OR=0) showed a significant reduction in Covid-19 patients compare to the HC. 
     At the genus level, significant reductions in relative abundances of  Bifidobacterium  P&lt;0.00001 (CI; 0.00-0.05, OR=0),  Clostridium  p-value 0.006 (CI; 0.00-0.57, OR=0),  Veillonella  P&lt;0.00001 (CI; 0.08-0.30 OR=0.16),  Ruminococcus  p-value 0.00007 (CI; 0.00-0.21, OR=0),  Sutterella  P&lt;0.00001 (CI; 0.01-0.08 OR=0.03),  Faecalibacterium  p-value 0.00007 (CI; 0.00-0.21, OR=0) and  Erysiplatoclostridium  p-value 0.03 (CI; 0.20-0.96, OR=0.45) and  Veillonella  P&lt;0.00001 (CI; 0.08-0.30 OR=0.16), were observed. 
     Findings 
     Significant alterations of the fecal microbiota was observed, in some cases, for the first time, with special baseline characteristics in Covid-19 positive patients. Except for a significantly higher relative abundance of Enterobacterales at order level in Covid-19 positive patients, there was a significant relative abundance reduction in  Bifidobacterium, Clostridium, Veillonella, Ruminococcus, Sutterella, Faecalibacterium, Erysiplatoclostridium,  and  Veillonella  at the species and genus levels, Bifidobacteriacae, Veilloneacellae, Sutterellacae, Prevotellaceae and Erysipelotrichaceae at the family level, Bifidobacteriales, Veillonellales, Burkholderiales, and Erysipelotrichales at the order level, Actinobacteria, Negativicutes, Betaproteobacteria, and Erysipelotrichia class level and more importantly, Proteobacteria and Actinobacteria at the phylum level. 
     There were significant reductions in species, genus, family, order, class, and phylum level between Covid-19 positive patients and the control population. Dysbiosis was largest for Actinobacteria and Proteobacteria, at the phylum level, in the Covid-19 positive populations and must be addressed accordingly. The only significant abundance noted was at Enterobacterales order. At the class level, a significant reduction in Actinobacteria, Negativicutes &amp; Betaproteobacteria was observed. At the order level, significant reductions in more bacteria, including, Bifidobacteriales, Veillonellales, Burkholderiales, and Erysipelotrichales, were observed. At the family levels, Bifidobacteriacae, Veilloneacellae, Sutterellacae,  Faecalibacterium,  Erysipelotrichales and Prevotellaceae showed a significant reduction in Covid-19 patients compared to the HC. At the genus level, significant reductions in relative abundances of  Bifidobacterium, Clostridium, Veillonella, Ruminococcus, Sutterella, Faecalibacterium,  and  Erysiplatoclostridium  were observed. 
     Interpretation 
     Covid -19 positive patients will have a considerable reduction in microbiota composition and baseline microbiome dysbiosis, levels of cytokines, and inflammatory markers. Addressing the effects of microbial diversity and consequently, suppression of the immunologic response (known as the cytokine storm) is key to prevent and treat the disease. The importance of reduction at the phylum level must be considered as a possibility of an important indicator for Covid-19 positive susceptible patients. Application of diet-based improvement of microbiome would also be considered as an important preventive measure. 
     Example 9:  Bifidobacterium  and  Faecalibacterium  Depletion, and Other Bacterial Composition Changes, as Potential Susceptibility Markers of SARS-Cov2 Severity 
     Objective: To compare gut microbiome diversity and composition in SARS-CoV-2 polymerase chain reaction (PCR)-confirmed positive patients whose symptoms ranged from severe to asymptomatic, and PCR-negative exposed healthy controls. 
     Summary of Design: Using a cross-sectional study design, shotgun next-generation sequencing (NGS) was sued to evaluate microbiome composition and diversity in patients with PCR-confirmed SARS-CoV-2 infections from March 2020 through January 2021 and SARS-CoV-2 PCR-negative exposed healthy controls. Patients were classified as being asymptomatic or having mild, moderate, or severe symptoms based on NIH criteria. Exposed healthy controls were individuals with prolonged or repeated close contact to patients with SARS-CoV-2 infection or their samples and consisted of family members of subjects or frontline healthcare worker. Microbiome diversity and composition were compared between patients and exposed-controls and across patient subgroups at all taxonomic levels. 
     Results: 52 patients and 20 controls were identified. Compared with controls, patients had significantly less bacterial diversity (p&lt;0.05) and a lower relative abundance of  Bifidobacterium, Faecalibacterium  and other bacteria at the genus level. Additionally, there was an inverse association between disease severity and  Bifidobacterium  (P=0.001) relative abundance and bacterial diversity. 
     Conclusion: Depletion of  Bifidobacteria  spp. And  Faecalibacterium  either before or after infection minimized its immune-dampening effects and allowed SARS-Cov-2 infection to become symptomatic. Thus, low bacterial diversity and low Bifidobacteria abundance is a susceptibility signature for vulnerability to symptoms associated with SARS-CoV-2 infection. 
     Patients with SARS-CoV-2 infection possess significantly less bacterial diversity and lower abundance of  Bifidobacteria  and  Faecalibacterium  and increased abundance of Baceriodies at the genus level compared with exposed-healthy controls. There is an inverse association between disease severity and abundance of  Bifidobacteria  and  Faecalibacterium  and direct association of severity and Bacteriodes abundance. 
     Additional Detail Regarding Study Design 
     Patients diagnosed with COVID-19 were recruited to the study following expression of interest or physician referral. Informed consent was obtained. 
     Patients aged ≥18 years of age, with RT-PCR-confirmed SARS-CoV-2 infection within one week of screening were eligible for enrolment. SARS-CoV-2 negative but exposed healthy controls included patients who were PCR-negative for SARS-CoV-2 and remained antibody-negative for between 3-6 months and asymptomatic for 6 months-1 year. SARS-CoV-2 negative exposed healthy controls were further required to meet the following criteria of they either shared a household with at least one symptomatic SARS-CoV-2-positive family member (e.g., sibling, parent, or child) or were a healthcare worker who had been repeatedly directly exposed to symptomatic SARS-CoV-2-positive patients for a minimum of six months or were exposed to numerous SARS-CoV-2-positive patient samples. Also, exposed healthy controls were those that, despite exposure to SARS-CoV-2, chose not to quarantine or take prophylaxis or treatment for COVID-19. 
     Patients undergoing treatment with total parenteral nutrition, or those with a history of significant gastrointestinal surgery (e.g. bariatric surgery, total colectomy with ileorectal anastomosis, proctocolectomy, postoperative stoma, ostomy, or ileoanal pouch) were excluded. 
     The study was conducted in accordance with ethical principles of the Declaration of Helsinki, the International Council for Harmonisation (ICH) Harmonised Tripartite Guideline for Good Clinical Practice (GCP), and the E and I Institutional Review Board (IRB) regulations. The clinical study protocol, a sample informed consent form, and other study-related documents were reviewed and approved by the Ethical and Independent Review. All patients provided written informed consent to participate in the study. 
     Symptomatic patients, as well as asymptomatic household contacts, whose SARS-CoV-2 infectivity was assessed by RT-PCR and exposed healthy controls were required to collect a stool sample. Patients were to collect one mL of fresh stool and place it directly in a Zymo Research DNA/RNA Shield fecal collection tube. Following fecal collection, individual subject DNA was extracted and purified with the Qiagen PowerFecal Pro DNA extraction kit. The isolated DNA was then quantitated utilizing the Quantus Fluorometer with the QuantFluor ONE dsDNA kit. After DNA quantification, the DNA was normalized, and libraries were prepared utilizing shotgun methodology with Illumina&#39;s Nextera Flex kit. Per the lab shotgun metagenomic processes, samples underwent tagmentation, amplification, indexing, and purification. After completing the NextSeq run, the raw data were streamed in real-time to Illumina&#39;s BaseSpace cloud for conversion to FASTQ files. The FASTQ data were compared to evaluate microbiome diversity in patients with PCR-confirmed SARS-CoV-2 infections and SARS-CoV-2 PCR-negative individuals. The COVID-19 patients had sought remote care in 2020 and 2021. The microbiome differences between the SARS-CoV-2 patients and SARS-CoV-2 exposed healthy controls were compared at all taxonomic levels. 
     The differences in relative abundance across taxa between the gut microbiome of SARS-CoV-2 infected patients and exposed healthy controls were assessed with One Codex&#39;s bioinformatics analysis pipeline using Jupyter notebook in Python. To evaluate the statistical significance of any variability noted between patients and exposed healthy controls at each taxonomic classification, ANOVA and t-test statistics were conducted by R version 3.6.1 (2019 Jul. 5), GraphPad, and SigmaPlot 12.0. Confidence intervals, and p-values were calculated for all comparisons in the statistical analysis, with P-values &lt;0.05 considered significant. Shannon and Simpson indices were calculated by the bioinformatics software One Codex by Python based notebook software. 
     Patient Characteristics 
     Demographic and clinical characteristics of patients (n=52) and exposed healthy controls (n=20) are presented in Table 19. Half (50%) of patients and 36% of exposed healthy controls were male. The median (SEM) age of patients was 53±2.54 years and of exposed healthy controls was 48±3.62 years. A total of 85% of patients were non-Hispanic White; 12.5% were Hispanic; and 2.5%, Black and 91% of exposed healthy controls were non-Hispanic White and 9% were Black. Nearly two-thirds of patients (62.5%) had underlying comorbidities considered risk factors for increased morbidity and mortality by CDC.1 Of the SARS-CoV-2 positive individuals, 57.7% had severe disease, 23.1% had moderate disease, and 11.5% had mild disease. The remaining 7.7% were asymptomatic. More than half (54.5%) of exposed controls had underlying comorbidities. The median (SEM) BMI of the 52 patients for whom data were available was 27.9+0.94 compared with 25.1+0.96 for the 20 exposed healthy controls. 
     Of the healthy exposed subjects, 16 were family members of one of the COVID-19 positive subjects, and 2 were Health Care workers with extensive, non-protected, exposure to subjects and 2 were laboratory personnel exposed to thousands of COVID-19 samples. Given the low number of health care worker subjects, statistics were not performed comparing similarity to family member groups. None of the patients or controls were vaccinated or on COVID-19 prophylaxis or treatment. 
     
       
         
           
               
               
               
               
               
               
               
               
               
             
               
                 TABLE 50 
               
               
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                 PCR 
                   
               
               
                 No 
                 Age 
                 Sex 
                 Ethnicity 
                 State 
                 Risk Factors 
                 BMI* 
                 Result 
                 Category 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 1 
                 21 
                 F 
                 W 
                 PA 
                 none 
                 24.8 
                 positive 
                 severe 
               
               
                 2 
                 71 
                 M 
                 H 
                 CA 
                 none 
                 18.4 
                 positive 
                 severe 
               
               
                 3 
                 86 
                 M 
                 W 
                 CA 
                 Alzheimer&#39;s 
                 21.1 
                 positive 
                 severe 
               
               
                 4 
                 18 
                 F 
                 W 
                 AZ 
                 none 
                 22 
                 positive 
                 severe 
               
               
                 5 
                 31 
                 M 
                 W 
                 CO 
                 overweight 
                 26.9 
                 positive 
                 severe 
               
               
                 6 
                 35 
                 M 
                 W 
                 CA 
                 obese 
                 30.7 
                 positive 
                 severe 
               
               
                 7 
                 43 
                 F 
                 W 
                 TX 
                 overweight 
                 25.8 
                 positive 
                 severe 
               
               
                 8 
                 70 
                 F 
                 W 
                 LO 
                 overweight 
                 25.8 
                 positive 
                 severe 
               
               
                 9 
                 56 
                 M 
                 W 
                 CA 
                 overweight 
                 27.3 
                 positive 
                 severe 
               
               
                 10 
                 44 
                 F 
                 W 
                 GA 
                 overweight 
                 27.8 
                 positive 
                 severe 
               
               
                 11 
                 53 
                 M 
                 W 
                 GA 
                 obese 
                 30.7 
                 positive 
                 severe 
               
               
                 12 
                 37 
                 M 
                 W 
                 FL 
                 hypertension 
                 23.8 
                 positive 
                 mild 
               
               
                 13 
                 60 
                 M 
                 W 
                 CO 
                 none 
                 22.7 
                 positive 
                 severe 
               
               
                 14 
                 69 
                 F 
                 W 
                 CO 
                 hypertension 
                 22.9 
                 positive 
                 severe 
               
               
                 15 
                 55 
                 M 
                 W 
                 NY 
                 obesity, HIV 
                 37.2 
                 positive 
                 severe 
               
               
                 16 
                 67 
                 F 
                 W 
                 IL 
                 hypertension, 
                 48.5 
                 positive 
                 severe 
               
               
                   
                   
                   
                   
                   
                 diabetes 
               
               
                   
                   
                   
                   
                   
                 mellitus, 
               
               
                   
                   
                   
                   
                   
                 obesity, 
               
               
                   
                   
                   
                   
                   
                 cerebral 
               
               
                   
                   
                   
                   
                   
                 aneurysm 
               
               
                 17 
                 66 
                 M 
                 W 
                 IL 
                 hypertension, 
                 33.2 
                 positive 
                 severe 
               
               
                   
                   
                   
                   
                   
                 obesity, 
               
               
                   
                   
                   
                   
                   
                 pulmonary 
               
               
                   
                   
                   
                   
                   
                 embolus 
               
               
                 18 
                 49 
                 F 
                 W 
                 AL 
                 overweight 
                 27.4 
                 positive 
                 severe 
               
               
                 19 
                 60 
                 M 
                 W 
                 CA 
                 hypertension, 
                 26.2 
                 positive 
                 severe 
               
               
                   
                   
                   
                   
                   
                 overweight 
               
               
                 20 
                 63 
                 F 
                 W 
                 GA 
                 hypertension, 
                 28.1 
                 positive 
                 severe 
               
               
                   
                   
                   
                   
                   
                 overweight 
               
               
                 21 
                 59 
                 F 
                 W 
                 GA 
                 hypertension 
                 NA 
                 positive 
                 severe 
               
               
                 22 
                 53 
                 F 
                 W 
                 CA 
                 none 
                 24.5 
                 positive 
                 severe 
               
               
                 23 
                 36 
                 M 
                 W 
                 OR 
                 obese 
                 30.7 
                 positive 
                 severe 
               
               
                 24 
                 10 
                 M 
                 W 
                 HI 
                 none 
                 14.5 
                 positive 
                 severe 
               
               
                 25 
                 52 
                 M 
                 W 
                 HI 
                 none 
                 24.1 
                 positive 
                 severe 
               
               
                 26 
                 55 
                 F 
                 W 
                 HI 
                 hypertension, 
                 25 
                 positive 
                 severe 
               
               
                   
                   
                   
                   
                   
                 overweight 
               
               
                 27 
                 61 
                 F 
                 W 
                 WA 
                 none 
                 23.8 
                 positive 
                 severe 
               
               
                 28 
                 72 
                 F 
                 W 
                 CA 
                 none 
                 19.5 
                 positive 
                 severe 
               
               
                 29 
                 62 
                 M 
                 W 
                 TN 
                 obese, 
                 31.5 
                 positive 
                 severe 
               
               
                   
                   
                   
                   
                   
                 hypertension 
               
               
                 30 
                 18 
                 M 
                 W 
                 AZ 
                 Crohn&#39;s 
                 15.1 
                 positive 
                 severe 
               
               
                 31 
                 23 
                 F 
                 W 
                 CA 
                 obesity 
                 30.9 
                 positive 
                 moderate 
               
               
                 32 
                 25 
                 F 
                 W 
                 CA 
                 obesity, asthma 
                 30.6 
                 positive 
                 moderate 
               
               
                 33 
                 24 
                 F 
                 W 
                 AZ 
                 asthma 
                 NA 
                 positive 
                 moderate 
               
               
                 34 
                 36 
                 F 
                 H 
                 AZ 
                 hypertension, 
                 25.4 
                 positive 
                 moderate 
               
               
                   
                   
                   
                   
                   
                 overweight 
               
               
                 35 
                 33 
                 M 
                 H 
                 CA 
                 none 
                 18.4 
                 positive 
                 moderate 
               
               
                 36 
                 63 
                 F 
                 H 
                 CA 
                 overweight 
                 NA 
                 positive 
                 moderate 
               
               
                 37 
                 65 
                 M 
                 W 
                 TX 
                 COPD 
                 NA 
                 positive 
                 moderate 
               
               
                 38 
                 75 
                 M 
                 W 
                 CA 
                 hypertension, 
                 48.5 
                 positive 
                 moderate 
               
               
                   
                   
                   
                   
                   
                 severe obesity 
               
               
                 39 
                 67 
                 M 
                 W 
                 CA 
                 obesity 
                 33.1 
                 positive 
                 moderate 
               
               
                 40 
                 70 
                 M 
                 W 
                 GA 
                 obesity 
                 30.7 
                 positive 
                 moderate 
               
               
                 41 
                 57 
                 F 
                 W 
                 TX 
                 obesity 
                 32 
                 positive 
                 moderate 
               
               
                 42 
                 69 
                 F 
                 W 
                 NE 
                 hypertension 
                 20.9 
                 positive 
                 moderate 
               
               
                 43 
                 19 
                 M 
                 W 
                 GA 
                 none 
                 23.5 
                 positive 
                 mild 
               
               
                 44 
                 36 
                 F 
                 W 
                 NY 
                 none 
                 18.6 
                 positive 
                 mi 
               
               
                 45 
                 35 
                 F 
                 W 
                 CA 
                 overweight 
                 28.5 
                 positive 
                 mild 
               
               
                 46 
                 53 
                 M 
                 W 
                 NE 
                 overweight 
                 29 
                 positive 
                 mild 
               
               
                 47 
                 38 
                 F 
                 1 
                 TN 
                 obese 
                 31.3 
                 positive 
                 mild 
               
               
                 48 
                 45 
                 F 
                 W 
                 PA 
                 hypertension 
                 23 
                 positive 
                 mild 
               
               
                 49 
                 22 
                 M 
                 W 
                 GA 
                 overweight 
                 25.8 
                 positive 
                 asymptomatic 
               
               
                 50 
                 48 
                 F 
                 H 
                 CA 
                 none 
                 20.4 
                 positive 
                 asymptomatic 
               
               
                 51 
                 67 
                 M 
                 W 
                 WI 
                 overweight 
                 28.5 
                 positive 
                 asymptomatic 
               
               
                 52 
                 53 
                 M 
                 W 
                 IL 
                 obese, 
                 35.3 
                 positive 
                 asymptomatic 
               
               
                   
                   
                   
                   
                   
                 diabetes 
               
               
                 53 
                 68 
                 F 
                 W 
                 WI 
                 overweight 
                 26.6 
                 negative 
                 n/a 
               
               
                 54 
                 58 
                 F 
                 H 
                 PA 
                 obesity, 
                 30 
                 negative 
                 n/a 
               
               
                   
                   
                   
                   
                   
                 exposed to + 
               
               
                   
                   
                   
                   
                   
                 daughter 
               
               
                 55 
                 51 
                 F 
                 W 
                 CA 
                 hypertension, 
                 25 
                 negative 
                 n/a 
               
               
                   
                   
                   
                   
                   
                 prediabetes, 
               
               
                   
                   
                   
                   
                   
                 exposed to + 
               
               
                   
                   
                   
                   
                   
                 son, + 
               
               
                   
                   
                   
                   
                   
                 husband, and + 
               
               
                   
                   
                   
                   
                   
                 daughter 
               
               
                 56 
                 63 
                 M 
                 W 
                 CA 
                 overweight 
                 26.4 
                 negative 
                 n/a 
               
               
                   
                   
                   
                   
                   
                 exposed to + 
               
               
                   
                   
                   
                   
                   
                 mother 
               
               
                 57 
                 29 
                 M 
                 W 
                 CA 
                 overweight: 
                 26.4 
                 negative 
                 n/a 
               
               
                   
                   
                   
                   
                   
                 exposed to + 
               
               
                   
                   
                   
                   
                   
                 brother 
               
               
                 58 
                 23 
                 F 
                 W 
                 CA 
                 overweight: 
                 26.4 
                 negative 
                 n/a 
               
               
                   
                   
                   
                   
                   
                 exposed to + 
               
               
                   
                   
                   
                   
                   
                 brother 
               
               
                 59 
                 62 
                 F 
                 W 
                 CA 
                 none:exposed 
                 17.4 
                 negative 
                 n/a 
               
               
                   
                   
                   
                   
                   
                 to (kissed) + 
               
               
                   
                   
                   
                   
                   
                 boyfriend 
               
               
                 60 
                 51 
                 F 
                 W 
                 CA 
                 none:exposed 
                 19.9 
                 negative 
                 n/a 
               
               
                   
                   
                   
                   
                   
                 to + son, + 
               
               
                   
                   
                   
                   
                   
                 husband, and + 
               
               
                   
                   
                   
                   
                   
                 daughter 
               
               
                 61 
                 16 
                 F 
                 W 
                 CA 
                 none:exposed 
                 21.2 
                 negative 
                 n/a 
               
               
                   
                   
                   
                   
                   
                 to + mom 
               
               
                 62 
                 26 
                 M 
                 B 
                 CA 
                 none:exposed 
                 19.5 
                 negative 
                 n/a 
               
               
                   
                   
                   
                   
                   
                 to + patients 
               
               
                 63 
                 56 
                 F 
                 W 
                 CA 
                 overweight: 
                 25.5 
                 negative 
                 n/a 
               
               
                   
                   
                   
                   
                   
                 exposed to + 
               
               
                   
                   
                   
                   
                   
                 patient 
               
               
                   
                   
                   
                   
                   
                 samples 
               
               
                 64 
                 53 
                 F 
                 W 
                 CA 
                 none:exposed 
                 24 
                 negative 
                 n/a 
               
               
                   
                   
                   
                   
                   
                 to workers, and + 
               
               
                   
                   
                   
                   
                   
                 patients and + 
               
               
                   
                   
                   
                   
                   
                 patient 
               
               
                   
                   
                   
                   
                   
                 samples 
               
               
                 65 
                 35 
                 M 
                 W 
                 KS 
                 none:exposed 
                 23.7 
                 negative 
                 n/a 
               
               
                   
                   
                   
                   
                   
                 to + wife 
               
               
                 66 
                 35 
                 M 
                 W 
                 CA 
                 none:exposed 
                 21.7 
                 negative 
                 n/a 
               
               
                   
                   
                   
                   
                   
                 to + roommate 
               
               
                 67 
                 64 
                 F 
                 H 
                 TN 
                 prediabetic, 
                 26 
                 negative 
                 n/a 
               
               
                   
                   
                   
                   
                   
                 overweight 
               
               
                 68 
                 43 
                 F 
                 W 
                 CA 
                 overweight, 
                 26.6 
                 negative 
                 n/a 
               
               
                   
                   
                   
                   
                   
                 exposed to + 
               
               
                   
                   
                   
                   
                   
                 patient 
               
               
                   
                   
                   
                   
                   
                 samples 
               
               
                 69 
                 45 
                 F 
                 W 
                 CA 
                 obese, exposed 
                 35.1 
                 negative 
                 n/a 
               
               
                   
                   
                   
                   
                   
                 to + patients 
               
               
                 70 
                 44 
                 M 
                 W 
                 CA 
                 overweight, 
                 27.2 
                 negative 
                 n/a 
               
               
                   
                   
                   
                   
                   
                 exposed to + 
               
               
                   
                   
                   
                   
                   
                 patients and + 
               
               
                   
                   
                   
                   
                   
                 patient 
               
               
                   
                   
                   
                   
                   
                 samples 
               
               
                 71 
                 52 
                 M 
                 W 
                 CA 
                 overweight, 
                 27.1 
                 negative 
                 n/a 
               
               
                   
                   
                   
                   
                   
                 exposed to + 
               
               
                   
                   
                   
                   
                   
                 patients 
               
               
                 72 
                 15 
                 F 
                 W 
                 AZ 
                 none: exposed 
                 16.9 
                 negative 
                 n/a 
               
               
                   
                   
                   
                   
                   
                 to + brother 
               
               
                   
               
               
                 *BMI, body mass index. 
               
            
           
         
       
     
     Gut Microbiome Diversity and Composition 
     Alpha diversity in patients and exposed healthy controls are shown in  FIG. 4A  (Difference in Shannon Index for genus and family levels [95% CI 0.11 to 1.67, P=0.026]) and  FIG. 4B  (Difference in Simpson Index [95% CI 0.0031 to 0.18, P=0.043]) suggesting that the gut microbiome of patients was less diverse than that of exposed healthy controls. 
     Microbiome analysis revealed significant differences between the two groups. Table 51 lists the bacteria that had significant or otherwise different abundance of bacteria, either comparing difference due to COVID-19 severity (center column) among COVID-19 positive subjects or difference due to COVID-19 positivity (right column) among all subjects. Patients with PCR-confirmed SARS-CoV-2 infection showed a significantly decreased abundance of  Bifidobacterium  spp.,  Clostridium, Faecali  NR,  Faecalibacterium prausnitzii, Ruminococcus,  and  Subdolingranulum  and increased abundance of Bacteriodes. (Table 51 right column). Table 51 left column shows the abundance of bacteria was affected by patient&#39;s level of symptom severity. A one-way ANOVA analysis revealed a significant relation between increased disease severity and decrease in abundance of  Bifidobacterium  spp.,  Faecali  NR,  Faecalibacterium prausnitzii, Ruminococcus,  and  Subdolingranulum  and increase in abundance of Bacteriodes and Dorea. 
     
       
         
           
               
               
               
             
               
                   
                 TABLE 51 
               
               
                   
                   
               
               
                   
                 P-value 
                 P-value 
               
               
                   
                 Does severity of 
                 Does COVID-19 
               
               
                   
                 COVID-19 affect 
                 positivity affect 
               
               
                   
                 bacterial 
                 bacterial 
               
               
                   
                 abundance? 
                 abundance? 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 
                   Akkermansia 
                 
                 0.0790 
                 0.6933 
               
               
                 Alistpies 
                 0.7860 
                 0.5922 
               
               
                 
                   Bacteroides 
                 
                     0.0380 ↑ 
                     0.0023 ↑ 
               
               
                 Bifido 
                     0.0010 ↓ 
                     0.0001 ↓ 
               
               
                 Blautia 
                 0.0800 
                 0.2859 
               
               
                 
                   Clostridium 
                 
                 0.8420 
                     0.0326 ↓ 
               
               
                 
                   Collinsella 
                 
                 0.8080 
                 0.9551 
               
               
                 
                   Coprococcus 
                 
                 0.2340 
                 0.0607 
               
               
                 
                   Dorea 
                 
                     0.0380 ↑ 
                 0.3731 
               
               
                 
                   Eubacterium 
                 
                 0.7580 
                 0.1825 
               
               
                 
                   Faecalibacterium 
                 
                     0.0100 ↓ 
                     0.0001 ↓ 
               
               
                 
                   Faecalibacterium prausnitzii 
                 
                     0.0070 ↓ 
                     0.0060 ↓ 
               
               
                 
                   Klebsiella 
                 
                 0.8780 
                 0.6280 
               
               
                 
                   Oscillobacter 
                 
                 0.8830 
                 0.6365 
               
               
                 
                   Parabacteroides 
                 
                 0.4070 
                 0.4734 
               
               
                 
                   Prevotella 
                 
                 0.6770 
                 0.9510 
               
               
                 
                   Roseburia 
                 
                 0.4030 
                 0.1557 
               
               
                 
                   Ruminococcus 
                 
                 0.0900 
                     0.0415 ↓ 
               
               
                 
                   Subdoligranulum 
                 
                 0.8970 
                     0.0058 ↓ 
               
               
                   
               
            
           
         
       
     
       FIG. 5  shows the relative abundance of Bifidobacteria for each subject, grouped by COVID-19 severity. Analyzed via one-way ANOVA with Dunnett&#39;s post-hoc (comparing all COVID-19 severities including asymptomatic to healthy controls), there was a significant (P=0.0002) association between severity and Bifidobacteria relative abundance, particularly for severe (P&lt;0.0001) and moderate severity (P=0.0031). 
     Discussion 
     Immune function and health could be enhanced by bacterial abundance. Interactions between the host and gut microbiota are complex, numerous, and bidirectional. Gut microbiota regulate the development and function of the innate and adaptive immune system, potentially allowing them to protect against COVID-19. The study&#39;s main findings are that COVID-19 severity and positivity correlate to decreased levels of the pro-immune  Bifidobacteria  and  Faecalibacteria  and decreased levels of bacterial diversity. This was the first one to compare exposed to SARS-CoV-2 healthy subjects with COVID-19 affected subjects. That is, the following parameters were controlled: COVID-19 exposure, and observing bacterial changes are the associated with development of COVID-19 positivity. 
     Bacterial diversity is known to inversely relate to presence of various common disorders. This study showed that bacterial diversity indices (Shannon and Simpson Index), show that higher diversity correlates (p&lt;0.05) to less COVID-19 severity, just as it reflects less vulnerability to many other disorders. 
     The genus  Bifidobacterium  has important immune functions, is a major component of the microbiome, and is frequently used in probiotics.  Bifidobacteria  are gram positive non-motile anaerobic rod-shaped bacteria.  Bifidobacteria  protect against intestinal epithelial cell damage and this protection is independent of their effects on tumor necrosis factor alpha (TNF-α) production. The exopolysaccharide coat which is a feature of some  Bifidobacteria  has been shown to play a significant role in this protective effect.  Bifidobacteria  also reduce cell damage by inhibiting TNF-α and macrophages. Also,  Bifidobacteria  increase Treg responses. These immune functions of  Bifidobacteria  could be critical for its COVID-protective effect. 
       Faecalibacterium  genus and  Faecalibacterium prausnitzii  species levels were also inversely related to COVID-19 severity in this analysis. Age and diabetes are risk factors for COVID-19, and  F. prausnitzii  levels decline markedly in elder and diabetic populations. In fact,  Faecalibacterium  is considered an “indicator” of human health. The “western” diet (consumption of more meat, animal fat, sugar, processed foods, and low fiber) reduces the level of  F. prausnitzii,  while a high-fiber (e.g. Mediterranean diet of vegetables and fruits) and low meat diet enhances the count of  F. prausnitzii.  Preliminary studies show that reduced use of this Mediterranean diet within the same country was associated with increased COVID-19 death. In conclusion, it was shown that  F. prausnitzii  levels correlate to COVID severity and prior studies show that reduced  F. prausnitzii  is associated with COVID-19 vulnerabilities including age, diabetes, obesity, and possibly diet. 
     Evidence has accumulated to support a beneficial effect from supplementation with  Bifidobacteria  in a number of disease states. The number of commensal  Bifidobacteria  has been shown to decrease with age, a major COVID-19 risk factor. This study found that there is significant relationship between disease severity and relative abundance of  Bifidobacterium  spp.; patients with a more severe course of viral infection having decreased  Bifidobacteria  levels. However, it should be noted that there are no definitive studies concerning what constitutes a normal baseline abundance of  Bifidobacteria  in a “healthy” individual. 
     Changes in other bacteria were observed, characterized, namely: genus  Bifidobacterium, Clostridium, Faecalibacterium  (especially species  F. prausnitzii ),  Subdolingranulum,  and  Ruminococcus  showed decreased in bacterial abundance due to COVID-19 positivity, while genus  Bacteroides  showed increased bacterial abundance. Similarly,  Bifidobacterium, Clostridium,  and  Faecalibacterium  showed decreased bacterial abundance with increased COVID-19 severity, while  Dorea  and  Bacteroides  showed increased abundance. 
     Innate immunity could be enhanced by increased bacterial level. COVID-19 pathology impacts include both direct effects from viral invasion and complex immunological responses including, in its most severe form, the so-called ‘cytokine storm.’ ( FIG. 6 ) The cytokine storm is based upon innate immunity which is also enhanced by  Bifidobacteria, Faecalibacteria,  and bacterial diversity. Steroid treatment has situational success in COVID-19, and it is based on controlling an overactive innate immunity. 
       FIG. 8  shows the proposed mechanism for cytokine storm and immune hyper-response in SARS-CoV-2 positive patients. In individuals infected with SARS-CoV-2, the macrophages become activated; these in turn activate T-cells, additional macrophages, and neutrophils—all of which release cytokines, including TNF-α. Bifidobacteria, when present in sufficient numbers, can bind to TNF-α and prevent the subsequent cytokine storm. Therefore, patients with bifidobacterial dysbiosis lack this line of defense which may lead to a cytokine storm. 
     The findings reported in the current study support the role of commensal  Bifidobacteria  in the severity of viral infection, suggesting the importance of microbiota in viral pathogenesis and their potential for use in treatment ( FIG. 8 ). 
     Conclusions 
     Given the cross-sectional study design, it is not possible to determine whether the differences in levels of  Bifidobacteria  observed between patients and exposed healthy controls preceded or followed infection. If the differences preceded SARS-CoV-2 infection and are a marker of susceptibility, then boosting  Bifidobacteria  levels is be expected to decrease the risk or severity of SARS-CoV-2 infection. If changes followed SARS-CoV-2 infection, then adjunctive repopulation of the gut microbiome and boosting of  Bifidobacteria  through supplements or fecal transplant can speed recovery and reduce organ damage; particularly for patients hospitalized with ‘severe’ disease (including children with SARS-CoV-2-related multisystem inflammatory syndrome) and those with persistent symptoms. 
     COVID-19 ranges in presentation from asymptomatic to fatal, and bacterial composition and diversity also demonstrate marked variation. The same changes observed due to COVID-19, namely drop in  Bifidobacterium  and/or  Faecalibacterium,  underly COVID-19 risk factors including obesity, old age, and diabetes. Thus, bacterial levels, especially for  Bifidobacterium  and/or  Faecalibacterium  may be markers that determine the response (asymptomatic, severe, fatal) to infection by the same SARS-COV2 virus. 
     In summary, in this study, findings from only COVID-exposed subjects, both healthy and COVID-19 positive, were presented, and show the level of various bacteria are significantly related to the severity of COVID-19 and SARS-CoV-2 positive patients possessed significantly less bacterial diversity. These findings suggest that  Bifidobacterium  and other probiotic supplementation or repopulation via fecal transplant may speed recovery and reduce organ damage, particularly for patients hospitalized with severe disease. The data also suggest that characterizing the microbiome may allow the ability to predict vulnerability and appropriately treat COVID-19 patients in a manner dependent on their microbiome. 
     Having thus described the invention, it should be apparent that numerous structural modifications and adaptations may be resorted to without departing from the scope and fair meaning of the instant invention as set forth herein above and described herein below by the claims. 
     While particular forms of the invention have been illustrated and described, it will also be apparent to those skilled in the art that various modifications can be made without departing from the spirit and scope of the invention. 
     Although the present invention has been described in considerable detail with reference to certain preferred embodiments, other embodiments are possible. The forgoing description is not intended to be exhaustive or to limit the invention to the precise form disclosed. Many modifications and variations are possible in light of the above teaching. The steps disclosed for the present methods, for example, are not intended to be limiting nor are they intended to indicate that each step is necessarily essential to the method, but instead are exemplary steps only. Therefore, the scope of the appended claims should not be limited to the description of preferred embodiments/methods contained in this disclosure. All references cited herein are incorporated by reference. Insofar as the description above and the accompanying drawings disclose any additional subject matter that is not within the scope of the claims below, the inventions are not dedicated to the public and the right to file one or more applications to claim such additional inventions is reserved.