Effect of Direct-from-blood Bacterial Testing on Antibiotic Administration and Clinical Outcomes: A Learning Healthcare Pragmatic Randomized Trial
Phase: NA
Conditions: Bloodstream Infection, Sepsis Bacterial, MRSA Bacteremia, Vancomycin
Sponsor: Vanderbilt University Medical Center

 
 
 
 
 
 
Effect of Direct-from-blood Bacterial Testing on Antibiotic 
Administration and Clinical Outcomes 
 
NCT06069206 
 
Protocol Version Date February 5, 2024 
 
Uploaded to ct.gov on January 26, 2026 
 
 
 
 
 
 
 
 
 

Study Protocol- v1.5 
Full Study Title: Effect of direct-from-blood bacterial testing on antibiotic administration and clinical 
outcomes: A learning healthcare pragmatic randomized trial 
Primary Investigators: David Gaston, MD, PhD and Romney Humphries, PhD, D(ADBMM), M(ASCP)  
 
 
Effect of direct-from-blood bacterial testing on antibiotic administration and 
clinical outcomes: A learning healthcare pragmatic randomized trial 
 
Version 1.5 
 February 5, 2024 
 
Lead Investigators: 
 
Matthew Semler, MD, MSc, Medical Director, Center for Learning Healthcare; Associate Director, 
Vanderbilt Medical Intensive Care Unit 
 
David Gaston, MD, PhD, Medical Director, Molecular Infectious Disease Laboratory; Assistant Professor, 
Pathology, Microbiology, and Immunology 
 
Romney Humphries, PhD, D(ADBMM), M(ASCP), Director, Division of Laboratory Medicine; Professor of 
Pathology, Microbiology, and Immunology 
 
Co-Investigators:  
 
Ritu Banerjee, MD, PhD, Medical Director, Pediatric Antimicrobial Stewardship Program; Professor of 
Pediatrics, Infectious Diseases  
 
Matthew Christensen, MD, Clinical Fellow, Department of Medicine, Division of Allergy, Pulmonary, and 
Critical Care 
 
Benjamin Ereshefsky, PharmD, BCIDP, Infectious Diseases Pharmacist, Department of Pharmaceutical 
Services 
 
Jodell Jackson, PhD, Manager of Translational Research, Vanderbilt Institute for Clinical & Translational 
Research  
 
George Nelson, MD, Medical Director, Adult Antimicrobial Stewardship Program; Associate Professor, 
Department of Medicine, Division of Infectious Diseases 
 
Edward Qian, MD, Clinical Fellow, Department of Medicine, Division of Allergy, Pulmonary, and Critical 
Care 

Study Protocol- v1.5 
Full Study Title: Effect of direct-from-blood bacterial testing on antibiotic administration and clinical 
outcomes: A learning healthcare pragmatic randomized trial 
Primary Investigators: David Gaston, MD, PhD and Romney Humphries, PhD, D(ADBMM), M(ASCP)  
 
 
Joanna Stollings, PharmD, FCCP, FCCM, BCPS, BCCCP, Department of Pharmaceutical Services 
 
Coordinating Center:  
 
Cheryl Gatto, PhD, Operations Director, Center for Learning Healthcare; Director, Vanderbilt Institute for 
Clinical & Translational Research 
 
Wesley Self, MD, MPH, Senior Vice President for Clinical Research, Director, Vanderbilt Institute for 
Clinical & Translational Research, Professor of Emergency Medicine 
 
Matthew Semler, MD, MSc, Medical Director, Center for Learning Healthcare; Associate Director, 
Vanderbilt Medical Intensive Care Unit  
 
Ariel Lewis, MPH, BSN, RN, Research Nurse Specialist, Center for Learning Healthcare 
Contents 
1.0 Study Summary ....................................................................................................................................... 5 
2.0 Background ............................................................................................................................................. 6 
2.1 Blood cultures versus direct-from-blood tests for identifying bacterial pathogens ........................... 6 
2.2 Empiric, broad-spectrum antibiotics for suspected bacterial infections of the bloodstream ............ 7 
2.3 Empiric vancomycin for MRSA bacteremia ......................................................................................... 7 
2.4 Overview of trial .................................................................................................................................. 8 
2.5 Rationale for trial ................................................................................................................................ 8 
3.0 Aims and Hypotheses .............................................................................................................................. 8 
4.0 Study Description .................................................................................................................................... 8 
5.0 Inclusion and Exclusion Criteria .............................................................................................................. 9 
5.1 
Inclusion Criteria ........................................................................................................................... 9 
5.2 Exclusion Criteria ................................................................................................................................. 9 
6.0 Enrollment and Randomization .............................................................................................................. 9 
6.1 Study Site ............................................................................................................................................ 9 
6.3 Enrollment ........................................................................................................................................... 9 
6.4 Consent ............................................................................................................................................. 10 
6.4.1 Minimal risk ................................................................................................................................ 11 
6.4.2 Practicability of obtaining written, informed consent ............................................................... 11 
6.5 Randomization .................................................................................................................................. 12 

Study Protocol- v1.5 
Full Study Title: Effect of direct-from-blood bacterial testing on antibiotic administration and clinical 
outcomes: A learning healthcare pragmatic randomized trial 
Primary Investigators: David Gaston, MD, PhD and Romney Humphries, PhD, D(ADBMM), M(ASCP)  
 
7.0 Study Procedures .................................................................................................................................. 12 
7.1 Blood Culture Group ......................................................................................................................... 12 
7.2 Direct from Blood Test Group (Intervention Group) ........................................................................ 12 
7.3 Co-interventions: Management of Vancomycin by Pharmacokinetics Service ................................ 13 
7.4 Blinding ............................................................................................................................................. 14 
8.0 Data Collection and Outcome Measures .............................................................................................. 14 
8.1 In-Hospital Outcomes ....................................................................................................................... 14 
8.2 Baseline Data .................................................................................................................................... 16 
8.3 Data from enrollment to hospital discharge ..................................................................................... 16 
8.4 Outcome Data ................................................................................................................................... 16 
9.0 Risks and Benefits ................................................................................................................................. 16 
10.0 Safety Monitoring and Adverse Events ............................................................................................... 17 
10.1 Adverse Event Definitions ............................................................................................................... 17 
10.2 Monitoring for Adverse Events ....................................................................................................... 18 
10.3 Recording and Reporting Adverse Events ....................................................................................... 18 
10.4 Clinical Outcomes that may be Exempt from Adverse Event Recording and Reporting ................ 19 
10.5 Unanticipated Problems involving Risks to Subjects or Others ...................................................... 19 
11.0 Study Withdrawal/Discontinuation .................................................................................................... 20 
12.0 Statistical Considerations .................................................................................................................... 20 
13.0 Privacy and Confidentiality ................................................................................................................. 21 
14.0 Follow-up and Record Retention ........................................................................................................ 22 
15.0 References .......................................................................................................................................... 23 
16.0 Appendix ............................................................................................................................................. 27 
16.1 Enrollment Pause ............................................................................................................................ 27 
 
 
 
 

Study Protocol- v1.5 
Full Study Title: Effect of direct-from-blood bacterial testing on antibiotic administration and clinical 
outcomes: A learning healthcare pragmatic randomized trial 
Primary Investigators: David Gaston, MD, PhD and Romney Humphries, PhD, D(ADBMM), M(ASCP)  
 
1.0 Study Summary 
Title  
Effect of direct-from-blood bacterial testing on antibiotic 
administration and clinical outcomes: A learning healthcare 
pragmatic randomized trial 
Coordinating Center 
Center for Learning Healthcare, Vanderbilt Institute for Clinical 
and Translational Research (VICTR), Vanderbilt University 
Medical Center (VUMC)  
Sponsor 
T2 Biosystems 
Background 
Bacterial blood stream infections are common and life-
threatening.  Bloodstream infections have historically been 
identified using blood cultures, which often take 24-72 hours 
to result and are imperfectly sensitive.  Diagnostic tests that 
can rapidly detect bacteria directly from whole blood samples 
without relying on growth in culture are increasingly used in 
clinical care. Use of direct-from-blood bacterial testing has 
been hypothesized to decrease exposure to unnecessary 
antibiotics and shorten the time to appropriate antibiotic 
therapy. More evidence is needed to fully understand the 
effects of use of direct-from-blood bacterial testing, compared 
to blood cultures alone, on antibiotic administration and 
clinical outcomes. 
Aims  
Primary Aim: Evaluate the effect of using direct-from-blood 
testing, compared to blood cultures alone, on the time to last 
dose of intravenous vancomycin among adult patients 
presenting to the emergency department with suspected 
infection and empiric initiation of vancomycin. 
 
Secondary Aim: Evaluate the effect of using direct-from-blood 
testing, compared to blood cultures alone, on time to systemic 
anti-pseudomonal beta-lactam antibiotic among adult patients 
presenting to the emergency department with suspected 
infection and empiric initiation of vancomycin. 
Primary Hypothesis  
We hypothesize that using direct-from-blood testing, 
compared with blood cultures alone, will decrease the time to 
the time to last dose of intravenous vancomycin. 
Study Design 
Single-center, parallel-group, non-blinded, pragmatic, 
randomized clinical trial 
Inclusion Criteria 
• 
Patient is located in the Emergency Department at 
Vanderbilt University Hospital 
• 
≤ 12 hours from patient presentation to the 
Emergency Department at Vanderbilt University 
Hospital 
• 
Age ≥ 18 years 
• 
Clinician has ordered blood cultures 

Study Protocol- v1.5 
Full Study Title: Effect of direct-from-blood bacterial testing on antibiotic administration and clinical 
outcomes: A learning healthcare pragmatic randomized trial 
Primary Investigators: David Gaston, MD, PhD and Romney Humphries, PhD, D(ADBMM), M(ASCP)  
 
• 
Clinician has ordered intravenous vancomycin 
Exclusion Criteria 
• 
Patient is known to be a prisoner 
• 
Patient is known to be pregnant 
• 
Patient is known to have received 2 or more doses of 
vancomycin since presentation to the Vanderbilt ED 
• 
Patient is known to have a positive bacterial culture in 
the previous 7 days 
• 
Patient is known to have an infection for which at least 
7 days of intravenous vancomycin would routinely be 
administered regardless of bacterial testing results 
(e.g., skin and soft tissue infection) 
 
Randomization 
For patients who meet eligibility criteria, a computerized 
randomization within the electronic health record will assign 
patients in a 1:1 ratio to the direct-from-blood testing group or 
the blood culture group. 
Interventions  
Direct-from-blood testing Group.  In addition to blood 
cultures, patients will receive direct-from-blood testing using 
the T2Bacteria® Panel. 
Blood culture Group. Patients will receive blood cultures and 
will not receive direct-from-blood testing. 
Primary Outcome 
Time to last dose of intravenous vancomycin 
Secondary Outcome 
Time to last dose of systemic anti-pseudomonal beta-lactam 
antibiotic 
Analysis 
The primary analysis will be an intention-to-treat comparison 
of patients randomized to the direct-from-blood testing group 
versus patients randomized to the blood culture group with 
regard to the primary outcome of time to last dose of 
intravenous vancomycin using a Cox proportional hazards 
model. 
Sample Size  
The trial will include 500 patients (250 patients per group). 
Expected Duration  
12 months of enrollment 
 
2.0 Background 
 
2.1 Blood cultures versus direct-from-blood tests for identifying bacterial pathogens  
Bacterial infections of the bloodstream are a major cause of morbidity and mortality and are recognized 
as a major public health problem worldwide.1 Bacteria in the bloodstream have historically been 
identified using blood cultures.  Blood cultures, however, are limited by prolonged time to full 
identification - often 24-72 hours for bacteria2–5- and imperfect sensitivity that depends on the volume 
of the sample and a patient’s prior exposure to antibiotics.6  Because of these limitations, there has 
been increasing interest in using rapid diagnostic tests that identify bacteria directly from whole blood 

Study Protocol- v1.5 
Full Study Title: Effect of direct-from-blood bacterial testing on antibiotic administration and clinical 
outcomes: A learning healthcare pragmatic randomized trial 
Primary Investigators: David Gaston, MD, PhD and Romney Humphries, PhD, D(ADBMM), M(ASCP)  
 
samples without relying on growth in culture, referred to as “direct-from-blood” tests, in addition to 
standard blood cultures, to guide early therapeutic management of patients with suspected 
bloodstream infections .7–9   
 
One such direct-from-blood test is the T2Bacteria® Panel.  This panel’s accuracy and performance as a 
direct-from-blood test for bacterial pathogens has been described in previous studies.10–13 For this test, 
whole blood in a standard EDTA tube is used to amplify microbial cell-associated DNA for detection on a 
dedicated instrument platform in a clinical laboratory. This panel detects five bacterial pathogens: 
Staphylococcus aureus, Enterococcus faecium, Klebsiella pneumoniae, Pseudomonas aeruginosa, and 
Escherichia coli. These five bacterial pathogens account for approximately 50% of organisms from 
positive blood cultures, are known for their antibiotic resistance mechanisms and nosocomial spread, 
and are leading causes of sepsis.14–17 In a recent prospective, multicenter study, this direct-from-blood 
test demonstrated a sensitivity and specificity of 90% for these 5 organisms.  The negative predictive 
value was 99.7%.  Results were available within 4 to 8 hours.10 Additionally, a recent meta-analysis of 
largely observational studies reported a faster transition to targeted microbial therapy and de-escalation 
of empirical microbial therapy, as well as a shorter duration of intensive care unit stay and hospital stay 
for patients who received this direct-from-blood test.17  
 
2.2 Empiric, broad-spectrum antibiotics for suspected bacterial infections of the 
bloodstream 
Early administration of antimicrobial therapy is a fundamental component of the management of adults 
presenting to the hospital with a suspected bloodstream infection or sepsis.18 However, a major 
predictor of length of stay and mortality in these patients is administration of appropriate and targeted 
antimicrobial therapy.18 Because blood cultures frequently take 24-72 hours to result, patients are 
typically treated with empiric, broad-spectrum antibiotics.19  Thus, patients are commonly exposed to 
unnecessary antibiotics without evidence of infection or with evidence of infection requiring narrow 
antibiotic selection.  In a meta-analysis of sepsis studies, empirical antibiotic therapy was inappropriate 
for the organism that ultimately grew in culture in almost half of patients.18   
 
2.3 Empiric vancomycin for MRSA bacteremia  
Current guidelines recommend the use of empiric intravenous vancomycin as coverage for methicillin-
resistant S. aureus (MRSA) bacteremia.20  In a multicenter, prospective surveillance study of adults 
hospitalized with community-acquired pneumonia, approximately one third of patients received empiric 
anti-MRSA antibiotics (vancomycin or linezolid), despite only 2.7% of ICU patients and 0.7% of patients 
overall growing MRSA in culture.21 At Vanderbilt, vancomycin is the most commonly used antimicrobial 
and, as a result, the antimicrobial that generates the most cost for the health system.  Vancomycin 
requires careful monitoring due to its narrow therapeutic range and high risk of toxicity.22 Up to two-
thirds of patients receiving high dose vancomycin develop acute kidney injury.23,24 Vancomycin can also 
cause bone marrow suppression, linear IgA bullous dermatosis, anaphylaxis, and life-threatening 
hypersensitivity reactions.25–28 Administration of vancomycin to patients who do not have MRSA can 
lead to avoidable adverse drug events and costs, as well as drive antimicrobial resistance.  

Study Protocol- v1.5 
Full Study Title: Effect of direct-from-blood bacterial testing on antibiotic administration and clinical 
outcomes: A learning healthcare pragmatic randomized trial 
Primary Investigators: David Gaston, MD, PhD and Romney Humphries, PhD, D(ADBMM), M(ASCP)  
 
 
 
2.4 Overview of trial  
We will conduct a pragmatic, randomized clinical trial examining the effect of using direct-from-blood 
testing for bacterial pathogens, compared to using blood cultures alone, on antimicrobial receipt and 
clinical outcomes for adults presenting to the hospital with suspected infection who have been initiated 
on empiric therapy with intravenous vancomycin.  
 
2.5 Rationale for trial   
The accuracy and performance characteristics of direct-from-blood testing for bacterial pathogens are 
well-described.7,10–12,17 Direct-from-blood testing has been adopted into clinical care at health systems 
across the United States.  Several studies have also suggested the importance of pairing rapid diagnostic 
testing with active antimicrobial stewardship efforts to achieve antibiotic de-escalation.7–9,29 However, 
the effect of using direct-from-blood testing as a part of clinical care on antibiotic administration and 
clinical outcomes remains unclear. A rigorous, real-world, randomized trial is required to compare the 
effects of direct-from-blood testing to blood cultures alone on the de-escalation of empirical antibiotic 
therapy, transition to targeted antimicrobial therapy, antimicrobial stewardship efforts, and clinical 
outcomes.7,8,11,17,30,31 Before considering implementation of direct-from-blood testing across the health 
system, Vanderbilt seeks to better understand how the use of direct-from-blood testing for bacterial 
pathogens impacts use of antimicrobials, antimicrobial stewardship, health system resources, and costs.  
Therefore, this pragmatic, randomized clinical trial is designed to compare the use of direct-from-blood 
testing for bacterial pathogens to the use of blood cultures alone within clinical care.  
3.0 Aims and Hypotheses 
Study Aims 
Primary Aim: Evaluate the effect of using direct-from-blood testing, compared to blood cultures alone, 
on the time to last dose of intravenous vancomycin among adult patients presenting to the emergency 
department with suspected infection initiated on empiric vancomycin. 
 
Secondary Aim: Evaluate the effect of using direct-from-blood testing, compared to blood cultures 
alone, on time to last dose of systemic anti-pseudomonal beta-lactam antibiotic among adult patients 
presenting to the emergency department with suspected infection initiated on empiric vancomycin.  
 
Study Hypotheses 
We hypothesize that using direct-from-blood testing, compared to using blood cultures alone, will 
decrease the time from randomization to time to last dose of intravenous vancomycin among adult 
patients presenting to the emergency department with suspected infection initiated on empiric 
vancomycin. 
4.0 Study Description 
To address these aims, we will perform a single center, parallel-group, non-blinded, randomized clinical 
trial comparing the use of direct-from-blood testing for bacterial pathogens to blood cultures alone. The 

Study Protocol- v1.5 
Full Study Title: Effect of direct-from-blood bacterial testing on antibiotic administration and clinical 
outcomes: A learning healthcare pragmatic randomized trial 
Primary Investigators: David Gaston, MD, PhD and Romney Humphries, PhD, D(ADBMM), M(ASCP)  
 
study population will be 500 adult patients presenting to the Emergency Department at Vanderbilt 
University Medical Center with suspected infection for whom collection of blood cultures and empiric 
initiation of intravenous vancomycin are ordered within 12 hours of presentation to the Emergency 
Department.  Patients in the direct-from-blood testing group will receive both blood cultures and direct-
from-blood testing for bacterial pathogens.  Patients in the blood cultures group will receive blood 
cultures and will not receive direct-from-blood testing for bacterial pathogens.  The primary outcome 
will be the time to last dose of intravenous vancomycin.  The secondary outcome will be the time to 
definitive antibacterial therapy.  We will also report exploratory antimicrobial stewardship outcomes, 
safety outcomes, and clinical outcomes. 
5.0 Inclusion and Exclusion Criteria 
5.1 Inclusion Criteria 
• 
Patient is located in the Emergency Department at Vanderbilt University Hospital 
• 
≤ 12 hours from patient presentation to the Emergency Department at Vanderbilt University 
Hospital 
• 
Age ≥ 18 years 
• 
Clinician has ordered blood cultures 
• 
Clinician has ordered intravenous vancomycin 
 
5.2 Exclusion Criteria 
• 
Patient is known to be a prisoner 
• 
Patient is known to be pregnant 
• 
Patient is known to have received 2 or more doses of vancomycin since presentation to the 
Vanderbilt ED Patient is known to have a positive bacterial culture in the previous 7 days 
• 
Patient is known to have an infection for which at least 7 days of intravenous vancomycin would 
routinely be administered regardless of bacterial testing results (e.g., skin and soft tissue 
infection, etc.) 
6.0 Enrollment and Randomization 
6.1 Study Site 
Vanderbilt University Adult Hospital at Vanderbilt University Medical Center; Nashville, Tennessee, USA 
 
6.3 Enrollment  
At the time that a treating clinician in the Emergency Department at Vanderbilt University Adult Hospital 
initiates orders for blood cultures and intravenous vancomycin for a patient who meets all inclusion 
criteria, an advisor within the electronic health record (details in section 7) will prompt treating 
clinicians to record whether the patient meets any exclusion criteria.  If the treating clinician confirms 
that the patient does not meet any exclusion criteria, the patient will be enrolled and randomized.  
Enrollment times will be flexible to accommodate clinical staff. For patients who are determined to be 

Study Protocol- v1.5 
Full Study Title: Effect of direct-from-blood bacterial testing on antibiotic administration and clinical 
outcomes: A learning healthcare pragmatic randomized trial 
Primary Investigators: David Gaston, MD, PhD and Romney Humphries, PhD, D(ADBMM), M(ASCP)  
 
ineligible, the advisor will track the number and reasons for exclusion.  The time of randomization will 
be defined as “time zero” on “study day 0.”  
 
 
Figure 1: Enrollment Schema  
 
6.4 Consent  
 
Because this study involves minimal incremental risk, the study would not adversely affect the welfare 
or privacy rights of the participant, and obtaining informed consent would be impracticable, we will 
request a waiver of informed consent. Numerous previous randomized trials comparing two clinically 
available approaches to diagnosis of acute infection have been completed with a waiver of informed 
consent.29,32–34 
 

Study Protocol- v1.5 
Full Study Title: Effect of direct-from-blood bacterial testing on antibiotic administration and clinical 
outcomes: A learning healthcare pragmatic randomized trial 
Primary Investigators: David Gaston, MD, PhD and Romney Humphries, PhD, D(ADBMM), M(ASCP)  
 
6.4.1 Minimal risk  
This study compares two standard-of-care laboratory methods for detecting bacterial pathogens in 
blood samples.  The technology used in both laboratory methods is approved by the U.S. Food and Drug 
Administration, available for use in routine clinical care, and used in current clinical care in health 
systems across the U.S.  In current clinical care, the technology a health system uses to detect bacterial 
pathogens in the blood is based primarily on the preferences of health system leaders and laboratory 
administrators because no large, randomized trials or evidence-based guidelines support the use of one 
method over the other and clinicians, patients, and families generally do not determine which 
laboratory methods a health system uses to identify bacterial pathogens in blood samples.  As such, 
determining which laboratory method for detecting bacterial pathogens in blood samples a patient 
receives (blood cultures alone vs blood cultures plus direct-from-blood testing) as part of this research 
study poses minimal incremental risk compared to clinical care outside of the study.  The total volume of 
blood drawn is approximately 4 mL greater with the use of direct-from-blood testing and blood cultures, 
compared to blood cultures alone.  This volume of blood is similar to that normally drawn during a 
routine medical examination. Because a patient not participating in this study could experience any 
potential risks and benefits of use of direct-from-blood testing or use of blood cultures alone if 
presenting for care to a health system that primarily uses direct-from-blood testing or a health system 
that primarily uses blood cultures alone, a primary risk of the research that would not be present in 
clinical care is the potential for loss of confidentiality from the use of protected health information for 
research.  All data that will be used for the study are collected in the electronic health record as part of 
routine clinical care.  The extensive procedures the study takes to prevent loss of confidentiality are 
detailed in Section 13. 
 
6.4.2 Practicability of obtaining written, informed consent 
Obtaining written informed consent prior to enrollment in this study would be impracticable. Patients 
presenting to an emergency department with a suspected blood stream infection are experiencing a 
medical emergency. Blood for cultures or direct-from-blood testing must be obtained prior to 
administration of antibiotics and each hour of delay in the initiation of antibiotics increases mortality by 
7%.35  The average time between initiation of an order for an intravenous antibiotic and its 
administration is 28 minutes in the Emergency Department at Vanderbilt. Obtaining prospective written 
informed consent during this interval is impracticable and risks delaying antibiotic administration.  
Moreover, patients presenting to the Emergency Department with suspected infection requiring empiric 
intravenous antibiotics are frequently unconscious or delirious, and a legally authorized representative 
(LAR) is frequently not present.  Because the trial determines the laboratory method for detecting 
bacterial pathogens in the initial blood sample drawn before antibiotic administration in the Emergency 
Department but defers all subsequent aspects of treatment to clinicians (e.g., choice of antibiotics, 
timing of discontinuation of antibiotics), the primary study procedure will be completed within 1 hour of 
meeting eligibility criteria. While additional attempts to collect the sample for direct-from-blood testing 
may be made for up to 12 hours following randomization if an adequate sample is not initially drawn for 
patients in the intervention arm, consent would still not be feasible or practicable in this instance as 1) 

Study Protocol- v1.5 
Full Study Title: Effect of direct-from-blood bacterial testing on antibiotic administration and clinical 
outcomes: A learning healthcare pragmatic randomized trial 
Primary Investigators: David Gaston, MD, PhD and Romney Humphries, PhD, D(ADBMM), M(ASCP)  
 
consent would not be obtained for patients in usual care and thus 2) the study could be biased if 
patients differentially consented to participate in the study across study arms.   
 
6.5 Randomization  
Eligible patients will be randomized in a 1:1 ratio to the direct-from-blood test group or the blood 
culture alone group.  A series of study group assignments will be generated by computerized 
randomization using EPIC functionality.  Simple randomization without stratification, permuted blocks, 
or minimization will be used. 
7.0 Study Procedures 
 
7.1 Blood Culture Group 
For patients assigned to the blood culture group, the blood cultures ordered at the time of eligibility 
assessment will be collected, performed, and reported as in routine clinical care.  The blood culture 
testing will be performed in the clinical laboratory at Vanderbilt.  At Vanderbilt, blood culture results are 
updated intermittently as results become available. Negative blood cultures, similar to other tests 
without a critical result, are uploaded in the electronic health record for review by the clinician.  
Following a gram stain for a positive blood culture, the ePlex/Roche test is used.  This tests for 95% of 
the most common bacterial pathogens recovered in blood cultures.36 The results of the ePlex/Roche test 
on the blood culture are reported in the electronic health record along with an electronic notification 
(alert) to the clinical team.  Identification of selected organisms (e.g., S. aureus, Enterococcus and S. 
lugdunensis) are also alerted to the Infectious Disease fellows.  Blood containing bacteria is plated onto 
culture media to definitively identify organisms and characterize antibiotic susceptibility, if indicated. 
These results are also communicated to the clinical team in the electronic health record.  Patients will 
have access to these results in their MyHealth at Vanderbilt portal. 
 
7.2 Direct from Blood Test Group (Intervention Group) 
For patients assigned to the direct-from-blood test group, in addition to having blood cultures collected, 
performed, and reported in the process described above, direct-from-blood testing for bacterial 
pathogens will be performed.  The order for blood cultures will be accompanied by an order for direct-
from-blood testing for bacterial pathogens.  As the blood is being collected for blood cultures, clinical 
personnel will collect an additional 4 mL for direct-from-blood testing for bacterial pathogens as 
possible. If an adequate direct-from-blood test sample is not drawn (e.g. incorrect tube or insufficient 
sample volume, missed or cancelled lab order, etc.) additional attempts to collect the sample for direct-
from-blood testing may be made for up to 12 hours following randomization. Direct-from-blood testing 
for bacterial pathogens will be performed in the clinical laboratory.  Direct-from-blood testing will use 
the T2Bacteria® Panel.  Results of direct-from-blood testing for bacterial pathogens will be reported in 
the following ways: 
1. The results will be reported in the electronic health record in the same section on microbiology 
in which blood culture results are reported.  The report will contain a description of the 

Study Protocol- v1.5 
Full Study Title: Effect of direct-from-blood bacterial testing on antibiotic administration and clinical 
outcomes: A learning healthcare pragmatic randomized trial 
Primary Investigators: David Gaston, MD, PhD and Romney Humphries, PhD, D(ADBMM), M(ASCP)  
 
performance characteristics of the direct-from-blood testing and an interpretation of the 
findings. Patients will have access to these results in their MyHealth at Vanderbilt portal.  
2. The results will be sent to the treating clinicians on the patient’s primary team via a text page at 
the time the results populate into the patient’s electronic health record.  The text page will 
include the results for all five pathogens identified by the panel.  Treating clinicians will also be 
directed to information regarding interpreting the direct-from-blood testing results. 
3. For patients with direct-from-blood testing that is negative for S. aureus who continue to have 
an active order for intravenous vancomycin, an interruptive best practice alert (BPA) will remind 
treating clinicians of the result of the direct-from-blood testing and prompt the clinician to 
select a reason for continuing vancomycin if continued (e.g., known or suspected infection with 
a pathogen for which vancomycin is standard of care; severe or serious allergy to alternative 
antimicrobials; surgical prophylaxis; other). This BPA does not result in any automatic action and 
does not limit the autonomy of the care provider in the clinical decision-making process. All 
aspects of the patient’s treatment remain at the clinician’s discretion.  
4. For patients receiving intravenous vancomycin for whom the pharmacokinetics services is 
consulting, the pharmacokinetics service will incorporate the results of the direct-from-blood 
testing into their recommendations and communications with the treating clinicians (as they 
would standard blood culture results as well). 
 
Clinicians and patients may use these results, in addition to information about the patient’s 
demographics, chronic conditions, acute conditions, vital signs, other laboratory results, and concurrent 
treatments to help inform decisions.  Neither the blood cultures nor the direct-from-blood test will serve 
as the sole basis for diagnosis, treatment, or other management decisions. 
 
7.3 Co-interventions: Management of Vancomycin by Pharmacokinetics Service  
For patients in both trial groups of the current study, management of intravenous vancomycin by the 
pharmacokinetics service will occur as it does in routine clinical care.  At Vanderbilt, adult patients with 
an active order for intravenous vancomycin are followed by the pharmacokinetics service to determine 
the dosing regimen of vancomycin to maximize safety and therapeutic efficacy. The typical workflow 
consists of:  
• 
Assessing patient information necessary to dose and monitor the antibiotic. 
• 
Entering order for serum creatinine if one has not been obtained. 
• 
Entering order for dosage regimen based on pharmacokinetic principles. 
• 
Entering order for peak, trough, and/or random levels, as applicable. 
• 
Performing follow-up monitoring and/or dose adjustments, as needed. 
• 
Documenting a consult note in the electronic medical record upon initial consultation and daily 
until the protocol is discontinued.  
• 
Notifying the provider for further instructions if: 
o Adverse reactions or evidence of hypersensitivity. 

Study Protocol- v1.5 
Full Study Title: Effect of direct-from-blood bacterial testing on antibiotic administration and clinical 
outcomes: A learning healthcare pragmatic randomized trial 
Primary Investigators: David Gaston, MD, PhD and Romney Humphries, PhD, D(ADBMM), M(ASCP)  
 
o Systemic concentration of specified medication that is two times above the upper limit 
or if the concentration is significantly below the lower limit of the therapeutic range as 
defined by VUMC. 
o Increases in serum creatinine two times the patient’s baseline serum creatinine value.  
 
7.4 Blinding  
This will be an unblinded study. Given that this study evaluates the use of diagnostic testing to provide 
guidance to clinicians, it is not feasible to blind clinicians in this study.  As patients will receive the results 
of the diagnostic tests through communication with their clinicians and directly through access to their 
electronic health record, it is not feasible to blind patients in this study.  
 
8.0 Data Collection and Outcome Measures 
 
8.1 In-Hospital Outcomes   
 
Primary Outcome  
The primary outcome is time to last dose of intravenous vancomycin, defined as the time between 
randomization and the start time for the last dose of intravenous vancomycin received by the patient 
within 14 days of randomization.  
 
Secondary Outcome 
The secondary outcome is time to last dose of systemic anti-pseudomonal beta-lactam antibiotic, 
defined as the time between randomization and start time of the last dose of systemic anti-
pseudomonal beta-lactam antibiotic received by the patient within 14 days of randomization. 
 
Exploratory Outcomes 
 
Exploratory Antimicrobial Stewardship Outcomes: 
1. Total number of doses of intravenous vancomycin received between randomization and 14 days 
after randomization. 
2. Total number of days on which gram-positive antibacterial therapy was received between 
randomization and 14 days after randomization. 
3. Total number of days on which gram-negative antibacterial therapy was received between 
randomization and 14 days after randomization. 
4. Time to receipt of antibacterial therapy with effective coverage for blood stream infections 
identified by final blood culture results collected prior to or at the same time as randomization, 
assessed at 14 days after randomization.  
5. Proportion of patients who experienced Clostridioides difficile infection between randomization 
and hospital discharge or 28 days after randomization, whichever occurs first.  
 

Study Protocol- v1.5 
Full Study Title: Effect of direct-from-blood bacterial testing on antibiotic administration and clinical 
outcomes: A learning healthcare pragmatic randomized trial 
Primary Investigators: David Gaston, MD, PhD and Romney Humphries, PhD, D(ADBMM), M(ASCP)  
 
Exploratory Safety Outcomes: 
1. Proportion of patients who experienced an allergic reaction to antimicrobial therapy between 
randomization and 14 days after randomization 
2. Proportion of patients for whom vancomycin was discontinued between randomization and 72 
hours for whom any culture from the 24 hours prior to or 24 hours after randomization grew 
methicillin resistant S. aureus.  
3. Proportion of patients for whom all anti-staphylococcal therapy was discontinued between 
randomization and 72 hours for whom any culture from the 24 hours prior to or 24 hours after 
randomization grew S. aureus.  
4. Proportion of patients for whom all anti-pseudomonal therapy was discontinued between 
randomization and 72 hours for whom any culture from the 24 hours prior to or 24 hours after 
randomization grew P. aeruginosa 
 
Exploratory Clinical Outcomes: 
1. Highest stage of acute kidney injury by KDGIO creatinine criteria between randomization and 14 
days after randomization  
2. Receipt of renal replacement therapy between randomization and 14 days after randomization  
3. Lowest platelet count between randomization and 14 days after randomization 
4. Hospital-free days to day 28, defined as the number of calendar days alive and free of 
hospitalization between randomization and 28 days after randomization with outcome 
assessment censored at hospital discharge 
5. ICU-free days to day 28, defined as the number of calendar days alive and free of intensive care 
unit admission between randomization and 28 days after randomization with outcome 
assessment censored at hospital discharge 
6. All-cause, in-hospital mortality to day 28  
 
 
Process Measures 
 
Antimicrobial Stewardship Process Measures: 
1. Time from randomization to a positive test for bacteria in the blood (either direct-from-blood 
test or bacterial culture) 
2. Time from randomization to direct-from-blood test result (in direct-from-blood test group) 
3. Time from randomization to blood culture results (in blood culture group) 
4. Concordance between direct-from-blood result and blood culture result (in direct-from-blood 
test group) 
5. Receipt of non-vancomycin antimicrobial therapy for MRSA bacteremia in the 14 days following 
randomization 
6. Number of consultations to Infectious Disease in the 14 days following randomization 
7. Number of supratherapeutic vancomycin levels in the 14 days following randomization 
8. Number of patients followed by the Pharmacokinetics team.  
 

Study Protocol- v1.5 
Full Study Title: Effect of direct-from-blood bacterial testing on antibiotic administration and clinical 
outcomes: A learning healthcare pragmatic randomized trial 
Primary Investigators: David Gaston, MD, PhD and Romney Humphries, PhD, D(ADBMM), M(ASCP)  
 
8.2 Baseline Data  
We will collect demographic characteristics of the patients and details of their acute and chronic clinical 
conditions to describe the trial population at baseline.  Data collected will include: age, sex, race, 
ethnicity, body mass index, sepsis, sequential organ failure assessment score, suspected source of 
infection, presence of chronic kidney disease, presence of end-stage renal disease on renal replacement 
therapy, presence of acute kidney injury at enrollment, baseline vital signs (e.g., temperature, heart 
rate), baseline laboratory values (e.g., white blood cell count), comorbidities (e.g., Charlson comorbidity 
index), and time from presentation to the ED to enrollment. 
 
8.3 Data from enrollment to hospital discharge   
We will collect data on the diagnostic tests and treatments patients receive and their clinical condition 
from enrollment to hospital discharge.  Data collected will include: collection and results of cultures, 
collection and results of direct-from-blood testing for bacterial pathogens, receipt of antimicrobial 
medications, dose of antimicrobial medications, vital signs (e.g., temperature, heart rate), laboratory 
values (e.g., white blood cell count), organ function (e.g., sequential organ failure assessment score), 
location (e.g., emergency department, hospital ward, intensive care unit). 
 
8.4 Outcome Data  
We will collect data on the trial outcomes.  Data collected will include: timing and dose of antimicrobial 
therapy, antimicrobial sensitivities of organisms identified, allergic reactions to antimicrobials, receipt of 
organ support (e.g., kidney replacement therapy, mechanical ventilation), duration of intensive care unit 
admission, duration of hospital admission, and death.  
9.0 Risks and Benefits 
At this time, there is no reason to believe that participation in this study comparing two laboratory 
methods for identifying bacterial pathogens in blood samples would expose patients to greater medical 
risks or benefits than those experienced by acutely ill patients receiving blood tests for bacterial 
pathogens as a part of routine care. This study has the potential to provide a direct benefit to patients 
by decreasing the time to treatment with effective antimicrobial therapy and decreasing patients’ 
exposure to unnecessary antimicrobial therapy and associated side effects. However, whether such 
benefits exist is currently unknown.  
 
A potential risk to patients participating in this study involves the collection of protected health 
information (PHI). To limit the associated risks of inadvertent disclosure of PHI, the minimum amount of 
PHI necessary for study conduct will be collected. After collection, the data will be stored in a secure 
online database only accessible by the investigators and relevant key study personnel (KSP). Once the 
data collection period is complete, a de-identified research database will be created for data analysis.  

Study Protocol- v1.5 
Full Study Title: Effect of direct-from-blood bacterial testing on antibiotic administration and clinical 
outcomes: A learning healthcare pragmatic randomized trial 
Primary Investigators: David Gaston, MD, PhD and Romney Humphries, PhD, D(ADBMM), M(ASCP)  
 
10.0 Safety Monitoring and Adverse Events 
 
10.1 Adverse Event Definitions 
 
Adverse Event – An adverse event will be defined as any untoward or unfavorable medical occurrence in 
a human subject temporally associated with the subject’s participation in the research, whether or not 
considered related to the subject’s participation in the research. Any adverse event occurring during the 
research will be classified according to the following characteristics: 
 
● Seriousness – An adverse event will be considered “serious” if it: 
o Results in death 
o Is life-threatening (defined as placing the patient at immediate risk of death) 
o Results in inpatient hospitalization or prolongation of existing hospitalization 
o Results in a persistent or significant disability or incapacity 
o Results in a congenital anomaly or birth defect or  
o Based upon appropriate medical judgment, may jeopardize the patient’s health, and 
may require medical or surgical intervention to prevent one of the other outcomes 
listed in this definition. 
 
● Unexpectedness – An adverse event will be considered “unexpected” if the nature, severity, or 
frequency is neither consistent with: 
o The known or foreseeable risk of adverse events associated with the procedures 
involved in the research that are described in the protocol-related documents, such as 
the IRB-approved research protocol; nor 
o The expected natural progression of any underlying disease, disorder, or condition of 
the subject experiencing the adverse event and the subject’s predisposing risk factor 
profile for the adverse event. 
 
● Relatedness – The strength of the relationship of an adverse event to a study intervention or 
study procedure will be defined as follows:  
o Definitely Related: The adverse event follows (1) a reasonable, temporal sequence from 
a study procedure AND (2) cannot be explained by the known characteristics of the 
patient’s clinical state or other therapies AND (3) evaluation of the patient’s clinical 
state indicates to the investigator that the experience is definitely related to study 
procedures.  
o Probably or Possibly Related: The adverse event meets some but not all of the above 
criteria for “Definitely Related”. 
o Probably Not Related: The adverse event occurred while the patient was on the study 
but can reasonably be explained by the known characteristics of the patient’s clinical 
state or other therapies.  
o Definitely Not Related: The adverse event is definitely produced by the patient’s clinical 
state or by other modes of therapy administered to the patient.  
o Uncertain Relationship: The adverse event does not fit in any of the above categories. 

Study Protocol- v1.5 
Full Study Title: Effect of direct-from-blood bacterial testing on antibiotic administration and clinical 
outcomes: A learning healthcare pragmatic randomized trial 
Primary Investigators: David Gaston, MD, PhD and Romney Humphries, PhD, D(ADBMM), M(ASCP)  
 
 
10.2 Monitoring for Adverse Events 
The time interval during which patients will be monitored for the occurrence of adverse events begins at 
randomization and ends at the first of hospital discharge or 14 days. Adverse events occurring before 
randomization or after hospital discharge or 14 days will not be collected. The principal investigator (PI) 
will have primary responsibility for overseeing the monitoring, assessment, and reporting of adverse 
events. Site study personnel will evaluate for the occurrence of adverse events by review of the 
electronic health record and by communication with treating clinicians. Site study personnel will 
evaluate for the occurrence of adverse events by manual review of the electronic health record during 
initial data collection and during final data collection. Study personnel will also communicate regularly 
with the treating clinicians in the study environments to solicit information about any potential adverse 
events.  If study personnel at a site identify a potential adverse event, the PI will be immediately 
notified. The PI will assess the seriousness, unexpectedness, and relatedness of the potential adverse 
event. The PI at the site will determine whether the event qualifies for recording and reporting. 
 
10.3 Recording and Reporting Adverse Events 
The following types of adverse events will be recorded and reported: 
● Adverse events that are Serious and Definitely Related, Probably or Possibly Related, or of 
Uncertain Relationship. 
● Adverse events that are Unexpected and Definitely Related, Probably or Possibly Related, or of 
Uncertain Relationship.  
 
Adverse events that do not meet the above criteria will not be recorded or reported. Adverse events 
that the PI at a site assesses to meet the above criteria for recording and reporting will be entered into 
the adverse event electronic case report form in the trial database. The PI at the site will record an 
assessment of each characteristic for the adverse event, including seriousness, unexpectedness, and 
relatedness.  
For any adverse event that is serious AND unexpected, and definitely related, probably or possibly 
related, or of uncertain relationship, the PI will report the adverse event to the coordinating center 
within 24 hours of becoming aware of the adverse event. For any other adverse event requiring 
recording and reporting, the PI will report the adverse event to the coordinating center within 72 hours 
of becoming aware of the adverse event. The coordinating center will coordinate with the PI to obtain 
information about the adverse event regarding each characteristic for the adverse event, including 
seriousness, expectedness, and relatedness. The PI will be responsible for making final determinations 
regarding seriousness and unexpectedness. Upon formal reporting, the IRB will be responsible for 
making final determinations regarding relatedness. 
 
For adverse events that meet the above criteria for recording and reporting, the PI will notify the IRB 
and the sponsor in accordance with the following reporting plan: 
 
Characteristics of the Adverse Event 
Reporting Period 

Study Protocol- v1.5 
Full Study Title: Effect of direct-from-blood bacterial testing on antibiotic administration and clinical 
outcomes: A learning healthcare pragmatic randomized trial 
Primary Investigators: David Gaston, MD, PhD and Romney Humphries, PhD, D(ADBMM), M(ASCP)  
 
Fatal or life-threatening (and therefore serious), 
unexpected, and definitely related, probably or 
possibility related, or of uncertain relationship. 
Report to the IRB and sponsor within 7 days after 
notification of the event. 
Serious but non-fatal and non-life-threatening, 
unexpected, and definitely related, probably or 
possibly related, or of uncertain relationship. 
Report to IRB and sponsor within 15 days of 
notification of the event. 
 
10.4 Clinical Outcomes that may be Exempt from Adverse Event Recording and Reporting 
In this study of hospitalized patients with advanced illness at high risk for death and other adverse 
outcomes due to their underlying advanced illness, clinical outcomes, including death, will be 
systematically collected and analyzed for all patients. The primary, secondary, and exploratory 
outcomes will be recorded and reported as clinical outcomes and not as adverse events unless treating 
clinicians or site investigators believe the event is Definitely Related or Probably or Possibly Related to 
the study intervention or study procedures. This approach – considering death and other outcomes as 
clinical outcomes rather than adverse events and systemically collecting these clinical outcomes for 
analysis – is common in trials among patients with advanced illness. This approach ensures 
comprehensive data on death and other outcomes for all patients, rather than relying on sporadic 
adverse event reporting to identify these important events.  The following events are examples of study-
specific clinical outcomes that would not be recorded and reported as adverse events unless treating 
clinicians or site investigators believe the event was Definitely Related or Probably or Possibly Related to 
the study intervention or study procedures: 
● Death (all deaths occurring prior to hospital discharge or 28 days will be recorded) 
● Duration of ICU admission 
● Duration of hospitalization 
 
10.5 Unanticipated Problems involving Risks to Subjects or Others 
Investigators must also report to the coordinating center Unanticipated Problems Involving Risks to 
Subjects or Others (“Unanticipated Problems”), regardless of severity, associated with study procedures 
within 24 hours of the site investigator becoming aware of the Unanticipated Problem. An 
Unanticipated Problem is defined as any incident, experience, or outcome that meets all of the following 
criteria:  
● Unexpected (in terms of nature, severity, or frequency) given (a) the research procedures that 
are described in the protocol-related documents, such as the IRB-approved research protocol; 
and (b) the characteristics of the subject population being studied; AND 
● Definitely Related or Probably or Possibly Related to participation in the research (as defined 
above in the section on characteristics of adverse events); AND  
● Suggests that the research places subjects or others at a greater risk of harm (including physical, 
psychological, economic, or social harm) than was previously known or recognized.  
 
If any study personnel become aware of an event that may represent an Unanticipated Problem, they 
will immediately contact the PI. The PI will assess whether the event represents an Unanticipated 
Problem by applying the criteria described above. If the PI determines that the event represents an 

Study Protocol- v1.5 
Full Study Title: Effect of direct-from-blood bacterial testing on antibiotic administration and clinical 
outcomes: A learning healthcare pragmatic randomized trial 
Primary Investigators: David Gaston, MD, PhD and Romney Humphries, PhD, D(ADBMM), M(ASCP)  
 
Unanticipated Problem, the PI will record the Unanticipated Problem in the Unanticipated Problem 
electronic case report form in the trial database.  The PI will obtain information about the Unanticipated 
Problem and report the Unanticipated Problem to the IRB and sponsor within 15 days of becoming 
aware of the Unanticipated Problem. 
 
11.0 Study Withdrawal/Discontinuation 
Patients can be withdrawn from study participation in the following circumstances: 
• 
The investigator decides that the patient should be withdrawn for safety considerations. 
• 
There is a significant protocol violation in the judgment of the PI such that continuing the study 
would place the patient at undue risk. 
 
The reason and date of every withdrawal will be recorded in the patient study records. For patients in 
the direct-from-blood testing group, if they withdraw from the study prior to blood collection for the 
direct from blood test, the test will not be completed.  Once the blood for direct-from-blood testing has 
been sent to the laboratory, the order will not be discontinued, and the results will not be removed 
from the medical record.  Patients who withdraw from the study will be followed via medical record 
review to evaluate safety parameters but study personnel will not directly interact with a patient after 
withdrawal.  Follow-up will be performed for all patients who discontinue due to an adverse event or 
any other safety parameter.   If a patient experiences an adverse event meeting criteria reporting 
criteria above, the patient will be followed until the event has resolved, is deemed chronic and stable, or 
as long as clinically appropriate.  This follow-up will be documented in the patient study record.   
12.0 Statistical Considerations 
 
Sample size considerations 
The planned sample size for this trial is 500 patients. In a prior study in the same study location among a 
similar patient population who underwent blood culturing without direct-from-blood bacterial testing, 
the median time from randomization to discontinuation of intravenous vancomycin was 48 hours (IQR 
24 to 96).  Assuming a statistical power of 80%, a two-sided alpha of 0.05, and no missing data for the 
primary outcome, we calculated that detecting a 0.5 day (12 hour) difference in the primary outcome of 
time to discontinuation of intravenous vancomycin would require enrolling 500 patients (250 patients in 
each group). 
 
Trial profile:  
We will present a Consolidated Standards of Reporting Trials diagram to detail the movement of 
patients through the study. This diagram will include total number of patients meeting inclusion criteria, 
number excluded and reason for exclusion, number enrolled and randomized in the study, number 
followed, and number analyzed. 
 
Baseline Characteristics:  

Study Protocol- v1.5 
Full Study Title: Effect of direct-from-blood bacterial testing on antibiotic administration and clinical 
outcomes: A learning healthcare pragmatic randomized trial 
Primary Investigators: David Gaston, MD, PhD and Romney Humphries, PhD, D(ADBMM), M(ASCP)  
 
We will summarize in a Table the distribution of baseline variables across the study arms. Categorical 
variables will be reported as frequencies and percentages and continuous variables as either means with 
SDs or medians with interquartile ranges. 
 
Primary Analysis of the Primary Outcome: 
The primary analysis will be an intention-to-treat comparison of patients randomized to the direct-from-
blood testing group versus patients randomized to the blood culture alone group with regard to the 
primary outcome.  The primary outcome will be compared between trial groups using a Cox 
proportional hazards model. 
 
Secondary Analyses of the Primary Outcome: 
We will perform an intention-to-treat comparison of the primary outcome between groups using a 
multivariable Cox proportional hazard model adjusting for baseline characteristics, which will be 
prespecified in a statistical analysis plan. 
 
Analyses of Secondary and Exploratory Outcomes 
We will perform intention-to-treat comparisons of the secondary and exploratory outcomes between 
trial groups.  Categorical outcomes will be compared with a Chi-square test, continuous or ordinal 
outcomes will be compared with a Wilcoxan rank sum test, and time-to-event outcomes will be 
compared between groups using Kaplan-Meier curves or Cox proportional hazards models. 
 
Heterogeneity of Treatment Effect 
To evaluate whether pre-specified baseline variables modify the effect of trial group assignment on the 
primary outcome, we will perform Cox proportional hazards modelling with the primary outcome as the 
dependent variable and independent variables of the study group, the proposed effect modifier, and the 
interaction between the two. To account for non-linear relationships, continuous variables will be 
analyzed using restricted cubic splines with between 3 and 5 knots. Forest plots will be used to 
graphically display the adjusted analyses, and locally weighted regression or partial effects plots will be 
used to portray the association between continuous covariates and the outcome. A full list of 
prespecified variables for evaluating effect modification will be outlined in the detailed Statistical 
Analysis Plan prior to conclusion of enrollment. 
13.0 Privacy and Confidentiality  
At no time during the course of this study, its analysis, or its publication will patient identifiers be 
revealed outside the study team. The minimum necessary data containing patient or provider identities 
will be collected. As quickly as feasible, all data collected will be uploaded into a password-protected 
computerized database maintained within a secure, web-based application for building and managing 
online databases or stored on secure servers with user-level access control. All patients will be assigned 
a unique study number for use in the computerized database. At the time of publication all identifiers 
will be removed. 

Study Protocol- v1.5 
Full Study Title: Effect of direct-from-blood bacterial testing on antibiotic administration and clinical 
outcomes: A learning healthcare pragmatic randomized trial 
Primary Investigators: David Gaston, MD, PhD and Romney Humphries, PhD, D(ADBMM), M(ASCP)  
 
14.0 Follow-up and Record Retention 
Patients will be followed for 28 days or until hospital discharge, whichever occurs first. Records will be 
retained compliant with institutional, federal, and local regulations. PHI will be stored in a secure online 
database. Only relevant Key Study Personnel will have access to this database containing PHI.  Once the 
data collection period is complete, a de-identified research database will be used for analysis. 
Additionally, per the VUMC contract with T2 Biosystems, the study team will provide a de-identified 
data set through a secure, encrypted messaging system at the conclusion of data analysis, as 
appropriate.  Secondary use of the data will be with IRB approval.  The dataset may be made available 
outside of the study team on reasonable request with approval from an authorized Institutional Review 
Board and concurrence with the study team that the data are fit for purpose.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Study Protocol- v1.5 
Full Study Title: Effect of direct-from-blood bacterial testing on antibiotic administration and clinical 
outcomes: A learning healthcare pragmatic randomized trial 
Primary Investigators: David Gaston, MD, PhD and Romney Humphries, PhD, D(ADBMM), M(ASCP)  
 
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Study Protocol- v1.5 
Full Study Title: Effect of direct-from-blood bacterial testing on antibiotic administration and clinical 
outcomes: A learning healthcare pragmatic randomized trial 
Primary Investigators: David Gaston, MD, PhD and Romney Humphries, PhD, D(ADBMM), M(ASCP)  
 
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Study Protocol- v1.5 
Full Study Title: Effect of direct-from-blood bacterial testing on antibiotic administration and clinical 
outcomes: A learning healthcare pragmatic randomized trial 
Primary Investigators: David Gaston, MD, PhD and Romney Humphries, PhD, D(ADBMM), M(ASCP)  
 
23. Tongsai S, Koomanachai P. The safety and efficacy of high versus low vancomycin trough levels in 
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Study Protocol- v1.5 
Full Study Title: Effect of direct-from-blood bacterial testing on antibiotic administration and clinical 
outcomes: A learning healthcare pragmatic randomized trial 
Primary Investigators: David Gaston, MD, PhD and Romney Humphries, PhD, D(ADBMM), M(ASCP)  
 
35. Liu VX, Fielding-Singh V, Greene JD, et al. The Timing of Early Antibiotics and Hospital Mortality in 
Sepsis. Am J Respir Crit Care Med. 2017;196(7):856-863. doi:10.1164/rccm.201609-1848OC 
36. GenMark Diagnostics. Blood Culture Identification (BCID) Panels. Published 2023. Accessed July 3, 
2023. https://www.genmarkdx.com/panels/eplex-panels/bcid-panels/ 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Study Protocol- v1.5 
Full Study Title: Effect of direct-from-blood bacterial testing on antibiotic administration and clinical 
outcomes: A learning healthcare pragmatic randomized trial 
Primary Investigators: David Gaston, MD, PhD and Romney Humphries, PhD, D(ADBMM), M(ASCP)  
 
16.0 Appendix 
16.1 Enrollment Pause  
On December 25, 2023, study enrollment was paused (36 patients had been enrolled in the trial). The 
study team deemed this necessary due to logistical challenges in receiving an adequate direct-from-
blood test sample volume for patients in the intervention arm, as well as to evaluate the performance of 
the instrument that runs the direct-from-blood test samples. All components of trial procedures were 
evaluated at this time. The trial will resume enrollment upon implementation of procedures to ensure 
intervention fidelity.  
 
 

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