Endocrine, Metabolic and Microbiome Influence on the Post-acute Sequelae SARS-CoV-2 (PASC)
Conditions: Covid19
Sponsor: The University of Texas Medical Branch, Galveston

The Endocrine, Metabolic and Microbiome Influence on the Post-COVID Syndrome 
PI: Randall Urban, MD  
 
 
 
 
 
 
 
 
3-24-22 
1 
 
 
Title:  Endocrine, Metabolic and Microbiome Influence on the Post-COVID Syndrome 
 
Principal Investigator: Randall Urban, M.D., Internal Medicine, UTMB 
 
Co-I: Melinda Sheffield-Moore, Ph.D., Internal Medicine, UTMB  
Co-I: Rick Pyles, Ph.D., Pediatrics, UTMB 
Co-I: Elena Volpi, M.D, Ph.D., Internal Medicine, UTMB 
Co-I: Matthew Dacso, M.D., Internal Medicine, UTMB 
Co-I: Mohammed Zaidan, M.D., Internal Medicine, UTMB 
Co-I: Traver Wright, Ph.D., Internal Medicine, UTMB & Health and Kinesiology, TAMU 
 
 
Institutions: The University of Texas Medical Branch at Galveston (UTMB) 
 
           Texas A&M University (TAMU) 
 
 
 
Background and Significance 
 
The onset of the COVID-19 pandemic has led to a subset of patients that, once recovered from 
the acute infection, also experience an intractable and debilitating set of lingering symptoms 
termed post-COVID syndrome, also known as post-acute sequelae of SARS CoV-2 (PASC). The 
most common symptoms include anxiety, shortness of breath, continued loss of the sense of 
smell and taste, loss of appetite with subsequent weight loss, sleep difficulties, severe fatigue, 
cognitive dysfunction (foggy brain) and increased frailty. These patients frequently present to the 
emergency room looking for symptom management because they are unable to perform normal 
activities of daily living and maintain job performance. The mechanisms behind this syndrome 
have proven elusive because patients symptom severity and treatment regimen including different 
requirements for hospitalization, supplemental oxygen, dexamethasone and remdesivir. Thus, it 
is critical to characterize the baseline endocrine, metabolic, inflammatory and microbiome 
alterations in PASC patients to better identify and manage the symptoms to prevent potential long-
term health consequences.  
UTMB has established a post-COVID clinic for management of PASC patients, but it is recognized 
that a more complete clinical picture of the underlying mechanisms driving these lingering 
symptoms is needed. UTMB’s post-COVID clinic has seen over 150 patients to date with a waiting 
list of over 200 patients who wish to be treated. Thus, it is clear that there is great demand for 
understanding the mechanisms and potential treatments for the long-term debilitating effects of 
PASC. 
Persistent and long-lasting health problems are common in patients after COVID-19 infection. In a 
recent study of patients that had been hospitalized with COVID-19, two months after discharge, 87% 
reported at least one lingering symptom (joint pain, fatigue, breathing issues, etc), more than 50% reported 

 
2
more than three lingering issues, and over 40% reported a reduction in their of quality of life. Another study 
found that at 1-month after hospitalization for COVID-19, 74% reported persistent issues related to 
shortness of breath and a decrease in both physical and mental health. Preliminary data from our Post-
COVID Recovery clinic agree with these two recent reports.  In our study, 1 1/2 months after COVID-19 
diagnosis, patients reported on average 10 of the 18 common symptoms (with 90% having chest pain, 
87% dyspnea, 75% fatigue, and 90% with cognitive changes). While the previous studies examined 
patients that had severe COVID-19 infections, >50% of our patients were never hospitalized, yet have 
numerous persistent symptoms. This has serious implications for the ability of patients to return to work, 
downstream effects on mental health due to sometimes drastic lifestyle and work capacity changes, and 
the ability to engage in activities or hobbies enjoyed prior to COVID-19 illness.  
Notably, the cluster of symptoms associated with PASC include profound fatigue and cognitive 
dysfunction, which are strikingly consistent with a syndrome that our clinical research team has 
described in patients after traumatic brain injury (TBI) designated BIAFAC (Brain Injury Associated 
Fatigue and Altered Cognition). Over the last 12 months we have reported the characteristics of 
BIAFAC syndrome (Urban 2020; Yuen et al. 2020). In particular, TBI patients with BIAFAC present 
with lingering and profoundly debilitating symptoms including severe fatigue, cognitive dysfunction 
(foggy brain), sleep disturbances, and the inability to perform activities of daily living that persist 
for years post-injury. Mechanistically we have explored the role of the gut microbiome discovering 
altered communities in TBI patients in long-term care facilities compared with controls (Urban et 
al. 2020). We also established that many TBI patients with BIAFAC also present with abnormal 
growth hormone (GH) secretion, and when treated with recombinant GH, a majority of patients 
have significant improvement of both fatigue and impaired cognition (Wright et al. 2020).  While 
studies are underway to understand the details of the mechanism causing BIAFAC and why GH 
treatment alleviates symptoms in these patients, we are intrigued that the symptom phenotype of 
PASC patients overlaps with many BIAFAC symptoms. It is possible that PASC symptoms may 
be addressed through similar treatment strategies including the potential for prebiotic/probiotic 
enhancement of microbiome health. 
In the current pilot proposal, we will characterize the baseline endocrine, metabolic, inflammatory 
and gut microbiome alterations in PASC patients and compare those to our extensive database 
of BIAFAC patients and normal controls. From this critical baseline data, we will develop carefully 
defined clinical research trials that will test potential treatments for alleviating the syndrome. We 
hypothesize that an imbalanced endocrine axis stemming from COVID infection leads to 
metabolic, inflammatory and microbial dysregulation resulting in the onset of persistent PASC 
symptoms.   
 
Specific Aims 
 
Specific Aim 1: Characterize baseline measures of endocrine function, metabolism, 
inflammation, and composition of the gut and nasal microbiome of patients reporting PASC 
symptoms.  
 
Specific Aim 2: Assess neuropsychological measures of fatigue, sleep, and cognition for 
patients reporting PASC symptoms. 

 
3
 
Specific Aim 3: Compare physiological and neuropsychological measures of PASC patients to 
our extensive database of BIAFAC patients and normal controls. 
 
Specific Aim 4: Characterize the microbiome of PASC patients and compare to: a)our database 
of healthy control subjects, b) our database of symptomatic BIAFAC patients, c) new collected 
samples of patients with a history of COVID who did not develop PASC.  
 
 
Experimental Design and Methods 
 
Subjects. We will study subjects (aged 18 - 80 years) with a history of COVID (n=16M/16F) that 
have been seen in the UTMB COVID clinic for ongoing symptom management and subjects (aged 
18 – 80 years) with a history of COVID (n=16M/16F) who did not develop PASC. Potentially 
eligible patients may also self-refer to the study from surrounding post-COVID clinics or 
clinicaltrials.gov.  Subjects with a history of COVID and no PASC will be recruited by placement 
of flyers around the community as well as through UTMB announcements.   
 
(See Protection of Human Subjects - Recruitment Methods and Consenting Process for detailed 
information regarding recruitment and consenting).   
 
 
 
COVID non-PASC Control Subjects 
 
Interested subjects with a previous diagnosis of COVID without a history of PASC will undergo a 
phone pre-screen. If they are interested, they will be scheduled for a formal consenting and 
medical screening at the UTMB Clinical Research Center (CRC). Subjects will report to the UTMB 
Institute for Translational Sciences (ITS) Clinical Research Center (CRC) for the medical 
screening and one study visit. The study visit will consist of a glucagon stimulation test (GST) to 
assess growth hormone status; questionnaires assessing health status, mood and quality of life, 
diet, sleep, and gastrointestinal health. Blood samples will be drawn to measure amino acid levels, 
hormones, and metabolites. Fecal samples will be collected to analyze the GI microbiome.  
 
Screening Procedures:  All subjects consenting will undergo the following procedures. 
Screenings will take place at the UTMB ITS Clinical Research Center (ITS-CRC).   

 
4
 
• Vital signs. Vital signs will be collected at screening. This will 
include height, weight, blood pressure, pulse, respirations, and 
temperature.  
 
• Urine Screening Labs. Urine will be collected at screening and sent 
to the UTMB clinical laboratory for basic urinalysis. Results will be 
entered into the UTMB medical chart.  
 
- 
Urinalysis 
• Urine Pregnancy Test. Urine will be collected from female subjects 
for a urine pregnancy test to be performed at the UTMB ITS-CRC 
as point of care testing (POCT). Women who are post-menopausal 
will be exempt from this test. 
 
• COVID test. A PCR test using a nasal swab will be used to ensure 
that the subject is negative for active COVID virus before beginning study. Subjects with negative 
PCR COVID tests within 2 weeks of screening date will not be retested.  
 
• Screening Blood Labs.  For initial screening, a 20 ml of blood will be drawn using a butterfly needle 
from a peripheral vein in the arm. Blood will be sent to the UTMB clinical laboratory for analysis 
and results entered into the subjects UTMB medical chart. Laboratory results will include:  
 
- 
Complete blood count with differential (CBC w/ diff) 
- 
Comprehensive metabolic panel (CMP) 
- 
Lipid Panel 
- 
Hepatitis B surface antigen 
- 
Hepatitis C virus antibody 
- 
HIV screen 
 
• History and Physical.  Subjects will undergo a medical history and physical examination by a study 
nurse, medical student or physician. Recent (within 3 months) clinical history and physical may 
be used in place.  
 
 
 
Study Procedures: 
 
Visit A 
 
• Vital signs. Vital signs will be collected. This will include height, weight, blood pressure, pulse, 
respirations, and temperature. Vitals will be collected both sitting and standing.  
 
• Urine. A clean catch urine will be obtained. Urine will be frozen at -80C and used to measure 
proteinuria, urinary microbiome signatures and renal function.   
 

 
5
• Phlebotomy.  Subjects will have a catheter placed into a peripheral vein in their arm (0.9% sodium 
chloride set at TKO) for the glucagon stimulation test. The IV will be discontinued when the 
glucagon stimulation test is complete.  
• Blood draw. Up to 20 ml of blood (2-serum separator top (5ml); 1-lithium heparin (6ml); 1-K-EDTA 
(4ml)) will be collected from fasted (at least 10 hours) subjects for analysis of hormones, amino 
acids and metabolites.  
 
An additional 20 ml of blood will be collected for the following labs to be sent to the UTMB 
clinical laboratory for analysis (all labs run at UTMB will be documented in the subject’s 
UTMB medical chart) An additional 3.5 ml blood will be collected and frozen as a back up 
in case of a problem with labs sent to ARUP.: 
• Insulin-like Growth Factor 1 (IGF-1) 
• Follicle Stimulating Hormone (FSH) 
• Sex Hormone Binding Globulin (SHBG) 
• Total Testosterone 
• Free Testosterone 
• Prolactin 
• Thyroid Stimulating Hormone (TSH) 
• Free T4 
• High Sensitivity CRP 
• Vitamin B12 
• Vitamin D 
 
 
• Glucagon stimulation test.  Subjects will have a glucagon stimulation test. After the IV is placed, 
3.5 ml of blood will be collected for the baseline (time: 0 minutes) to test for levels of human growth 
hormone. 1 mg glucagon (for subjects over 90 kg, 1.5 mg glucagon) will be injected IM in the 
deltoid muscle of the subject. Additional blood samples (3.5 mL) will be collected to test for levels 
of human growth hormone at time points: 90 minutes, 120 minutes, 150 minutes, and 180 minutes. 
All blood samples will be sent to UTMB clinical laboratory for analysis and entered into subjects 
UTMB medical chart.   
 
• GI microbiome. The GI microbiome will be assessed using a self-collected fecal sample. Subjects 
will provide a fecal sample as described below. Samples will be stored at room temperature until 
transferred to UTMB (on/+ 5 days after collection). Subjects will be given a tutorial on how to 
properly collect the fecal sample. They will be given a handout from the manufacturer of the 
collection kit with step by step instructions. They will also be shown a video of the collection 
process supplied by the kit manufacturer.  
 
https://www.youtube.com/watch?v=ytr_hmJdHqM&feature=youtu.be&rel=0 
 
Fecal specimens will be collected using the OMNIgene-Gut® (OMR-200) collection kit from 
DNA genotek (please see product inserts attached as pdf files). To assist in collection, the 
additional toilet accessory will be employed where appropriate (OM-AC1). The 
manufacturer’s instructions will guide sample collection. Briefly, fecal material will be 
collected by catch on a kit-provided tissue or toilet hat that has been wiped with a 

 
6
disinfecting wipe prior to use. The study participant’s fecal material will be transferred using 
the kit included spatula into the yellow top of a collection tube with the purple cap 
unscrewed. The tube will then be re-capped tightly and shaken for a minimum of 30 seconds 
to mix feces with kit included stabilization liquid. Storage recommendations as per 
manufacturer allow up to 60 days at room temperature or up to several months at -20°C or 
-80°C in the kit provided collection tube. Samples will be labeled with the subject’s unique 
code and stored at room temperature until transported to University of Texas Medical 
Branch (UTMB). Samples will be processed and analyzed by qPCR as described below.  
 
Nucleic acids will be extracted from batched fecal specimens using our optimized workflow 
that includes use of the DNeasy PowerMicrobiome kit (2600-50-1, Qiagen). If necessary, 
samples will be diluted/mixed 1:1 with sterile PBS to yield a pipettable liquid slurry. For each 
sample, 100 µl of the fecal liquid fraction will be mixed with 200 µl of kit-provided lysis buffer 
into a commercially available tube containing 0.1 mm glass beads (2600-50-BT, Qiagen). 
Samples will then be heated at 55°C for 30 minutes. Subsequently, samples will be 
homogenized for 5 minutes at 30 Hz using a Tissue Lyser instrument (Qiagen). Samples 
then will be centrifuged at 13,000 x g for 1 minute at room temperature. The liquid fraction 
will be placed into a sterile microtube along with 50 µl of IRS solution (2600-50-2, Qiagen) 
and incubated at 4°C for 5 minutes. The sample will then be centrifuged as before and the 
liquid fraction taken for extraction of DNA using a MagNAPure 96 automated nucleic acid 
extraction platform (Roche). Purified DNA will be used for next-generation sequencing and 
qPCR analyses to create molecular profiles for each microbiome. Residual DNA will be 
archived at -20°C.  
 
An additional collection will be performed to collect viable fecal material. Fecal samples will 
be collected using the spatula included in the OMNIgene-Gut kit used in the first collection. 
Briefly, after the participant will transfer fecal material to the yellow top of the collection tube 
for the first collection, then they will scoop another small amount of fecal material with the 
same spatula and transfer it to a prepared cryogenic freezer tube containing sterile PBS 
(without Ca/Mg) and 10% sterile glycerol. The tube will be closed and shaken for a minimum 
of 30 seconds to mix with the stabilization liquid. Samples will be labeled with the subject’s 
unique code and stored at 4°C until transported to University of Texas Medical Branch. 
Samples will then be frozen at -80°C and stored until the molecular analysis is complete. 
After initial analysis, if the sample is considered useful (representative of the average non-
PASC control subject), it may be thawed, diluted in saline and used either to mix in a 
mathematical ratio to create a synthetic community or to be directly transplanted into germ 
free mice. If the sample is considered not useful (not representative of the average non-
PASC control subject) for fecal microbiome transplant into germ free mice, the sample will 
be destroyed.  
 
 
• Questionnaires.  Questionnaires will be completed to assess perceptual fatigue, symptoms 
associated with growth hormone deficiency, depression, sleep, general quality of life, diet and 
gastrointestinal symptoms. 

 
7
 
o EuroQOL-5 Dimensions (EQ-5D) is a 5 item assessment of health status measuring 
mobility, self-care, usual activity, discomfort or pain and depression or anxiety. 
  
o Medical Outcome Study - Short Form 36 (MOS-SF-36) is a questionnaire developed by 
RAND Health to measure patient self-reported quality of life. Eight subscales are 
measured including physical function, role limitations due to physical health, role 
limitations due to emotional problems, energy/fatigue, emotional wellbeing, social 
functioning, pain and general health. Scores for each subscale range from 0 - 100, with 
100 being better perceived health and 0 being worst perceived health.  
 
o Hospital Anxiety and Depression Scale (HADS) is a 14 item assessment which 
includes 7 questions for anxiety and 7 questions for depression, which are summed 
separately for a total score for each. HADS classifies scores 0-7 as normal; 8-10 as 
borderline abnormal (borderline case); 11-21 as abnormal case.  
 
o Impact of Event Scale – Revised (IES-R) is a 22-item self-report measure that assesses 
subjective distress caused by traumatic events. Respondents are asked to identify a 
specific stressful life event and then indicate how much they were distressed or bothered 
during the past seven days by each "difficulty" listed. Items are rated on a 5-point scale 
ranging from 0 ("not at all") to 4 ("extremely"). The IES-R yields a total score (ranging from 
0 to 88) and subscale scores can also be calculated for the Intrusion, Avoidance, and 
Hyperarousal subscales. 
 
o Activities of Daily Living (ADL) (Katz, 1970) is an index to assess functional status of 
basic activities of independent living. There are six functions measured, bathing, dressing, 
toileting, transferring, continence and feeding. Summary scores range from 0 (low 
function, severe functional impairment) to 6 (full function). ADL will be assessed as 
perceived pre-COVID and current.  
 
o Instrumental Activities of Daily Living (IADL) (Lawton, 1969) is an instrument to access 
independent living skills. These skills are considered more complex than the basic 
activities measured by the Katz ADL scale.  There are eight categories, ability to use a 
telephone, shopping, food preparation, housekeeping, laundry, mode of transportation, 
responsibility for own medications, ability to handle finances. Summary scores range from 
0 (low function, dependent) to 8 (high function, independent).  
 
o Pittsburgh Sleep Quality Index (PSQI) is a self-rated questionnaire which assesses 
sleep quality and disturbances over a 1 month-time interval. Minimum Score = 0 (better); 
Maximum Score = 21 (worse). Interpretation: Total < 5 associated with good sleep quality. 
Total > 5 associated with poor sleep quality. PSQI will be assessed as perceived pre-
COVID and current.  
 
o Automated Self-Administered 24 hour dietary recall (ASA-24) will be used to record 
two 24 hour dietary periods.  
 
o Multidimensional Fatigue Symptom Inventory (MFSI) will also be used to measure 
perceptual fatigue.  Fatigue symptoms will be measured using the 30-item 

 
8
Multidimensional Fatigue Symptom Inventory – Short Form, a validated measure that 
yields one overall score of total fatigue (range -24-96, with higher scores indicating more 
fatigue) and five subscales (general, physical, emotional, mental, vigor). With the exception 
of the vigor subscale, higher scores indicate greater fatigue.  
 
o Assessment of Growth Hormone Deficiency in Adults (QoL-AGHDA) Symptoms of 
growth hormone deficiency will be measured in subjects using the QoL-AGHDA. This 25-
item questionnaire measures specific symptoms associated with growth hormone 
deficiency, with a score range of 0 to 25, with a higher score indicating worse symptoms. 
 
o The Beck Depression Inventory-II (BDI-II) is one of the most widely used self-report 
inventories to assess depressive symptom severity in adolescent and adult populations 
(clinical and non-clinical). The BDI-II is comprised of 21 individual items reflecting specific 
cognitive, affective, and physical symptoms of depression.  Each item includes four 
statements that vary in the description of symptom of severity.  Scores range from 0 to 3, 
with a score of “3” indicating a severe symptoms and a score of “0” indicating an absence 
of concern with that particular aspect of depressive symptomology. The total score is the 
sum of all endorsed statements.  If more than one statement from a given item is chosen 
by the respondent, only the statement of greatest severity is scored.  The maximum total 
score is 63.  The BDI-II Manual designates the following raw score classifications 
depression severity: ≤13 = minimal; 14-19 = mild; 20-28 = moderate; ≥ 29 = severe.  
 
o Gastrointestinal Symptom Rating Scale (GSRS). Gastrointestinal symptoms will be 
monitored using the 15-item GSRS. Each question has a seven-point Likert-type scale 
where 1 represents absence of symptoms and 7 represents very troublesome symptoms. 
 
 
PASC Subjects 
Interested subjects with a 
previous 
diagnosis 
of 
COVID will undergo a 
phone 
pre-screen 
including the Brief Fatigue 
Inventory (BFI) questions 
1-3. If they score ≥ 3 on 
any BFI questions 1-3, and 
are 
interested 
in 
participating in the study, 
they will be scheduled for 
a formal consenting and 
medical screening at the 
UTMB Clinical Research 
Center (CRC). Subjects 
will report to the UTMB 
Institute for Translational Sciences (ITS) Clinical Research Center  (CRC) for the medical 
screening and then for three separate study visits (Visits A, B and C). The study visits will consist 
of measurement of glucose tolerance, growth hormone and cortisol production, resting energy 
expenditure, functional testing including hand grip strength, gait measurements and 6 minute walk 
test, fatigue measurements, cognitive function assessment and questionnaires of health status, 
mood and quality of life, diet, sleep, and gastrointestinal health. Blood sampling for measurement 

 
9
of amino acid levels, hormones, and metabolites will be drawn. In addition, fecal and oral samples 
will be collected for analysis of the GI and oral microbiomes. Visits A, B and C will be at least 7 
days apart.  All in person visits we will follow UTMB in-person procedures for safety.  
 
Screening Procedures:  All subjects consenting will undergo the following procedures. 
Screenings will take place at the UTMB ITS Clinical Research Center (ITS-CRC).   
 
• Vital signs. Vital signs will be collected at screening. This will include height, weight, blood 
pressure, pulse, respirations, and temperature.  
 
• Urine Screening Labs. Urine will be collected at screening for the following labs. The labs will be 
sent to the UTMB clinical laboratory for analysis and entered into the UTMB medical chart.  
 
- 
Urinalysis 
• Urine Pregnancy Test. Urine will be collected from female subjects for a urine pregnancy test to 
be performed at the UTMB ITS-CRC as point of care testing (POCT). Women who are post-
menopausal will be exempt from this test. 
 
• COVID test. A PCR test using a nasal swab will be used to check ensure that the subject is 
negative for active COVID virus before beginning study. Subjects with negative PCR COVID tests 
within 2 weeks of screening date will not be retested.  
 
• Screening Blood Labs.  Subjects will have the following labs drawn for screening. Approximately 
20 ml of blood will be drawn for screening labs listed below using a butterfly needle from a 
peripheral vein in the arm. The labs will be sent to the UTMB clinical laboratory for analysis and 
entered into the subjects UTMB medical chart:  
 
- 
Complete blood count with differential (CBC w/ diff) 
- 
Comprehensive metabolic panel (CMP) 
- 
Lipid Panel 
- 
Hepatitis B surface antigen 
- 
Hepatitis C virus antibody 
- 
HIV screen 
 
• History and Physical.  Subjects will undergo a medical history and physical examination by a study 
nurse, medical student or physician. Recent (within 3 months) clinical history and physical may 
be used in place.  
 
 
 
Study Procedures: 
 
Visit A 
 
• Vital signs. Vital signs will be collected. This will include height, weight, blood pressure, pulse, 
respirations, and temperature. Vitals will be collected both sitting and standing.  
 

 
10 
• Urine. A clean catch urine will be obtained. Urine will be frozen at -80C and used to measure 
proteinuria, urinary microbiome signatures and renal function.   
 
• Phlebotomy.  Subjects will have a catheter placed into a peripheral vein in their arm (0.9% sodium 
chloride set at TKO) for the oral glucose tolerance test (OGTT). The IV will be discontinued when 
the OGTT is complete.  
 
• Blood draw. Up to 20 ml of blood (2-serum separator top (5ml); 1-lithium heparin (6ml); 1-K-EDTA 
(4ml)) will be collected from fasted (at least 10 hours) subjects for analysis of hormones, amino 
acids and metabolites.  
 
An additional 20 ml of blood will be collected for the following labs to be sent to the UTMB 
clinical laboratory for analysis (all labs run at UTMB will be documented in the subject’s 
UTMB medical chart) An additional 3.5 ml blood will be collected and frozen as a back up 
in case of a problem with labs sent to ARUP.: 
• Insulin-like Growth Factor 1 (IGF-1) 
• Follicle Stimulating Hormone (FSH) 
• Sex Hormone Binding Globulin (SHBG) 
• Total Testosterone 
• Free Testosterone 
• Prolactin 
• Thyroid Stimulating Hormone (TSH) 
• Free T4 
• High Sensitivity CRP 
• Vitamin B12 
• Vitamin D 
 
• Oral Glucose Tolerance Test (OGTT).  Glucose tolerance will be determined using a standard 
OGTT and a breath test. After an overnight fast (water allowed), an antecubital venous IV is placed 
for the collection of blood samples. During the baseline blood collection (~5 ml), a simultaneous 
breath sample is collected by having the subjects breathe into a breath collection bag fitted with 
one way valves. Following the baseline sample collection, a drink containing 75 g glucose and 
150 mg U-13C6-glucose is administered and consumed within 1 minute. From this point (t=0 
minutes) blood (~5 ml) and breath samples are collected at 30 min, 60 min, 90 min, 120 min. For 
the collection of breath samples, the subjects are instructed to breath normally, hold their breath 
for 3 seconds, and exhale completely into the provided collection bags. Subjects remain at rest 
throughout the OGTT.  Blood samples collected at specified time point will be analyzed for glucose 
and insulin at the UTMB clinical lab and will be documented in the subject’s UTMB medical chart. 
 
The ratios of 13CO2 to 12CO2 in single breath samples are measured using a UBiT-IR300 infrared 
spectrophotometer (Otsuka Electronics Co., Ltd, Hirakata, Osaka, CV ≤ 1.0 %). All breath results 
are calculated as per mille (‰) change of 13CO2 abundance from the baseline breath sample and 
expressed as ‰ delta over baseline (‰DOB). 
 
• GI microbiome. The GI microbiome will be assessed using a self-collected fecal sample. Subjects 
will provide a fecal sample as described below. Samples will be stored at room temperature until 
transferred to UTMB (on/+ 5 days after collection). Subjects will be given a tutorial on how to 
properly collect the fecal sample. They will be given a handout from the manufacturer of the 

 
11 
collection kit with step by step instructions. They will also be shown a video of the collection 
process supplied by the kit manufacturer.  
 
https://www.youtube.com/watch?v=ytr_hmJdHqM&feature=youtu.be&rel=0 
 
Fecal specimens will be collected using the OMNIgene-Gut® (OMR-200) collection kit from 
DNA genotek (please see product inserts attached as pdf files). To assist in collection, the 
additional toilet accessory will be employed where appropriate (OM-AC1). The 
manufacturer’s instructions will guide sample collection. Briefly, fecal material will be 
collected by catch on a kit-provided tissue or toilet hat that has been wiped with a 
disinfecting wipe prior to use. The study participant’s fecal material will be transferred using 
the kit included spatula into the yellow tube top of a collection tube with the purple cap 
unscrewed. The tube will then be re-capped tightly and shaken for a minimum of 30 seconds 
to mix feces with kit included stabilization liquid. Storage recommendations as per 
manufacturer allow up to 60 days at room temperature or up to several months at -20°C or 
-80°C in the kit provided collection tube. Samples will be labeled with the subject’s unique 
code and stored at room temperature until transported to University of Texas Medical 
Branch (UTMB). Samples will be processed and analyzed by qPCR as described below.  
 
Nucleic acids will be extracted from batched fecal specimens using our optimized workflow 
that includes use of the DNeasy PowerMicrobiome kit (2600-50-1, Qiagen). If necessary, 
samples will be diluted/mixed 1:1 with sterile PBS to yield a pipettable liquid slurry. For each 
sample, 100 µl of the fecal liquid fraction will be mixed with 200 µl of kit-provided lysis buffer 
into a commercially available tube containing 0.1 mm glass beads (2600-50-BT, Qiagen). 
Samples will then be heated at 55°C for 30 minutes. Subsequently, samples will be 
homogenized for 5 minutes at 30 Hz using a Tissue Lyser instrument (Qiagen). Samples 
then will be centrifuged at 13,000 x g for 1 minute at room temperature. The liquid fraction 
will then be placed into a sterile microtube along with 50 µl of IRS solution (2600-50-2, 
Qiagen) and incubated at 4°C for 5 minutes. The sample will then be centrifuged as before 
and the liquid fraction taken for extraction of DNA using a MagNAPure 96 automated nucleic 
acid extraction platform (Roche). Purified DNA will be used for next-generation sequencing 
and qPCR analyses to create molecular profiles for each microbiome. Residual DNA will be 
archived at -20°C.  
 
An additional collection will be performed to collect viable fecal material. Fecal samples will 
be collected using the spatula included in the OMNIgene-Gut kit used in the first collection. 
Briefly, after the participant will transfer fecal material to the yellow top of the collection tube 
for the first collection, then they will scoop another small amount of fecal material with the 
same spatula and transfer it to a prepared cryogenic freezer tube containing sterile PBS 
(without Ca/Mg) and 10% sterile glycerol. The tube will be closed and shaken for a minimum 
of 30 seconds to mix with the stabilization liquid. Samples will be labeled with the subject’s 
unique code and stored at 4°C until transported to University of Texas Medical Branch. 
Samples will then be frozen at -80°C and stored until the molecular analysis is complete. 

 
12 
After initial analysis, if the sample is considered useful (representative of the average PASC 
subject), it may be thawed, diluted in saline and used either to mix in a mathematical ratio 
to create a synthetic community or to be directly transplanted into germ free mice. If the 
sample is considered not useful (not representative of the average PASC subject) for fecal 
microbiome transplant into germ free mice, the sample will be destroyed.  
 
• Oral Microbiome. The oral microbiome will be assessed using saliva samples. Subjects will 
provide saliva samples as described below. Subjects will collect saliva samples at home. Samples 
will be stored at room temperature until transferred to UTMB (on/+ 10 days after collection). 
Subjects will be given a tutorial on how to properly collect the saliva sample. They will be provided 
a handout from the manufacturer of the collection kit with step by step instructions. They will also 
be directed to watch a video of the collection process supplied by the kit manufacturer.  
https://www.youtube.com/watch?v=N5K6LXmasK8&feature=youtu.be 
 
Saliva specimens will be collected using the Oragene-Discover® (OGR-500) collection kit 
from DNAgenotek (please see product inserts attached as pdf files). The manufacturer’s 
instructions will guide sample collection. Briefly, saliva will be collected by catch in a funnel 
that is attached the collection tube. The subject will spit into the funnel repeatedly until the 
saliva reaches the “fill line” marked on the tube, which is 2ml. The tube will then be re-
capped tightly. Once the top has clicked, the funnel will be unscrewed from the collection 
tube. The provided collection tube cap will be tightly secured and the tube will be shaken 
for a minimum of 5 seconds to mix saliva with kit included stabilization liquid. Storage 
recommendations as per manufacturer allow up to 5 years at room temperature or longer 
at -20°C or -80°C in the kit provided collection tube. Samples will be labeled with the 
subject’s unique code and stored at room temperature until transported to University of 
Texas Medical Branch (UTMB). 
 
During the study visit, an oral swab will be performed by research staff or by the subject 
under direction of the research staff. The sterile calcium alginate swab will be swiped 5 
times in each location listed: right cheek, left cheek, on the top of the tongue, under the 
tongue, on the roof of the mouth and along the gum lines. The swab will be immediately 
placed in a tube containing MagNAPure lysis buffer. The shaft of the swab will be snapped 
off and discarded leaving the head of the swab immersed in the lysis solution. The tube cap 
will be tightly secured and the tube will be shaken for a minimum of 5 seconds to saturate 
the head of the swab with the lysis buffer releasing the bacterial lysate into the fluid. 
Samples will be stored at -80C for subsequent batch DNA extraction on a MagNAPure96 
followed by microbiome profiling by NGS and/or qPCR. Samples will be labeled with the 
subject’s unique code. 
 
• Nasal Microbiome. During the study visit, a nasal swab will be performed by research staff or by 
the subject under direction of the research staff. The sterile calcium alginate swab will be inserted 

 
13 
into the left nare until resistance is met, rotated 5 times and then moved to the right nare and 
rotated 5 times. The swab will be immediately placed in a tube containing MagNAPure lysis 
buffer. The shaft of the swab will be snapped off and discarded leaving the head of the swab 
immersed in the lysis solution. The tube cap will be tightly secured and the tube will be shaken 
for a minimum of 5 seconds to saturate the head of the swab with the lysis buffer releasing the 
bacterial lysate into the fluid. Samples will be stored at -80C for subsequent batch DNA extraction 
on a MagNAPure96 followed by microbiome profiling by NGS and/or qPCR. Samples will be 
labeled with the subject’s unique code. 
 
• Vaginal Microbiome (women only). During the study visit, a vaginal swab will be self-collected by 
the subject under direction of the research staff. The sterile calcium alginate swab will be inserted 
into the mid-vaginal area and rotated for at least 20 seconds. The swab will be immediately 
placed in a tube containing PBS buffer. The shaft of the swab will be snapped off and discarded 
leaving the head of the swab immersed in the solution. The tube cap will be tightly secured and 
the tube will be shaken for a minimum of 5 seconds to saturate the head of the swab with the 
buffer. Samples will be placed on wet ice and processed sterilely to create aliquots for 
microbiome evaluations. Samples will be labeled with the subject’s unique code. This will be an 
optional procedure.  
 
• Oral Photograph. A photograph will be taken of the upper and lower mouth and used to assess 
for the presence of periodontal disease and general oral health by a licensed dentist, who will 
be IRB approved.  
 
• Post-COVID Recovery Clinic (PCRC) Questionnaires. A packet containing the PCRC battery of 
assessments that is currently being used in all PCRC participants will be provided to participants 
at Visit A.  They may fill the packet of information at Visit A or take home and bring back the 
completed packet to their Visit B appointment.  Questionnaires include: EQ-5D, MOS-SF-36 
Version 2, Hospital Anxiety and Depression Scale (HADS), Impact of Events Scale (IES-R), 
Activities of Daily Living ADL (pre-COVID, current), Instrumental Activities of Daily Living IADL 
(current), Pittsburgh Sleep Quality Index (PSQI, pre-COVID, current), and 2 days of 24 hour 
dietary recalls (ASA-24). 
 
o EuroQOL-5 Dimensions (EQ-5D) is a 5 item assessment of health status measuring 
mobility, self-care, usual activity, discomfort or pain and depression or anxiety. 
  
o Medical Outcome Study - Short Form 36 (MOS-SF-36) is a questionnaire developed by 
RAND Health to measure patient self-reported quality of life. Eight subscales are 
measured including physical function, role limitations due to physical health, role 
limitations due to emotional problems, energy/fatigue, emotional wellbeing, social 
functioning, pain and general health. Scores for each subscale range from 0 - 100, with 
100 being better perceived health and 0 being worst perceived health.  
 
o Hospital Anxiety and Depression Scale (HADS) is a 14 item assessment which 
includes 7 questions for anxiety and 7 questions for depression, which are summed 
separately for a total score for each. HADS classifies scores 0-7 as normal; 8-10 as 
borderline abnormal (borderline case); 11-21 as abnormal case.  
 

 
14 
o Impact of Event Scale – Revised (IES-R) is a 22-item self-report measure that assesses 
subjective distress caused by traumatic events. Respondents are asked to identify a 
specific stressful life event and then indicate how much they were distressed or bothered 
during the past seven days by each "difficulty" listed. Items are rated on a 5-point scale 
ranging from 0 ("not at all") to 4 ("extremely"). The IES-R yields a total score (ranging from 
0 to 88) and subscale scores can also be calculated for the Intrusion, Avoidance, and 
Hyperarousal subscales. 
 
o Activities of Daily Living (ADL) (Katz, 1970) is an index to assess functional status of 
basic activities of independent living. There are six functions measured, bathing, dressing, 
toileting, transferring, continence and feeding. Summary scores range from 0 (low 
function, severe functional impairment) to 6 (full function). ADL will be assessed as 
perceived pre-COVID and current.  
 
o Instrumental Activities of Daily Living (IADL) (Lawton, 1969) is an instrument to access 
independent living skills. These skills are considered more complex than the basic 
activities measured by the Katz ADL scale.  There are eight categories, ability to use a 
telephone, shopping, food preparation, housekeeping, laundry, mode of transportation, 
responsibility for own medications, ability to handle finances. Summary scores range from 
0 (low function, dependent) to 8 (high function, independent).  
 
o Pittsburgh Sleep Quality Index (PSQI) is a self-rated questionnaire which assesses 
sleep quality and disturbances over a 1 month-time interval. Minimum Score = 0 (better); 
Maximum Score = 21 (worse). Interpretation: Total < 5 associated with good sleep quality. 
Total > 5 associated with poor sleep quality. PSQI will be assessed as perceived pre-
COVID and current.  
 
o Automated Self-Administered 24 hour dietary recall (ASA-24) will be used to record 
two 24 hour dietary periods.  
 
• Gastrointestinal Symptom Rating Scale (GSRS). Gastrointestinal symptoms will be monitored 
using the 15-item GSRS. Each question has a seven-point Likert-type scale where 1 represents 
absence of symptoms and 7 represents very troublesome symptoms. 
 
• Functional assessments with wearable sensors. Functional capacity measures will be collected 
in (1) a standard laboratory setting and (2) in a simulated home setting (nearby empty room 
available for testing).  First, using a Zeno gait mat and instrumented insoles we will collect gait 
capacity metrics during functional assessment tests.  These tests will include the Short Physical 
Performance Battery, Timed Up and Go, and 2 Minute Step Test. Next participants will be given 
up to 30 minutes to rest.  Participants will complete the same battery of assessments at the 
simulated home setting while receiving direction and observation via videoconference. 
Questionnaires will be provided to ask participants about the acceptability, usability, and 
technology preferences after completion. 
 
• Handgrip Strength. Hand grip will be measured using a digital dynamometer (Jamar) three times 
on each hand.  The maximal value for each hand will be recorded. 
 

 
15 
 
Visit B 
 
• Vital signs. Vital signs will be collected. This will include height, weight, blood pressure, pulse, 
respirations, and temperature.  
 
• Phlebotomy.  Subjects will have a catheter placed into a peripheral vein in their arm (0.9% sodium 
chloride set at TKO) for the cortisol stimulation test. The IV will be discontinued when the cortisol 
stimulation test is complete.  
 
• Glucagon stimulation test.  Subjects will have a glucagon stimulation test. After the IV is placed, 
3.5 ml of blood will be collected for the baseline (time: 0 minutes) to test for levels of human growth 
hormone. 1 mg glucagon (for subjects over 90 kg, 1.5 mg glucagon) will be injected IM in the 
deltoid muscle of the subject. Additional blood samples (3.5 mL) will be collected to test for levels 
of human growth hormone at time points: 90 minutes, 120 minutes, 150 minutes, and 180 minutes. 
All blood samples will be sent to UTMB clinical laboratory for analysis and entered into subjects 
UTMB medical chart.   
 
Visit C 
 
• Vital signs. Vital signs will be collected. This will include height, weight, blood pressure, pulse, 
respirations, and temperature.  
 
• Indirect calorimetry for Resting Energy Expenditure (REE).  Expired gases will be collected and 
analyzed for O2 and CO2 concentrations for the determination of energy expenditure and substrate 
oxidation in subjects using the ITS-CRC indirect calorimetry system, Vmax Encore, Carefusion. The 
subject will be asked to lie down on a bed and the ventilated canopy hood will be placed over their 
head and shoulders. The room will be darkened and external noises will be eliminated, as much as 
possible. The subject will be asked to relax and remain awake but as still as possible for the testing 
period of 30 minutes.  
 
• ATCH (Cortrosyn) stimulation test.  Subjects will have an ATCH stimulation test. 3.5 ml of blood 
will be collected using a butterfly needle from a peripheral vein in the arm for the baseline (time: 
0 minutes) and analyzed for levels of cortisol. 0.25 mg Cortrosyn (1 vial) will be injected IM in the 
deltoid muscle of the subject. And additional blood samples (3.5 mL) will be collected at 30 and 
60 minutes after the injection to test for levels of cortisol.  All blood samples will be sent to UTMB 
clinical laboratory for analysis and entered into subjects UTMB medical chart.   
 
• Phlebotomy.  Subjects will have a catheter placed into a peripheral vein in their arm (0.9% sodium 
chloride set at TKO) for the ATCH stimulation test. The IV will be discontinued when testing is 
complete.  
 
• Cognitive Assessment. Subjects will undergo a cognitive assessment. 
 
Montreal Cognitive Assessment (MoCA). The Montreal Cognitive Assessment (MoCA) 
is a rapid assessment of cognition. The MoCA consists of 9 questions with the following 
subcategories: visuospatial/executive, naming, memory, language, abstraction, delayed 

 
16 
recall and orientation. The MoCA has been used extensively to detect cognitive impairment 
in many conditions, including head trauma. Version 8.1 will be used. 
 
• Modified 6-minute walk.  Walking performance will be assessed in subjects during 6 minutes of 
walking in long corridor hallways.  This is a standard test of walking performance that has been 
validated in similar studies. Subjects will be asked to walk at a 100% effort. Distance traveled for 
each 2-minute category will be recorded, as well as total distance.  
 
• Questionnaires.  Questionnaires will be completed to assess perceptual fatigue, symptoms 
associated with growth hormone deficiency, depression, and gastrointestinal symptoms. 
 
o Multidimensional Fatigue Symptom Inventory (MFSI) will also be used to measure 
perceptual fatigue.  Fatigue symptoms will be measured using the 30-item 
Multidimensional Fatigue Symptom Inventory – Short Form, a validated measure that 
yields one overall score of total fatigue (range -24-96, with higher scores indicating more 
fatigue) and five subscales (general, physical, emotional, mental, vigor). With the exception 
of the vigor subscale, higher scores indicate greater fatigue.  
 
o Assessment of Growth Hormone Deficiency in Adults (QoL-AGHDA) Symptoms of 
growth hormone deficiency will be measured in subjects using the QoL-AGHDA. This 25-
item questionnaire measures specific symptoms associated with growth hormone 
deficiency, with a score range of 0 to 25, with a higher score indicating worse symptoms. 
 
o The Beck Depression Inventory-II (BDI-II) is one of the most widely used self-report 
inventories to assess depressive symptom severity in adolescent and adult populations 
(clinical and non-clinical). The BDI-II is comprised of 21 individual items reflecting specific 
cognitive, affective, and physical symptoms of depression.  Each item includes four 
statements that vary in the description of symptom of severity.  Scores range from 0 to 3, 
with a score of “3” indicating a severe symptoms and a score of “0” indicating an absence 
of concern with that particular aspect of depressive symptomology. The total score is the 
sum of all endorsed statements.  If more than one statement from a given item is chosen 
by the respondent, only the statement of greatest severity is scored.  The maximum total 
score is 63.  The BDI-II Manual designates the following raw score classifications 
depression severity: ≤13 = minimal; 14-19 = mild; 20-28 = moderate; ≥ 29 = severe.  
 
Statistical Considerations  
 
In this observational study we intend to compare the microbiome community structures from the 
GI tract (fecal microbiome), oral, vaginal, and nasal mucosa sites in COVID long-haulers (subjects 
that meet our criteria for fatigue, altered cognition and are post-COVID-19) to community 
structures from our in house data as well as those published by others including the human 
microbiome project consortium. The observational hypothesis is that PASC subjects may have 
notable differences from normal subjects associated with the PASC sequelae described above. 
The study design is based on our past experiences with brain injury associated fatigue and altered 
cognition (BIAFAC) syndrome leading us to make practical group size selections of 32 
symptomatic PASC patients. The design will include equal numbers of males and females (n=16 
each). Because no publications are currently available describing changes to the microbiome in 
PASC patients, we are unable to complete specific power calculations. However, based on our 
previous clinical studies of BIAFAC patients with similar symptom presentation, we anticipate that 

 
17 
we would need 6 subjects per group to have an 80% probability of detecting significant differences 
in at least one of the previously identified BIAFAC organisms (two-sided, a=0.05). With the 
proposed subject numbers (n=32 completed subjects) we believe we will generate reasonable 
preliminary findings to identify differences in microbiome communities related to detection and/or 
abundance of specific organisms associated with specific PASC symptoms. 
 
For quantifiable outcomes such as abundances of individual species in the GI microbiome and 
symptom related outcomes, a standard repeated measure ANOVA model will be used. When 
possible, age and other covariates will be brought into the model to account for individual 
variability and group differences. Significance tests will be performed at the alpha=0.05 level, and 
multiple testing corrections will be used to account for potential Type I error rate inflation. 
Nonparametric, generalized linear, and transformed response models will be considered as 
necessary to alter the model to account for deviations from standard assumptions (normality, 
linearity, etc.). Current data support statistical outcomes being established with group sizes similar 
or slightly larger than our previous BIAFAC studies.    
 
The graph below is based on group difference in Alistipes onderdonkii that was associated with 
BIAFAC as an example of the data analysis for this study. Power calculations were performed 
using G*Power (two-groups, two-sided, a=0.05, Version 3.1.9.2, Germany). Effect size in this 
case was 1.184. Below the graph is a list of organisms we identified previously that we used to 
conduct past power analyses for similar studies. Please note that the intestinal and oral flora 
include an incredibly diverse system of organisms and at this time it is difficult to accurately predict 
which organisms will be included in potential microbial shifts. Therefore, additional organisms will 
be included in the microbiome comparisons between PASC patients and naïve control subjects. 
Further power analyses will be conducted using the data collected during this investigation.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Target 
n = 20 
(10/group) n=30 
n=40 
n= 50 
(25/group) 
Alistipes 
onderdonkii 
70.7% 
87.9% 95.4% 
98.4% 
Desulfovibrio 
60.4% 
79.3% 89.9% 
95.4% 
Prevotella spp 
97.5% 
99.8% 100.0% 100.0% 
Akkermansia 
64.8% 
83.2% 92.6% 
96.9% 
Odoribacter 
52.5% 
71.5% 83.8% 
91.2% 

 
18 
Ruminococcus 
bicirculans 
57.2% 
76.3% 87.7% 
93.9% 
Bacteroides 
vulgatus 
65.6% 
83.9% 93.1% 
97.2% 
Sutterella 
53.0% 
72.0% 84.2% 
91.5% 
Lachnoclostridium 
30.8% 
44.4% 56.4% 
66.4% 
Streptococcus 
salivarius 
27.8% 
40.2% 51.4% 
61.2% 
16s 
6.8% 
7.8% 
8.8% 
9.8% 
 
 
Study Timeline  
 
This is a 1 year project.  
 
 
 
 
 
Protection of Human Subjects 
Human Subjects 
 
Human subject involvement and characteristics.  We propose to study a total 32 subjects (16M/16F) 
with a history of COVID and PASC (males and females) and 32 subjects (16M/16F) with a history of 
COVID without development of PASC between the ages of 18 and 80. This COVID, non-PASC 
group has been requested by several recent reviewers based on recent published evidence that 
non-PASC COVID recoveries may associate with an altered fecal microbiome.  
 
See Protection of Human Subjects - Recruitment Methods and Consenting Process for detailed 
information regarding recruitment and consenting.  
 
COVID Non-PASC controls 
Inclusion criteria  
 
1. Male or female with a history of COVID with diagnosis confirmed by PCR test.  
2. Minimum of 6 months since diagnosis of COVID by PCR test.  
3. Ages 18 to 80 years. 
4. Participant is willing and able to give informed consent for participation in the study. 
 
Exclusion criteria  
 
1. Current COVID infection. 
2. Unable to walk unassisted. 
3. Significant heart, liver, kidney, blood or respiratory disease as determined by Principal 
Investigator.  
4. Uncontrolled diabetes mellitus.  
5. Any history of a recently (12 months) diagnosed cancer other than a skin cancer (excluding 
melanoma). 
6. Current alcohol or drug abuse. 
7. History of psychosis.  

 
19 
8. Pregnancy or become pregnant during the trial. 
9. Subjects who are being managed with narcotics will be excluded as the effects of central 
nervous system depressants may interfere with study test results. 
10. Other medical condition or medication administration deemed exclusionary by the study 
investigators. 
 
PASC subjects 
Inclusion criteria  
 
1. Male or female with a history of COVID with diagnosis confirmed by PCR test.  
2. Minimum of 6 months since diagnosis of COVID by PCR test.  
3. Ages 18 to 80 years. 
4. Score of 3 or higher on any question 1-3 of the Brief Fatigue Inventory (BFI) questionnaire. 
5. Participant is willing and able to give informed consent for participation in the study. 
 
Exclusion criteria  
 
1. Current COVID infection. 
2. Unable to walk unassisted. 
3. Significant heart, liver, kidney, blood or respiratory disease as determined by Principal 
Investigator.  
4. Uncontrolled diabetes mellitus.  
5. Any history of a recently (12 months) diagnosed cancer other than a skin cancer (excluding 
melanoma). 
6. Current alcohol or drug abuse. 
7. History of psychosis.  
8. Pregnancy or become pregnant during the trial. 
9. Subjects who are being managed with narcotics will be excluded as the effects of central 
nervous system depressants may interfere with study test results. 
10. Other medical condition or medication administration deemed exclusionary by the study 
investigators. 
 
Sources of materials. We may obtain blood, urine, fecal, saliva and questionnaire data from the 
subjects.  All data/samples will be collected solely for the purpose of experimentation. The blood 
will be used for screening and to measure metabolites, amino acids, hormones and measures of 
inflammation. Fecal, nasal and saliva samples will be used for determination of the GI, nasal and 
oral microbiomes. Questionnaire data will be used to assess quality of life. 
 
Recruitment Methods and Consenting Process  
 
a) Potential subjects may be patients of the investigators. 
 
b) 1. PASC subjects may be recruited from the UTMB post COVID clinic in one of the 
following ways: 
 
• In the clinic – potential subjects may be given a flyer along with Fast Fact sheet 
explaining the screening and study procedures in bulleted points. Potential subjects 
can read over the information with their families and decide if they would like to 

 
20 
enroll. If interested, the potential subject will contact the study team for a phone pre-
screening to go over eligibility requirements (pre-screening) for the study (see 
attached pre-screening sheet). If potential subject passes the pre-screening and is 
still interested, the potential subject will be asked to come to the ITS-CRC at a time 
that is mutually convenient for both the subject and the study team for consenting 
and medical screening.  
• Contacted by a post COVID clinic physician or nurse practitioner that they may be 
eligible for a research study for PASC patients and that the study team will contact 
them in a few days to see if they are interested. Contact may be in person or through 
phone call, by letter (see attached letter) by postal mail or myChart.  If interested, 
subjects will be sent the study Fast Fact sheet explaining the screening and study 
procedures in bulleted points. Potential subjects can read over the information with 
their families and decide if they would like to enroll.  If interested, the potential subject 
will contact the study team for a phone pre-screening to go over eligibility 
requirements (pre-screening) for the study (see attached pre-screening sheet). If 
potential subject passes the pre-screening and is still interested, the potential subject 
will be asked to come to the ITS-CRC at a time that is mutually convenient for both 
the subject and the study team for consenting and medical screening.  
 
2. COVID, non-PASC control subjects may be recruited by referral from UTMB clinicians 
and/or advertisement in both the UTMB daily announcements, researchmatch.org and word 
of mouth.  
 
3. Subjects in both groups may also self-refer to the study from surrounding clinics or from 
clinicaltrials.gov, flyers or UTMB announcements.  
 
c) All subjects will have privacy at each of their visits to the ITS-CRC. Consenting will take 
place at the ITS-CRC in a private room following the consent process checklist provided 
by the UTMB Office of Clinical Research. Subjects will be given a private room for each 
study visit.  
 
d) To minimize the possibility of coercion or undue influence all subjects will be given a copy of 
the approved consent forms and encouraged to discuss with their families prior to meeting 
the coordinator for the consenting process. 
 
e) A waiver of consent is requested for identification and prescreening of potential subjects. 
 
f) Vulnerable populations will be excluded from this study. 
 
Subject reimbursement for participation.   
 
• Non-PASC control subjects will receive $25 for screening and $50 for their study visit. If 
they complete the whole protocol, they will be reimbursed $75 for time and travel. Payment 
will be completed through Clincard. 
• PASC Subjects will receive $25 for screening and $50 for each of the three study visits. If 
they complete the entire protocol, they will be reimbursed $175 for time and travel. 
Payment will be completed through Clincard. 

 
21 
 
Potential Risks   
 
The potential risks for subjects enrolled in this project will be the following: 
 
Vital Signs. There are no known risks associated with this procedure.  
 
Urine Collection. There are no known risks associated with this procedure.  
 
Nasal Swab. Risks include pain and a chance for minor bleeding from the nose.  
 
Phlebotomy.  All patients will undergo periodic blood draws throughout the study. The risks of 
phlebotomy are pain, infection, bruising and introduction of blood borne pathogens. Aseptic 
technique will be used to minimize these risks. 
 
Blood draws. No more than 120ml will be collected for the entire study.  
 
Fasting. The risks of fasting include fatigue, headache, hypoglycemia and dehydration. 
 
Oral Glucose Tolerance Testing (OGTT).  Risks involved with the OGTT are related to phlebotomy. 
The risks of phlebotomy are pain and infection.  Aseptic technique will be used to minimize these 
risks.  
Oral Administration of Stable Isotope Labelled Glucose. There is no known risk to giving stable-
isotopic tracers orally.  They are frequently employed clinically and for research purposes in 
breath tests to measure various metabolic responses.  
 
Fecal Sampling:  There are no known risks associated with this procedure. 
 
Saliva collection: There are no known risks associated with this procedure.  
 
Vaginal Swab: There may be a risk of discomfort.  
 
Oral Photograph: There are no known risks associated with this procedure.  
 
Questionnaires:  There is no known risk to completing the questionnaires listed in this study. 
 
Functional Assessments: There is a small risk of falling during the functional assessments which 
could lead to injury. Additional risks may include muscle tightness, soreness, and/or fatigue. 
 
Handgrip testing and 6-minute walk:  The exercise tests may cause muscle soreness, cramps, 
dizziness, and possibly irregular heartbeats. The risks of the 6-minute walk include falling, 
elevated heart rate and elevated blood pressure. All subjects will be accompanied by a trained 
staff member.   
 
Glucagon Stimulation Testing (GST): The known side effects of the glucagon stimulation test are 
headache and nausea and possible vomiting. 
 

 
22 
Indirect calorimetry for Resting Energy Expenditure (REE): There are no known risks involved with 
these measurements. 
 
ATCH (Cortrosyn) Stimulation Testing: Side effects of cortrosyn include allergic reaction, rash, 
lightheadedness, fainting, high blood pressure, rapid or slow heartrate and swelling in the limbs. 
Bleeding, bruising or infection may occur at the injection site.  
 
Cognitive Testing: There are no known risks associated with this procedure. 
 
Confidentiality:  Divulgation of personal sensitive data is a general risk of clinical investigations 
involving human subjects. 
 
 
 
 
Adequacy of Protection Against Risks 
 
Recruitment and informed consent.  Subjects will be identified from the UTMB post COVID clinic, 
self-referred from surrounding post-COVID clinics, clinicaltrials.gov, flyers or UTMB 
announcements.   
1. Potentially eligible subjects may be informed about the study either during clinic visits or 
by a message from a post-COVID physician.  
• In the clinic – potential subjects may be given a flyer along with Fast Fact sheet 
explaining the screening and study procedures in bulleted points. Potential subjects 
can read over the information with their families and decide if they would like to 
enroll. If interested, the potential subject will contact the study team for a phone pre-
screening to go over eligibility requirements (pre-screening) for the study (see 
attached pre-screening sheet). If potential subject passes the pre-screening and is 
still interested, the potential subject will be asked to come to the ITS-CRC at a time 
that is mutually convenient for both the subject and the study team for consenting 
and medical screening.  
• Contacted by a post COVID clinic physician that they may be eligible for a research 
study for PASC patients (see attached letter) and that the study team will contact 
them in a few days to see if they are interested. Contact may be through postal mail 
or myChart.  If interested, subjects will be sent the study Fast Fact sheet explaining 
the screening and study procedures in bulleted points. Potential subjects can read 
over the information with their families and decide if they would like to enroll.  If 
interested, the potential subject will contact the study team for a phone pre-screening 
to go over eligibility requirements (pre-screening) for the study (see attached pre-
screening sheet). If potential subject passes the pre-screening and is still interested, 
the potential subject will be asked to come to the ITS-CRC at a time that is mutually 
convenient for both the subject and the study team for consenting and medical 
screening.  
2.   Potentially eligible subjects may contact the study team directly after viewing an IRB approved 
flyer or fast facts sheet or after viewing the study on clinicaltrials.gov, flyers or UTMB 
announcements.  

 
23 
Consenting will take place at the ITS-CRC in a private room following the consent process 
checklist provided by the UTMB Office of Clinical Research.  
 
Protections against risks.  All key personnel have completed and passed the certification exam of 
the NIH-mandated course on protection of human subjects. The screening tests will allow us to 
exclude a priori subjects with potentially higher risk of developing complications.  Randall Urban, MD 
will oversee the medical aspects of the experiments.  Protections against risks for all patients will be 
the following: 
 
Vital Signs. There are no known risks associated with this procedure.  
 
Urine Collection. There are no known risks associated with this procedure.  
 
Nasal Swab.  Care will be taken to minimize pain and/or bleeding.  
 
Phlebotomy.  The use of aseptic technique will be used to minimize the risk of infection, but will 
not eliminate it completely. Some patients may experience a sudden drop in blood pressure in 
response to the placement of catheters. Symptoms may include lightheadedness, nausea and 
possibly vomiting. There are no long-term effects associated with this response. In our 
experience, symptoms occur in 3.3% of our healthy subjects. All patients experiencing this 
response will be examined by a physician and given the option of discontinuing the study. If the 
physician, PI, study investigators, and patient find no reason to stop the study, the patient will be 
allowed to continue.  
 
Blood draws. No more than ~65 ml of blood will be drawn at any visit. This around 15% of a typical 
blood donation, and in adult individuals with normal blood count does not expose to the risk of 
anemia. Less than 120 ml will be drawn over the 1 month period of the study.  
 
Fasting.  All subjects will be made aware of the risks of overnight fasting. They will be advised to 
drink water the night before and the morning of their visits. All subjects will be fed a meal after 
the completion of their study visits.   
 
Oral Glucose Tolerance Test (OGTT). The risk are associated with phlebotomy. Care will be 
taken to minimize the risk of infection, but will not eliminate it completely.  
 
Oral Administration of Stable Isotope Labelled Glucose. There is no known risk to giving stable-
isotopic tracers orally. 
 
Fecal Sampling. There are no known risks associated with this procedure.  
 
Saliva collection: There are no known risks associated with this procedure.  
 
Vaginal Swab: A qualified member of the research team will fully explain the procedure for self-
collecting the vaginal swab safely.  
 
Oral Photograph: There are no known risks associated with this procedure.  
 
Questionnaires. There are no known risks associated with this procedure. 
 

 
24 
Functional Assessments.  A member of the research team will supervise all functional assessment 
measurements.   
 
Hand grip and 6-minute walk. Risk of muscle soreness or cramps can be reduced or avoided with 
an adequate warm-up. The subjects will be accompanied by a qualified staff member at all times.  
 
Glucagon Stimulation Test.  Subjects will be made aware of the risks of the test.  
 
Indirect calorimetry. There are no known risks associated with these measurements.  
 
ATCH (Cortrosyn) Stimulation Testing. Subjects will be made aware of the risks of the test.   
 
Cognitive Testing. There are no known risks associated with this procedure. 
 
Confidentiality.  Only the investigators, the study personnel, and the Medical Records department of 
the UTMB Hospital will have access to the subjects’ personal information.  All data containing the 
name of the patients will be locked in the PI office. To preserve confidentiality, immediately after 
enrollment each subject will be assigned a code by which each research sample will be identified for 
further analysis, thus avoiding identification by non-qualified individuals. 
 
 
Potential Benefits of the Proposed Research to the Subjects and Others 
 
No benefits are expected for the subjects.  The knowledge collected from the present studies may 
be of benefit to society, as these studies will help develop the scientific basis for characterizing 
PASC.   
 
Importance of Knowledge to be Gained 
 
This study will help characterize the symptoms that persist in patients after COVID.   
 
 
Data Safety and Monitoring Plan 
Study Title:  The Endocrine, Metabolic and Microbiome Influence on the Post-COVID Syndrome 
 
Type of Research Data or Events to be Monitored:   
Total subject accrual will be monitored as well as adverse events, protocol deviations, violations 
and unanticipated problems.  The risk level associated with this study is estimated to be minimum.  
 
Methods and Frequency of Analysis:  
Plan for Monitoring and Safety Review: 

 
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a.   Randall Urban, MD, the Principal Investigator is the individual responsible for monitoring the 
safety environment of the participants.  
b.   Thorough monitoring of the recruitment, enrollment, retention, informed consent process, 
adverse events, study procedures, and the evaluation of primary and secondary endpoints will be 
carried out.   
c.   Potential subjects will be informed of all procedures involved in the study.  If the subject agrees 
to participate, written consent will be obtained.   
d. Information will be reviewed on a weekly basis by the PI and the research team.  
Plan for Data Management: 
a. Study data collected at UTMB will be recorded on case report forms.  To preserve 
confidentiality, immediately after enrollment each subject will be assigned a code by which 
each research sample will be identified for further analysis to avoid identification by non-
qualified individuals.   Dr. Urban, the Principal Investigator, is the individual responsible for 
storage of data.  Recognizable personal subject information will be stored in Dr. Urban’s 
laboratory in a locked cabinet. 
 
b.   Data will be reviewed on a weekly basis by the PI and the research team. 
c.   The conditions that would necessitate early termination of the study include withdrawal of 
consent.  
d.   The PI with the aid of the Co-Investigators and the Study Coordinator will perform aggregate 
analysis of data and adverse events. 
 
Persons Responsible for Data Monitoring:  
The ultimate responsibility for data and safety monitoring, and submitting reports of unanticipated 
problems, adverse events, protocol deviations and protocol violations rests with the Principal 
Investigator, Randall Urban, MD. Every effort will be undertaken to monitor and minimize the 
risks to subjects.  Prior to obtaining informed consent, subjects will be encouraged to thoroughly 
read the informed consent form and ask questions regarding the outlined procedures and risks. 
To ensure ongoing protection of study subjects all procedures will be performed under the 
supervision of Randall Urban, M.D., or a study physician willing to provide backup medical 
coverage.  All study physicians will be familiar with the study protocol. 
 
Reporting Unanticipated Problems, Adverse Events, Protocol Deviations and Protocol 
Violations: 

 
26 
a.   Dr. Urban, the Principal Investigator, is the individual responsible for monitoring and reporting 
the occurrence of unanticipated problems, adverse events, protocol deviations and protocol 
violations throughout the study.    
b.   Anticipated Adverse Events, as discussed above will be included in the consent form.  All other 
anticipated adverse events will be addressed by the PI and study team. If an adverse event occurs 
Dr. Urban will be immediately notified and a note will be entered in to the subject’s medical chart 
using the grading scale listed below.  
c.   Adverse Event Grading Scale (UTMB – CRC Scale): 
0 = No Adverse Event or within normal limits 
1 = Mild Severity: Transient laboratory test alterations; discomforts noted but no disruption of daily 
activities; no therapy, or only symptomatic therapy required. 
2 = Moderate Severity: Laboratory test alterations indicating injury without long-term risk; 
discomfort sufficient to modify normal daily activity; specific therapy required (i.e., more than 
symptomatic). 
3 = Serious Severity: Laboratory test indicating a serious health threat or permanent injury; 
incapacity, inability to work, inability to perform normal daily activity; hospitalization required or 
prolonged; emergency treatment required; life-threatening events; death.     
The attribution scale assesses the relation of the event to the study procedures. The Principal 
Investigator will judge whether or not an adverse event is: 1) not related; 2) possibly related; 3) 
probably related; 4) definitely related to the study procedures and interventions. 
All unanticipated, serious, fatal and/or life-threatening adverse events will be reported to the IRB 
within 24 hours of occurrence.  The PI and UTMB Institutional Review Board (IRB) are 
responsible for determining whether modifications to the protocol and consent form are required. 
If a determination is made that participants are found to be exposed to excessive risks in 
relation to anticipated benefits, the study will be immediately suspended. Studies will not resume 
until modifications are made that are deemed to result in an acceptable risk/benefit ratio by the 
PI and IRB. Aggregate reports of adverse events will be prepared on an annual basis and 
forwarded to the IRB at annual review. 
 
 
Stopping Rules: 
The study would be stopped and re-evaluated if a subject had a loss of confidentiality.  
 
Procedures and Time Frames for Communicating Outcomes: 

 
27 
Monitoring Reports will be prepared and forwarded to the IRB on an annual basis.  
 
Precautions for Maintaining Data Integrity: 
Careful monitoring of the recruitment, enrollment, retention, adverse events, and study 
procedures will help to protect the safety of study subjects, the quality of data, and the integrity 
of the study. As part of the safety plan for this study, the PI will review individual study subject 
records as needed to ensure that appropriate mechanisms to protect the safety of study 
participants are being followed, that protocol requirements are being adhered to, and that data is 
accurate, complete, and secure. Subject records include consent forms, case report forms, flow 
of data forms, laboratory specimen records, inclusion/exclusion forms, and/or adverse event 
logs, and medical charts. The PI meets with the study team once per week to discuss ongoing 
study status.  
 
Records Retention 
The investigator shall retain the records for 15 years.  
 
Inclusion of Women and Minorities 
Women and men will be included. 
Minorities will be included in the proportion found in the general population (see Targeted 
Enrollment Table)  
 
Targeted/Planned Enrollment Table 
Study Title: The Endocrine, Metabolic and Microbiome Influence on the Post-COVID Syndrome 
 
Total Planned Enrollment: Up to 120 subjects 
Targeted/ Planned Enrollment: Number of Subjects 
Ethnic Category 
Sex/Gender 
Females 
Males 
Total 
Hispanic or Latino 
10 
10 
20 
Not Hispanic or Latino 
50 
50 
100 
Ethnic Category: Total of All Subjects * 
60 
6060 
120 

 
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Racial Categories 
 
American Indian/Alaska Native 
0 
0 
0 
Asian 
0 
0 
0 
Native Hawaiian or Other Pacific Islander  
0 
0 
0 
Black or African American  
10 
10 
20 
White 
50 
50 
100 
Racial Categories: Total of All Subjects * 
60 
60 
120 
 
Inclusion of Children  
Children will not be included in this study. The age range for inclusion is 18 to 80.  
 
Appendix A 
Dr. Wright has a dual appointment with Texas A&M University, Department of Health and 
Kinesiology and University of Texas Medical Branch at Galveston, Department of Internal 
Medicine.He will conduct the following study procedures at Texas A&M: data analysis, 
manuscript writing. No subjects will be enrolled at Texas A&M University. Texas A&M 
University will rely on UTMB IRB as the IRB of record for this study.