Impact of a Multiplex Respiratory PCR Test on Outcomes for Patients Presenting With Respiratory Illness in the Urgent Care Setting: A Hybrid-Effectiveness Quasi-Experimental Trial
Phase: NA
Conditions: Acute Respiratory Infections (ARIs)
Sponsor: BioMérieux

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Impact of a Multiplex Respiratory PCR Test on Outcomes for 
Patients Presenting with Respiratory Illness in the Urgent Care 
Setting: A Hybrid-Effectiveness Quasi-Experimental Trial  
BioMérieux-Initiated Research Project
 
Sponsor: 
BioMérieux 
1201 S 4800 W  
Salt Lake City, UT 84104 
1This document is the property of bioMérieux. All rights strictly reserved. Use, reproduction, issue, loan, 
or disclosure of the contents to third parties in any form whatsoever is not permitted without written 
authority from the proprietor except that this document may be disclosed to appropriate institutional 
review committees so long as they are requested to keep it confidential. 
The information provided in this document may not be used or made public without our explicit consent, 
and is to be regarded as a trade secret in that it contains unpublished results of private research which 
are used in our business and which gives an opportunity to obtain an advantage over competitors who do 
not know or use it, and/or as commercial or financial information that is privileged or confidential in that 
it contains valuable data or information which is used in our business and is of a type customarily held in 
strict confidence or regarded as privileged and not disclosed to any member of the public by the person 
to whom it belongs.  As such, it is protected from disclosure by the law of several countries such as the 
U.S. Freedom of Information Act. 
 
 

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Accountable & Approvers
Name 
Function
Signature/Date
Brianne Couturier
Director, Clinical Research Scientists
Andrea Prinzi 
Principal Investigator, Director, 
Scientists 
Contributors 
Name 
Function
Zoë Heins
Clinical Research Scientist
Alyssa Duenes
Associate Clinical Research Scientist 
Reviewers 
Name 
Function
Minkey Wungwattana
Field Medical Director, US Medical Affairs 
Brian Raux 
Medical Advisor, US Medical Affairs 
Barbara Powe
Senior Market Access Policy Strategy Manager
Stéphanie Pascual 
Disease State Scientist 
Informed 
Name 
Function
Charles Cooper 
Executive Vice President, Chief Medical Officer
John Osieki
VP Medical Affairs North America
John Hurst 
Sr Director, Field Medical Affairs
Spencer Hunt 
Sr Product Manager, Molecular Product Management
Anastasia Petrova 
Sr Product Manager, Molecular Product Management
Brett Barrett
Sr Market Shaping & Development Manager, Molecular Franchise

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Table of Modifications 
Revision Page(s) or chapter(s) modified
Description of the modification
1
All
Creation

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PROTOCOL APPROVAL SIGNATURE PAGE 
 
Protocol Number: BFD-BIR-24-006 
Full protocol name: Impact of a Multiplex Respiratory PCR on Outcomes for Patients Presenting with 
Respiratory Illness in the Urgent Care Setting: A Hybrid-Effectiveness Quasi-Experimental Trial 
Protocol Version: 1.0 
Protocol Date: 9/19/25 
 
This protocol has been read and approved by: 
SPONSOR: 
 
 
Brianne Couturier, PhD
Director, Global Medical Affairs
bioMérieux 
Add others as required. 
 
Date (dd/mmm/yyyy) 

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INVESTIGATOR PROTOCOL SIGNATURE PAGE 
 
I have read and understand this protocol and will conduct the study in accordance with this protocol, all 
attachments and amendments, applicable Food and Drug Administration regulations, HIPAA, IRB 
requirements, and the policies of the institutions where the study will take place.  
In my formal capacity as Investigator, my duties include ensuring the safety of the study subjects 
enrolled under my supervision and providing bioMérieux with complete and timely information, as 
outlined in the protocol.  It is understood that all information pertaining to the study will be held strictly 
confidential and that this confidentiality requirement applies to all study staff at this site. 
Protocol Number: BFD-BIR-24-006 
Protocol full name: Impact of a Multiplex Respiratory PCR on Outcomes for Patients Presenting with 
Respiratory Illness in the Urgent Care Setting: A Hybrid-Effectiveness Quasi-Experimental Trial 
Protocol Version: 1.0 
Protocol Date: 9/19/25 
Investigator: 
(Print Name)
 
(Signature)
Date (dd/mmm/yyyy) 
(Print Name)
 
(Signature)
Date (dd/mmm/yyyy) 
Upon signing, send a copy of this page to bioMérieux and retain a copy for your files 
 

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Table of Contents 
PROTOCOL APPROVAL SIGNATURE PAGE ..................................................................................................... 4 
INVESTIGATOR PROTOCOL SIGNATURE PAGE .............................................................................................. 5 
1. 
INTRODUCTION ..................................................................................................................................... 8 
2. 
DEFINITIONS .......................................................................................................................................... 9 
3. 
PRODUCT........................................................................................................................................... 100 
3.1 Product Name and Intended Use .................................................................................................... 100 
3.2 Product Description ........................................................................................................................ 100 
4. 
OBJECTIVES ....................................................................................................................................... 122 
5. 
BIOETHICS REQUIREMENTS .............................................................................................................. 133 
6. 
STUDY SCHEDULE .............................................................................................................................. 134 
6.1 Pre-Implementation ........................................................................................................................ 134 
6.2 Active Intervention ......................................................................................................................... 144 
6.3 Post-Implementation ...................................................................................................................... 155 
6.4 Study Schedule of Activities ............................................................................................................ 155 
7. 
METHODS .......................................................................................................................................... 155 
7.1 Site-Specific Information ................................................................................................................. 155 
7.1.1 Description of Study Sites …………………………………………………………………………………………………………16 
7.1.2 Site-Specific Data Collection………………………………………………………………………………………………………16 
7.2 Inclusion Criteria ............................................................................................................................... 18 
7.3 Exclusion Criteria ............................................................................................................................... 18 
7.4 Subject Enrollment ............................................................................................................................ 18 
7.5 Screen Failures .................................................................................................................................. 19 
7.6 Informed Consent ............................................................................................................................. 19 
7.7 Intervention Sample Collection and Handling ................................................................................ 200 
7.8 Quality Control Testing ................................................................................................................... 211 
7.9 Diagnostic and Treatment Algorithm .............................................................................................. 211 
7.10 Data Capture Plan ......................................................................................................................... 211 
7.11 Data Analysis Plan ........................................................................................................................... 22 
7.11.1 Sample Size and Power Calculation ............................................................................................. 22 
7.12 Statistical Analysis ……………………………………………………………………………………………………………………..22 
8. 
STUDY SUSPENSION OR EARLY TERMINATION ................................................................................... 25 
8.1 Procedure for Suspension or Early Termination ............................................................................... 25 
8.2 Procedure for Resuming the Clinical Investigation After Temporary Suspension ............................ 26 
8.3 Close Out Activities ........................................................................................................................... 26 
9. 
MATERIALS/EQUIPMENT .................................................................................................................... 26 
9.1 Provided by bioMérieux: ................................................................................................................... 26 

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9.2 Provided by Site: ............................................................................................................................... 26 
10. TRAINING REQUIREMENT ................................................................................................................... 27 
11. PROTOCOL DEVIATIONS ...................................................................................................................... 27 
12. ADVERSE EVENTS ................................................................................................................................ 27 
13. CLINICAL MONITORING ....................................................................................................................... 29 
13.1 Site Monitoring ............................................................................................................................... 29 
13.2 Monitoring Activities ....................................................................................................................... 29 
14. REFERENCES ...................................................................................................................................... 300 
15. APPENDICES ........................................................................................................................................ 33 
APPENDIX 1: BIOFIRE® SPOTFIRE® Respiratory/Sore Throat (R/ST) Panel  IFU ...................................... 34 
APPENDIX 2: BIOFIRE® SPOTFIRE® Respiratory/Sore Throat (R/ST) Panel Quick Guide ......................... 35 
APPENDIX 3: Study Flow Diagram ........................................................................................................... 36 
APPENDIX 4: Study Schedule of Activities .............................................................................................. 37 
APPENDIX 5: Study Data Table ................................................................................................................ 39 
APPENDIX 6: Provider Survey .................................................................................................................. 45 
APPENDIX 7: Patient Survey .................................................................................................................... 46 
APPENDIX 8: Diagnostic Treatment Algorithm ....................................................................................... 47 

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1.
INTRODUCTION
Antimicrobial resistance (AMR) is a critical public health challenge and a significant global burden.[1] In 
the United States, most antibiotics are prescribed in outpatient settings, with suspicion or confirmation 
of a respiratory infection serving as a primary driver of unnecessary antibiotic use. This use is deemed 
inappropriate in approximately 30% of cases [2]. This estimate differs based on setting and patient 
population. For example, a study assessing antibiotic use in a Veterans Affairs (VA) hospital 
demonstrated that 64.2% of patients received unnecessary antibiotics, with diagnoses such as bronchitis 
or nonspecific upper respiratory infections being the most likely to result in antibiotic use.[3] 
Importantly, antibiotic overuse is not without consequences, as it is associated with adverse events that 
frequently lead to emergency department (ED) visits.[2] Regardless of setting, acute respiratory 
conditions, such as acute bronchitis and rhinosinusitis are associated with the highest rates of 
inappropriate antibiotic prescribing.[4] In cases of pharyngitis, similar trends have been observed. A 
2020 study by Shapiro et al. revealed that while Group A streptococcus (GAS) pharyngitis prevalence is 
20-30% in children and 5-15% in adults, antibiotics were prescribed in 57% of cases across these groups. 
Notably, 50% of patients received antibiotics without prior GAS testing, even though laboratory testing 
is linked to more appropriate prescribing.[5] In a pediatric study, unnecessary antibiotic prescribing 
occurred in 58% of outpatient visits for bronchitis or bronchiolitis, with diagnostic uncertainty identified 
as a key factor driving inappropriate use.[6] 
 
The role of multiplex rapid diagnostic tests (mRDTs) in the inpatient setting has been the focus of many 
studies. Its role on the outpatient care setting remains less studied. In outpatient settings, prescribing 
behaviors are influenced by factors such as the lack of an established patient-provider relationship, time 
constraints, patient satisfaction, and medicolegal considerations.[7] Early evidence from studies 
implementing mRDTs suggests that molecular respiratory rapid diagnostic tests have the potential to 
positively impact patient care. In ED settings, studies have demonstrated benefits such as decreased test 
turnaround times (TAT), reduced lengths of stay, and more appropriate antiviral use.[8],[9],[10] 
However, the impact on antibiotic use and ED length of stay remains mixed, with clinical decision-
making and contextual factors playing crucial roles.[11],[12] 
 
The Centers for Disease Control and Prevention (CDC) provides core elements for outpatient antibiotic 
stewardship programs (ASP) [13], and studies performed in outpatient settings that leverage elements 
of these recommendations within multifactorial interventions have shown promise in improving 
stewardship outcomes. Gerber et al. demonstrated that clinician education combined with audit and 
feedback improved adherence to prescribing guidelines for bacterial acute respiratory tract 
infections.[14] Similarly, Finkelstein et al. highlighted the effectiveness of a physician behavior change 
strategy paired with parental education in reducing antibiotic use.[15] More recently, Stenehjem et al. 
demonstrated that a multifaceted stewardship approach—including education for both providers and 

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patients, electronic health record tools, benchmarking, and media campaigns—led to reduced antibiotic 
prescribing for respiratory infections in urgent care settings.[16] 
Performance metrics, such as those from the Healthcare Effectiveness Data and Information Set (HEDIS), 
play a vital role in evaluating antibiotic stewardship efforts. HEDIS measures, used by over 90% of U.S. 
health plans, help identify performance gaps. Analysis of 2008-2012 HEDIS data that described antibiotic 
prescribing for acute bronchitis demonstrated poor performance across insurance plans, with antibiotics 
prescribed in 80% of adult cases.[17] Recently, a newly validated HEDIS measure has been introduced to 
assess antibiotic use for respiratory conditions, emphasizing “measurement” (i.e., utilization, 
improvement) as a core element of outpatient stewardship.[18] This measure has broad support among 
antibiotic stewardship programs and health plan stakeholders and is used to evaluate care quality for 
over 200 million individuals. 
 
Implementation science is a relatively recent arm of clinical research that aims to increase the adoption 
and sustainability of evidence-based interventions.[19],[20] Years of research has demonstrated that 
innovation uptake relies on contextual factors in addition to effectiveness. To fully understand the 
impact of diagnostic innovation on clinical outcomes, it is important to consider these elements 
together. 
2. 
DEFINITIONS 
 
ABX: Antibiotics 
 
AMR: Antimicrobial Resistance 
 
ASP: Antibiotic Stewardship Programs 
 
CAHPS: Consumer Assessment of Healthcare Providers and Systems, a program developed by the 
Agency for Healthcare Research and Quality.  
 
CDC: Centers for Disease Control and Prevention 
 
CFR: Code of Federal Regulations 
 
CLIA: Clinical Laboratory Improvement Amendments 
 
DID: Difference-in-Differences 
 
DO: Doctor of Osteopathic Medicine 
 
ED: Emergency Department 
 
EMR: Electronic Medical Record 
 
GAS: Group A Streptococcus 
 
GCP: Good Clinical Practices 
 
GDP: Good Documentation Practices 
 
GLP: Good Laboratory Practices 
 
GMP: Good Manufacturing Practices 
 
HEDIS: Healthcare Effectiveness Data and Information Set, a set of standardized performance 
measures used to assess the quality of care provided by health plans.  

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IFU: Instructions for Use 
 
MD: Medical Doctor 
 
mRDT: Multiplex Rapid Diagnostic Tests 
 
NCQA: National Committee for Quality Assurance 
 
NP: Nurse Practitioner 
 
NPS: Nasopharyngeal Swab 
 
PA: Physician Assistant 
 
QC: Quality Control 
 
R: Respiratory 
 
PCR: Polymerase Chain Reaction 
 
SOC: Standard of Care 
 
ST: Sore Throat 
 
TAT: Turnaround Time 
 
TS: Throat Swab 
 
VA: Veterans Affairs 
3. 
PRODUCT  
3.1 Product Name and Intended Use 
The BIOFIRE® SPOTFIRE® Respiratory/Sore Throat (R/ST) Panel is a multiplexed polymerase chain 
reaction (PCR) test intended for use with the BIOFIRE® SPOTFIRE® System for the simultaneous, 
qualitative detection and identification of multiple respiratory viral and bacterial nucleic acids in 
nasopharyngeal swab (NPS) specimens obtained from individuals with signs and symptoms of 
respiratory tract infection, including COVID-19; (Respiratory menu) or in throat swab (TS) specimens 
from individuals with signs and symptoms of pharyngitis (Sore Throat menu). Time from test initiation to 
result is approximately 15 minutes.  
 
3.2 Product Description 
The SPOTFIRE R/ST Panel, designed for use with the SPOTFIRE System, is a PCR based sample-to-answer 
diagnostic test that simultaneously identifies nucleic acids from 15 different bacterial and viral 
organisms from nasopharyngeal swab (NPS) specimens, or 14 different bacterial and viral organisms 
from TS specimens, in transport media collected from individuals with signs and symptoms of 
respiratory infection or pharyngitis, respectively. The SPOTFIRE R/ST Panel uses a single instrument 
protocol with different reporting of analytes for the two sample types. Sample type is selected at the 
time of testing and the system’s software controls the analyte reporting based on the selected sample 
type. 
Nucleic acids from the viral and bacterial organisms identified by this test are generally detectable in 
NPS/TS specimens during the acute phase of infection. The detection and identification of specific viral 

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and bacterial nucleic acids from individuals exhibiting signs and symptoms of respiratory infection 
and/or pharyngitis are indicative of the presence of the identified microorganism and aids in diagnosis if 
used in conjunction with other clinical and epidemiological information, and laboratory findings. The 
results of this test should not be used as the sole basis for diagnosis, treatment, or other patient 
management decisions. 
Negative results in the setting of a respiratory illness and/or pharyngitis may be due to infection with 
pathogens that are not detected by this test, or a respiratory tract infection that may not be detected by 
an NPS or TS specimen. Positive results do not rule out the possibility of coinfection with other 
organisms. The agent(s) detected by the SPOTFIRE R/ST Panel may not be the definite cause of disease. 
At the clinician’s discretion, additional laboratory testing (e.g., bacterial and viral culture, 
immunofluorescence, and radiography) may be performed when evaluating a patient with possible 
respiratory tract infection and/or pharyngitis. 
The SPOTFIRE R/ST Panel is intended for use by non-laboratory trained healthcare professionals in a 
facility holding a CLIA Certificate of Waiver (in the United States) or it may also be used by trained 
medical and laboratory professionals in a laboratory setting or under the supervision of a trained 
laboratory professional. 
The SPOTFIRE R/ST Panel identifies and differentiates the following organism types and subtypes:  
 
 Respiratory Menu 
Sore Throat Menu  
Viruses 
Viruses  
Adenovirus 
Adenovirus 
Coronavirus SARS-CoV-2 
Coronavirus (seasonal)  
Coronavirus (seasonal) 
Human metapneumovirus 
Human metapneumovirus  
Human rhinovirus/enterovirus 
Human rhinovirus/enterovirus 
Influenza A virus  
Influenza A virus  
Influenza A virus/ H1-2009 
Influenza A virus/ H1-2009 
Influenza A virus/ H3 
Influenza A virus/ H3  
Influenza B virus 
Influenza B virus  
Parainfluenza virus 
Parainfluenza virus  
Respiratory syncytial virus  
Respiratory syncytial virus  
 
Bacteria  
Bacteria 
Bordetella parapertussis 
Chlamydia pneumoniae 
Bordetella pertussis 
Mycoplasma pneumoniae  
Chlamydia pneumoniae 
Streptococcus dysgalactiae (Group C/G 
Strep) 

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Mycoplasma pneumoniae  
Streptococcus pyogenes (Group A Strep) 
4. 
OBJECTIVES 
This research study will use a hybrid effectiveness-implementation type 1 design to assess effectiveness 
of the Spotfire R/ST assay in the urgent care setting. A hybrid effectiveness-implementation type 1 study 
primarily focuses on the effectiveness of an intervention (e.g., use of Spotfire R/ST) but also 
simultaneously allows for the collection of data on the barriers and facilitators of implementing the 
Spotfire R/ST test in the real-world urgent care setting.  
Primary Research Aims of the study are as follows: 
Aim 1: Assess the impact of the Spotfire R/ST assay on antibiotic prescribing practices, , patient, and 
workflow in the urgent care setting.  
Aim 2: Identify barriers and facilitators to the adoption and implementation of Spotfire R/ST in the urgent 
care setting. 
In addition to clinical impact data, the study aims to also address the following questions:  
1. What barriers and facilitators affect the real-world implementation of the Spotfire R/ST assay 
when combined with provider and patient education?  
2. What challenges arise in delivering the Spotfire R/ST intervention (i.e., Spotfire R/ST + medical 
education and patient education), and how might they inform broader implementation?  
3. What modifications to practice could enhance Spotfire R/ST adoption and effectiveness?  
4. What implementation strategies are most promising? 
 
Primary outcome: Antibiotic utilization for respiratory conditions (HEDIS AXR) – The % of episodes for 
patients one year and older with a diagnosis of a respiratory condition that results in an antibiotic 
prescribing event. (Note: while the HEDIS measure includes all patients greater than 3 months of age, 
AFC’s electronic health record (EHR) stores age as whole years. Excluding patients with a reported age of 
0 from the baseline data resulted in only 0.03% of observations lost.)  
Secondary outcomes:  
 
Effectiveness and utilization outcomes:  
o Percentage of episodes for patients one year and older with a diagnosis of acute 
bronchitis/bronchiolitis that did not result in an antibiotic prescribing event (i.e., the 
percentage of episodes that were not prescribed an antibiotic) (HEDIS AAB)  

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o Percentage of episodes for members 3 years of age and older with a diagnosis of 
pharyngitis, dispensed an antibiotic and received a group A streptococcus test for the 
episode (HEDIS CWP)  
o Percentage of episodes for patients 3 years of age or older with a positive Group A 
Strep (GAS) test and prescribed an antibiotic   
o Percentage of patients with a positive influenza test who receive an antiviral for 
influenza  
o Percentage of patients without a positive influenza test with a clinical diagnosis of 
influenza who receive an antiviral for influenza  
o CAHPS clinician and group survey (patient satisfaction)  
o Workflow (specific measures to be determined from pre-intervention qualitative 
survey with AFC urgent care providers)  
o Length of patient visit (from check-in to discharge)  
 
Implementation outcomes:  
o Reach, effectiveness, adoption, implementation, maintenance (RE-AIM) outcomes 
5. 
BIOETHICS REQUIREMENTS 
Pursuant to 21 Code of Federal Regulations (CFR) 56, the study must be registered with and obtain IRB 
approval or waiver prior to beginning testing. The IRB letter must be provided to bioMérieux, Inc. and a 
copy retained at the site. No Resistance Consulting Group will be primarily responsible for obtaining this 
documentation and managing ongoing communications with the IRB as needed.  
The BIOFIRE SPOTFIRE system and panels will be manufactured in compliance with Good Manufacturing 
Practices (GMPs). The study will be conducted in compliance with Good Clinical Practices (GCPs), Good 
Documentation Practices (GDPs), and Good Laboratory Practices (GLPs).  
Protected Health Information (PHI) must not be provided to bioMérieux. Electronic Medical Record (EMR) 
and survey data shared with bioMérieux must be deidentified and linked by a study-specific identifier as 
specified in Data Capture Plan. 
 
 
6. 
STUDY SCHEDULE 
6.1 Pre-Implementation 

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The pre-implementation portion of the study, including the development and administration of the 
provider pre-implementation survey, development of the urgent care stewardship intervention, and work 
with the onsite clinical champion to understand clinic workflow, will take an estimated total of 4 weeks. 
Additionally, during this period, a bioMérieux Field Application Specialist will install the SPOTFIRE system 
at the intervention site and provide training of on-site users. Concurrently, members of the bioMérieux 
Global Medical Affairs and US Medical Affairs teams will provide general and non-product focused 
educational content on respiratory diagnosis and ASP/antibiotic prescribing best practices. This content 
will be reviewed and adapted by the AFC team and will be reviewed with the AFC Providers at the 
intervention site prior to the start of the active intervention phase.   
Based on the results of the provider pre-survey, site visit, and discussions with the clinical champion, 
members of the AFC and bioMérieux research teams will design an AMS intervention bundle to be 
implemented with the Spotfire assay at the intervention site. This intervention will likely include a bi-
weekly audit and feedback process providing peer comparison on antibiotic prescribing to providers, as 
well as the development of a diagnostic testing and prescribing algorithm designed by AFC to be used by 
providers in the intervention clinic. Additional AMS or implementation science elements may be included 
as needed based on the results of the pre-implementation work (i.e., based on barriers and facilitators to 
adoption that are identified during the pre-intervention period).  
6.2 Active Intervention  
Patients at both the intervention and control sites will be prospectively enrolled in the study over the 
course of 12 weeks during peak respiratory season  
At the intervention site, subjects meeting the inclusion and exclusion criteria, and who provide consent 
to participate in the study, will have a respiratory sample tested on the SPOTFIRE R/ST Panel, with results 
used by the treating clinician to guide management. At the end of the visit, enrolled patients will take a 
satisfaction survey, based on the AHRQ-validated CAHPS survey. Providers at the intervention site will 
participate in the AMS and implementation science interventions designed in the pre-implementation 
phase.  Clinicians will be encouraged to perform intervention testing only before considering SOC testing 
for enrolled patients. 
Patients who do not meet inclusion and exclusion criteria or those who do meet inclusion and exclusion 
criteria but decline participation in the study will receive SOC antigen testing as per standard clinic 
procedure. Additionally, due to the high volume of patients presenting to the clinics, a monthly enrollment 
cap will be set to ensure even distribution of patients across the enrollment period. Patients meeting 
inclusion and exclusion criteria who present to the clinic after monthly enrollment targets have been met 
will not be enrolled in the study and will be treated according to SOC.   
In the instance that providers choose to run SOC testing on intervention group patients (in addition to 
intervention SPOTFIRE R/ST testing), this data will be collected and analyzed along with all other data 

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listed in the table below. Clinicians will be encouraged to perform intervention testing before considering 
SOC testing for enrolled patients.  
At the control site and at the discretion of the treating clinician, patients will have their respiratory 
samples tested on the clinic’s usual standard of care (SOC) respiratory antigen tests, with results used by 
the treating clinician to guide management.  Patients at the control site who meet inclusion and exclusion 
criteria and agree to participate will also take the CAHPS-based satisfaction survey.  
See appendix 3 for study flow diagram. 
6.3 Post-Implementation  
After patient enrollment is completed at the end of January 2026, the medical records of participating 
subjects (from both the control and interventional sites) will be collated into a report by an AFC 
bioinformatics team who will extract/collate the relevant data points required for results analysis. 
6.4 Study Schedule of Activities 
See Appendix 4 for full schedule of activities 
7. 
METHODS 
7.1 Site-Specific Information
 

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7.1.1 Description of Study Sites 
Two urgent care sites within the American Family Care (AFC) Alabama system were selected for 
participation. Sites were chosen based in part on baseline comparability (e.g., similar baseline 
demographics, prescribing rates, policies and procedures, standard of care methods) and geographic 
distance apart, to reduce contamination of the intervention. Additionally, the baseline antibiotic 
prescribing rate for each site was compared to confirm that the parallel trends assumption (required for 
a difference-in-differences analysis) was met. Based on these criteria, the Gardendale and Grove AFC 
sites were selected. In the baseline period (October 2024 – January 2025), the average antibiotic 
prescribing rate for Gardendale was 58.0% [95% CI: 56.4%, 59.6%], and the average antibiotic 
prescribing rate for Grove was 77.4% [95% CI: 75.6%, 79.1%]. The correlation coefficient between the 
monthly average antibiotic prescribing rate for the two sites was 0.56. Due to the high rate of 
prescribing at Grove, this was identified as the intervention site. 
Figure 1: Antibiotic prescribing trends at Gardendale and Grove urgent care sites during the baseline 
period.
7.1.2 Site-Specific Data Collection
The following information will be collected from each site:
Information collected via EMR: 
o
See Study Data Table in Appendix 5

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Information Collected via provider survey: 
o See Appendix 6 
 
Information collected via patient survey: 
o See Appendix 7 
 
General Site Information to be collected:  
 
 
Implementation Outcomes 
Domain 
Measurement 
Reach 
Quantitative:
 
Total number of consistently employed (non-
float) employees that prescribe antimicrobials at 
the intervention site  
 
Proportion of staff members who participate in 
the pre-intervention educational session(s) 
Qualitative 
 
Pre-survey to understand attitudes around both 
prescribing practices and respiratory mPCR 
diagnostics, willingness to adopt new 
technologies and processes/ participate in 
implementation program. 
Effectiveness  
Quantitative:
 
See primary and secondary outcomes for the 
study (e.g., antibiotic prescribing rates) 
Qualitative: 
Patient satisfaction scores at the control and 
intervention sites
Adoption  
Quantitative: 
 
Porportion of staff at the intervention site who 
participate in the education session  
 
Results of audit and feedback process pre/post in 
the intervention site 
Qualitative 
 
Provider satisfaction, willingness to adopt new 
technology for respiratory infection diagnosis 
Implementation 
Acceptability (mixed methods) 
 
The information from the pre survey will be used 
to inform the bundled intervention and make 
modifications as necessary during the passive 

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implementation period. Pre and post survey 
results will be compared to assess 
implementation success 
7.2 Inclusion Criteria 
 
Patients older than 1 year  
 
Patients presenting with signs and symptoms of acute respiratory illness including but not limited 
to bronchitis, bronchiolitis, rhinitis, pharyngitis, tonsillitis, and laryngitis 
 
First visit for respiratory indication within the study period 
 
7.3 Exclusion Criteria 
 
Patients younger than 1 year  
 
Patients for which there is a clinical suspicion of pneumonia (i.e., severe illness on presentation 
requiring hospitalization or not eligible for urgent care) 
 
Patients experiencing symptoms for > 10 days  
 
Patients with significant respiratory comorbidities, including COPD, emphysema, pulmonary 
fibrosis, diseases causing chronic respiratory tract inflammation, and other conditions  
 
Patients with other significant oropharyngeal or upper respiratory comorbidities or conditions, 
including but not limited to nasal deformations, peritonsillar abscess  
 
Had a previous visit for respiratory indication within the study period  
 
7.4 Subject Enrollment  
Subjects who consent to participate shall be assigned a unique Subject Identification Number to de-
identify their information. This Subject Identification Number will be captured on the Screening & 
Enrollment Log and used to identify them on all source documents thereafter. 
A record of all screened subjects will be maintained in a Screening/Enrollment Log. The date of 
screening, results of screening (included or not) and, if not eligible, the primary reason for excluding the 
subject, enrollment status (enrolled or not) and, if not enrolled, the primary reason will be recorded. 
Please note, for the purposes of the study population a subject is considered enrolled at the time in 
which the informed consent is signed. 
 
7.5 Screen Failures 

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Subjects that do not meet the eligibility criteria for this study will be considered as screen failures, 
including subjects who withdraw consent prior to or during specimen collection, and subjects who are 
withdrawn from the study prior to or during specimen collection by the Investigator for any reason.  
If a subject signs the Informed consent form (ICF) but is found ineligible for inclusion in the study prior to 
or during the specimen collection procedure, the subject will not be advanced any further in this clinical 
investigation and will receive appropriate alternative care as identified by the Investigator. The subject’s 
signed ICF and completed inclusion/exclusion criteria will be retained by the Investigator and the subject 
will be notified of their ineligibility. 
Screening data, including the reason for exclusion, demographics, informed consent date and adverse 
event, if applicable, will be collected. The subject’s informed consent date, demographics, and reason 
for exclusion will be documented in the Case Report Form (CRF) and Screening & Enrollment Log. 
7.6 Informed Consent 
The ICF conforms to 21 CFR Part 50, Protection of Human Subjects.  Each subject must read, understand, 
have an opportunity to have any questions addressed, and sign this form prior to being enrolled in the 
study. The ICF does not contain exculpatory language. 
The Principal Investigator (PI), or a site representative who is appropriately qualified under national law 
and who has been trained on the protocol, will approach subjects who are potential candidates for 
participation. They will explain and verify that the subject understands the nature and scope of the 
study, the procedures to be performed as part of the study, the potential risks and benefits of 
participation, implications, expected duration, possible treatment alternatives, and follow-up measures 
if the participation of the subject in the clinical investigation is discontinued. The subject’s rights and the 
applicable damage compensation system will also be explained, and any questions the subject has will 
be answered. The study will be explained to the subject in lay terms and adequate time will be allowed 
for the subject to ask questions.  
Interested subjects will be invited to participate in the study and will be asked to provide written 
informed consent prior to initiation of any study-related procedures. Subjects will be assured that they 
may withdraw from the study at any time and for any reason, without repercussion. If the subject agrees 
to participate, the ICF must be signed and dated by the subject and by the person who obtained 
informed consent.  
The general process for obtaining informed consent shall: 
 
 
avoid any coercion of or undue influence of subjects to participate; 
 
not waive or appear to waive subject’s legal rights; 

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use native language that is non-technical and understandable to the subject or his/her legal 
representative; and 
 
include personally dated signatures of the subject and the Investigator or a designee responsible for 
conducting the informed consent process. 
 
The ICF that is used must be approved by the IRB. A dated and signed copy of the ICF will be given to the 
subject and the original dated and signed consent will be placed in the study binder kept by NRCG.  
 
If new information becomes available that can significantly affect a subject’s future health and medical 
care, that information shall be provided to the subjects affected in written form. If relevant, all affected 
subjects shall be asked to confirm their continuing informed consent in writing. 
 
A signed ICF must be obtained from every subject before any study-related procedures beyond Standard 
of Care (SOC) are performed. 
Failure to obtain a signed and dated informed consent form prior to performing study-related procedures 
constitutes a protocol violation, which must be reported in accordance with all applicable regulations. 
7.7 Intervention Sample Collection and Handling 
 
Sample Requirements 
o Specimen Type 
 
NPS collected according to standard technique and immediately placed in 3 mL 
of transport media. 
 
TS collected according to standard technique and immediately placed in 1 mL of 
transport media.   
 
Detailed NPS and TS specimen collection instructions can be found in the 
SPOTFIRE R/ST Panel Quick Guide. 
o Minimum Sample Volume 
 
0.3 mL (300 µL) 
 
Sample Storage 
o Patient samples will be labeled and stored per IFU and clinic standard practice after 
testing has been initiated. Once test results, and a re-test is confirmed as unnecessary, 
patient samples will be destroyed per clinic procedures.  
 
Sample Testing 
o All NP or throat swabs collected for the SPOTFIRE R/ST panel will be tested at the 
collection sites. All specimens should be tested according to the instructions provided 
with the BIOFIRE SPOTFIRE R/ST Panel test by qualified and trained staff. 
 
Testing Results 
o All SPOTFIRE R/ST results will be uploaded into the approproate area of the patient’s 
EMR for provider review. Printouts/documentation, including all results (e.g. test 
records and output files) will be maintained by the intervention site. 

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7.8 Quality Control Testing 
All testing will be accompanied by adequate quality control to verify the accuracy of the test procedure 
and to monitor performance of various components. bioMérieux will provide quality control from Maine 
Molecular Quality Controls, Inc., specifically the SPOTFIRE® RSP Negative Control and SPOTFIRE® RSP 
Positive Control M42638. QC tests should be performed as indicated in the IFU. Any unexpected QC results 
should be repeated. If the repeat testing still reports unexpected results, the use of the module should be 
paused, and the site should notify bioMérieux immediately.  
Positive and negative QC testing should be performed when: 
 
Training a new operator  
 
Receiving a new shipment or lot of SPOTFIRE R/ST Panels test kits  
 
Receiving a new SPOTFIRE control station or module 
A negative QC test should be run monthly to monitor for environmental contamination or more frequently 
if contamination is suspected. All QC testing and results should be logged in the study binder.  
7.9 Diagnostic and Treatment Algorithm 
 
Using evidence-based medicine and the SPOTFIRE R/ST IFU, AFC will develop a treatment 
algorithm to assist treating providers in deciding which test the patient’s specimen should be 
run on. Prior to study initiation, AFC will also develop a treatment algorithm based on relevant 
specialty treatment guidelines (CDC/IDSA/Etc.) which will assist the providers in how best to 
treat the pathogen(s) identified by the SPOTFIRE R/ST test. 
 
See Appendix 7 for developed treatment algorithm. 
 
7.10 Data Capture Plan 
 
All subjects enrolled in the study at the interventional clinic will be de-identified and assigned a 
single numerical identifier chosen by NRCG. Samples will be numbered incrementally and 
sequentially, e.g., 001, 002, 003… NRCG will be responsible for maintaining a study enrollment log 
linking enrolled patient EMR information to patient de-identified study number. No protected 
health information (PHI) will be provided to bioMérieux. See data table in section 7.1 for a 
comprehensive list of all information extracted from patient medical records.  
 
Study-related EMR data will be extracted by the AFC Bioinformatics team for review on a regular 
basis. The AFC study team will review each dataset to ensure compliance with applicable laws, 
regulations, completeness, consistency, and accuracy, including a review for missing values and 
potential deviations from the study protocol. If discrepancies or deviations are identified during 
this review, AFC will notify bioMérieux and NRCG staff. NRCG will be responsible for evaluating 

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the issue, documenting any protocol deviations, and initiating corrective and preventive actions 
as needed. NRCG will involve AFC and bioMérieux staff when necessary.   
 
Patient surveys will be collected at both control and intervention sites via an iPad (or similar tablet 
device) and securely transferred and stored via bioMérieux-housed CFR-compliant Microsoft 
Forms. Provider surveys will only be collected at the intervention site. They will be built and 
administered within Microsoft Forms and distributed to providers via email.  
 
7.11 Data Analysis Plan 
7.11.1 Sample Size and Power Calculation  
Based on current literature, a decrease of 10% or more in antibiotic prescribing is considered a clinically 
meaningful decrease. Additionally, National Committee for Quality Assurance (NCQA) considers a 
detectable difference for most HEDIS measures to be 10 percentage points (102024 Quality Rating 
System Measure Technical Specifications). The sample size for this study was calculated using existing 
AFC data on prescribing rates and patient volume, anticipating an effect size of 10%. A review of 
historical NCQA HEDIS reports demonstrates that the median AXR (inappropriate prescribing for 
respiratory infections) and median URI (appropriate prescribing) are approximately 26% and 73%, 
respectively. The Urgent Care Association estimates that the median patient volume in US urgent cares 
is 56 patients a day, 45% of which are due to respiratory illness. Preliminary data of antibiotic 
prescribing in the baseline period were utilized to simulate the sample size required to observe a 10% 
decrease in antibiotic prescribing at the intervention site using a DID approach. A sample size of at least 
350 patients from each site was estimated to be sufficient (approximately 80% power), assuming active 
intervention results are as stable as the baseline period. To account for attrition, we will aim to enroll 
400 patients at both the intervention and control sites.  
7.12 Statistical Analysis  
Data analysis will be performed by bioMérieux.  
This study employs a pre-post design with a difference-in-differences (DID) analysis to estimate the 
treatment effect of implementing the Spotfire R/ST assay paired with training and education. The DID 
approach compares changes in outcomes over time between the intervention and control groups, 
accounting for baseline differences and secular trends. 
The DID model will estimate the causal effect of the intervention (Spotfire) using an interaction term 
between time (pre- vs. post-intervention) and treatment group (Spotfire vs. standard of care):  
Y=
+
[Time]+
[Intervention]+
[Time*Intervention]+
[Covariates]+  
 

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Linear regression will be used for continuous outcomes, model will be adjusted for differing types of 
outcomes as necessary (e.g., dichotomous outcome = logistic regression, count data or rates = poisson 
and/or negative binomial) 
Covariates will adjust for potential confounders such as demographic and site-specific factors, as 
appropriate. Likelihood ratio testing will evaluate covariates for confounding effects, ensuring the final 
model includes only relevant predictors. 
Several key assumptions must be met in order to determine appropriateness of DID analysis. The 
common shocks assumption holds that other phenomena occurring at the same time or after the start 
of treatment will equally affect the treatment and comparison groups. The parallel trends assumption 
says that, although treatment and comparison groups may have different levels of the outcome prior to 
the start of treatment, their trends in pretreatment outcomes should be the same. The parallel trends 
assumption implies that, absent treatment, outcomes for the treatment and comparison groups are 
expected to change at the same rate. Thus, any difference in the differences in outcomes between 
groups can be attributed to the intervention, rather than to differential pre-existing trends in outcomes. 
The stable composition assumption states that the populations in treatment and control groups must 
remain consistent across pre- and post-intervention periods and the assumption of no spillover effects 
states intervention's impact on one group does not influence outcomes in the other group. 
The below checklist shares elements of DID analysis that will be tested prior to the final analysis of the 
model, and how each deviation will be addressed, if necessary.  
Confirm that
How to test
What to do if violated 
Data exists on study outcomes 
for at least one observation 
Direct observation 
N/A 

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period among groups exposed 
and not exposed to an 
intervention, both before and 
after intervention was 
implemented 
Trends in outcome 
performance prior to an 
intervention are “parallel” 
between treatment and 
comparison groups 
Test equivalence of linear trends 
between treatment and 
comparison groups prior to 
intervention by testing the 
significance of the interaction 
term between the time trend 
and the treatment group 
If multiple comparison groups 
are available, match treatment 
to comparison units 
Baseline outcome levels are 
unrelated to expectations for 
changes in outcomes 
For both treatment and 
comparison groups, test 
whether baseline outcome is 
correlated with change in 
performance across the study 
period
If multiple comparison groups 
are available, match treatment 
to comparison units 
Violations to standard statistical 
assumptions are appropriately 
addressed 
Test for violations of 
homoscedasticity of standard 
errors. (Breusch and Pagan 
1979; Drukker 2003) 
Permutation tests or clustered 
standard errors will likely result 
in the most accurate statistical 
inference when using 
difference-indifference analysis
Events or factors other than 
treatment, occurring at the 
time of treatment, do not 
differentially affect outcomes 
for treatment and comparison 
groups
Not directly testable
N/A 
The composition of treatment 
and comparison groups does 
not change over the course of 
the study 
Test for difference in observed 
covariates between treatment 
and comparison rates before 
and after the intervention. Test 
for differential drop-out rates 
between treatment and 
comparison groups. (Hausman 
and Wise 1979)
Control for differences in 
observed covariates between 
treatment and comparison 
rates before and after the 
intervention 
Treatment does not “spill-over” 
from treatment group to 
comparison group 
Test whether comparison group 
experiences deviation from 
existing trend concurrent with 
intervention
If multiple comparison groups 
are available, choose 
alternative comparison group 
that is not subject to spillovers

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Table adapted from HSR: Health Services Research 50:4 (August 2015) 
Secondary analysis will apply mixed-methods concepts to combine qualitative survey findings with 
quantitative outcomes to explore associations and identify barriers and facilitators to implementation. 
Subgroup analyses will evaluate intervention effects across demographic and clinical subcategories to 
detect heterogeneity of treatment effects. Specifically, a subgroup analysis of asthma patients will be 
conducted.  
Metrics specific to the intervention site will be described using RE-AIM dimensions: Reach (proportion of 
eligible providers and patients exposed to the intervention), Effectiveness (impact on prescribing and 
satisfaction outcomes), Adoption (proportion of sites/providers implementing the intervention), 
Implementation fidelity (extent to which the intervention was delivered as intended), and Maintenance 
(sustainability of the intervention over time). 
8. 
STUDY SUSPENSION OR EARLY TERMINATION 
8.1 Procedure for Suspension or Early Termination 
The Sponsor may suspend or prematurely terminate this clinical investigation either at an individual 
investigational site or at all sites. A Principal Investigator, IRB, or regulatory authority may suspend or 
prematurely terminate participation in this clinical investigation at the investigational sites for which they 
are responsible. 
If suspicion of an unacceptable risk to subjects arises during the clinical investigation, or when so 
instructed by the IRB or regulatory authorities, the Sponsor will suspend the clinical investigation while 
the risk is assessed. If an unacceptable risk is confirmed, the Sponsor will terminate the clinical 
investigation. 
The Sponsor will consider terminating or suspending the participation of a particular investigational site 
or Investigator if monitoring or auditing identifies serious or repeated deviations on the part of an 
Investigator. 
If suspension or premature termination occurs, the terminating party will justify its decision in writing and 
promptly inform the other parties with whom they are in direct communication. 
If suspension or premature termination occurs: 
The Sponsor will remain responsible for providing resources to fulfill the obligations from this protocol 
and existing agreements for following the subjects enrolled in the clinical investigation, and the site’s 
Principal Investigator or authorized designee will promptly inform the enrolled subjects at his/her 
investigational site. 

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If the study is terminated, bioMérieux must comply with all applicable government regulations. If 
discontinuation of the study should occur, the Principal Investigator must return all clinical investigation 
materials, if applicable, to the Sponsor and provide a written statement to the IRB explaining the reasons 
for the premature termination.  
8.2 Procedure for Resuming the Clinical Investigation After Temporary Suspension 
When the Sponsor concludes an analysis of the reason(s) for the suspension, implements the necessary 
corrective actions, and decides to lift the temporary suspension, the Sponsor will inform the relevant 
parties of the rationale and provide them with the relevant data supporting this decision. Concurrence 
will be obtained from the IRBs and, where appropriate, regulatory authorities before the clinical 
investigation resumes. If subjects have been informed of the suspension, the Principal Investigator or 
authorized designee will inform them of the reasons for resumption. 
8.3 Close Out Activities 
Close-out activities will be conducted to ensure that the Principal Investigator's records are accurate and 
complete, all documents needed for the Sponsor's files are retrieved, remaining clinical investigation 
materials are disposed of, previously identified issues have been resolved, and all parties are notified. 
9. 
MATERIALS/EQUIPMENT 
9.1 Provided by bioMérieux:  
 
15 kits (each containing 30 pouches and 30 Sample Preparation Reagent Kits) 
 
1 BIOFIRE® SPOTFIRE® Control Station  
 
2 BIOFIRE® SPOTFIRE® Modules  
 
BIOFIRE® Pouch Loading Station  
 
SPOTFIRE® RSP Negative Control M42738 (sufficient quantity for duration of study) 
 
SPOTFIRE® RSP Positive Control M42638 (sufficient quantity for duration of study) 
 
9.2 Provided by Site:  
Supplies necessary for testing that are not specifically listed in Section 9.1 are the responsibility of the 
testing site. Supplies and equipment used, but not provided by bioMérieux, Inc., must be within 
specifications and records must be available upon request (e.g., refrigerator and incubator temperatures, 
pipettors within calibration, media within expiration dates, etc.). Examples of such materials include, but 
are not limited to:  
 
Material to be used for the collection of NPS and TS samples, as defined in the IFU 
 
10% bleach or similar disinfectant for cleaning of the Pouch Loading Station and SPOTFIRE system 
 
2 tablets for patient surveys 

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10. TRAINING REQUIREMENT 
Training on the protocol, data capture method, and the conduct of the study will be provided by a 
representative of bioMérieux’s Global Medical Affairs department for site personnel involved in the study.  
 
Training on the BIOFIRE SPOTFIRE system and panels will be performed by a representative of bioMérieux 
field service team. 
All training of site and study staff should be logged as deemed appropriate in the study binder. 
11. PROTOCOL DEVIATIONS 
Conformance to the protocol is essential to the quality and integrity of the clinical study. Every effort 
should be made to avoid any deviation from the clinical protocol. A protocol deviation is an event whereby 
the clinical investigator or site personnel did not conduct the study according to the protocol. 
Under emergency circumstances, deviations from the protocol to protect the rights, safety and well-being 
of human subjects may proceed without prior approval of the Sponsor and the IRB. Such deviations shall 
be documented and reported to the Sponsor in the EDC within 5 days of knowledge. The IRB should be 
notified of the deviation as required by IRB reporting guidance. 
12. ADVERSE EVENTS 
1. Adverse Events, Serious Adverse Events, and Serious Incidents 
a. Definitions 
i. 
Adverse Event (AE): 
Any untoward medical occurrence, unintended disease or injury, or any 
untoward clinical signs (including abnormal laboratory findings) in a subject or 
user, whether directly related to the investigational device or not. For IVD-MD 
studies, AEs may arise from: 
 
Procedures associated with sample collection, i.e.: Nasopharyngeal 
swab. 
 
Psychological distress due to study participation or communication of 
results, 
 
Inaccurate, delayed, or misinterpreted results that may impact patient 
management.       
       
ii. 
Serious Adverse Event (SAE): 
Any untoward medical occurrence that: 

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Results in death, 
 
Is life-threatening, 
 
Required inpatient hospitalization or prolongation of existing 
hospitalization, 
 
Results in persistent or significant disability/incapacity, 
 
Is a congenital anomaly/birth defect, or 
 
Represents a serious deterioration in the health of the subject or user, 
which may include: 
 
A life-threatening illness or injury, 
 
Permanent impairment of a body structure or a body function, 
 
Hospitalization or prolongation of patient management in hospital, 
 
Medical or surgical intervention to prevent life-threatening illness or 
permanent impairment. 
 
iii. 
Serious Incident: 
In the context of an IVD-MD, a serious incident is any incident that directly or 
indirectly led, might have led, or might lead to death, serious deterioration in 
health of a subject, user, or other person or a serious public health threat. This 
includes incidents resulting from device malfunction, user error due to 
ergonomic or design issues, or incorrect, delayed, or missed results that have 
significant clinical consequences. 
iv. 
Field Safety Corrective Action (FSCA) 
A corrective action taken for technical or medical reasons to prevent or reduce 
the risk of a serious incident related to an IVD-MD already in use. 
  
b. Recording of Adverse Event 
All AEs will be recorded in the Case Report Form (CRF) from the time the Informed 
Consent Form is obtained until the end of the study. The investigator will document 
onset date/time, resolution date/time, severity, seriousness, relationship to the 
SPOTFIRE R/ST or related study procedures (e.g.: procedure discomfort), action taken 
and outcome. 
  
c. Reporting of Serious Adverse Events and Serious Incidents 
 
 
 

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i. 
To the Sponsor  
All SAEs and serious incidents, regardless of their relationship to the SPOTFIRE 
R/ST must be reported to the sponsor within 24 hours of the investigator’s first 
awareness. 
 
ii. 
To Regulatory Authorities 
The sponsor is responsible for timely reporting of SAE and FSCA to competent 
authorities with IVRD Art.82 and applicable national requirements. 
iii. 
To Ethics Committees/IRBs 
The investigator must report SAEs and serious incidents in compliance with 
applicable local requirements and EC/IRB policies. 
d. Causality and Expectedness Assessment 
Causality (relationship to the SPOTFIRE R/ST or study related procedures) will be 
assessed by the investigator using predefined categories (e.g., not related, unlikely 
related, possibly related, probably related, definitely related). bioMérieux will 
determine whether the event was expected or unexpected based on Device Risk 
Analysis and Instructions for Use (IFU). 
e. Follow-up of Adverse Event and Serious Incidents 
All AEs, SAEs, and serious incidents will be followed until resolution, stabilization, or 
return to baseline, or until they can no longer be reasonably monitored. 
13. CLINICAL MONITORING 
13.1 Site Monitoring 
The study will be monitored to ensure that it is conducted in conformance with the agreed upon Clinical 
Monitoring Plan to assess continued compliance with the protocol, recognized Good Clinical Practices, 
FDA’s IDE guidance documents, and federal regulations outlined in 21 CFR Part 812. In addition, the 
monitor verifies that study records are adequately maintained, that data are reported in a satisfactory 
manner with respect to timeliness, adequacy, and accuracy, and that the Investigator continues to have 
sufficient staff and facilities to conduct the study safely and effectively.  
The study may also be subject to a quality assurance audit by the Sponsor or its designees, as well as 
inspection by appropriate regulatory authorities. It is important that the Investigator and relevant study 
personnel are available during monitoring visits or audits and that sufficient time is devoted to the 
process. 
13.2 Monitoring Activities 

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Onsite monitoring visits will include Site Initiation Visit and Interim Monitoring Visit. 
The intervention site’s Investigator will allocate adequate time for monitoring activities. The Investigator 
will also ensure that the monitor or other compliance or quality assurance reviewer is given access to 
the study-related documents and study-related facilities and has adequate space to conduct the 
monitoring visit. 
Monitoring visits will be documented on monitoring visit reports as per Monitoring Plan, and will aim to 
verify that:  
o Compliance with the clinical protocol and applicable regulations is being maintained 
o Enrolled subjects are eligible for study participation 
o Signed and dated IRB approved ICF’s have been obtained from each prospective subject 
o Source data is verified and signed-off upon as accurate 
o Subject records are accurate and complete 
o Subject withdrawal has been documented (if applicable) 
o The Investigator and site staff are informed and knowledgeable of all relevant document 
updates concerning the clinical investigation 
o Only authorized individuals are performing study-related functions 
o Adequacy of staffing and facilities 
o All adverse events are reported to the Sponsor (if applicable) 
o All serious and unanticipated adverse device events are reported to the Sponsor and the 
IRB 
o All other required IRB reports, notifications, applications, submissions, and 
correspondence are maintained in the Investigator’s files and are accurate 
o Corrective and preventive actions have been implemented (if applicable) 
o Maintenance and calibration of equipment relevant to clinical assessments is 
appropriately performed and documented 
14. REFERENCES 
1. Naghavi M, Vollset SE, Ikuta KS, et al. Global burden of bacterial antimicrobial resistance 
1990&#x2013;2021: a systematic analysis with forecasts to 2050. The Lancet 2024;404(10459):1199-
226 doi: 10.1016/S0140-6736(24)01867-1.  
2. King LM, Bartoces M, Fleming-Dutra KE, Roberts RM, Hicks LA. Changes in US Outpatient Antibiotic 
Prescriptions From 2011-2016. Clin Infect Dis 2020;70(3):370-77 doi: 10.1093/cid/ciz225.  
3. Schroeck JL, Ruh CA, Sellick JA, Jr., Ott MC, Mattappallil A, Mergenhagen KA. Factors associated 
with antibiotic misuse in outpatient treatment for upper respiratory tract infections. Antimicrob 
Agents Chemother 2015;59(7):3848-52 doi: 10.1128/aac.00652-15 [published Online First: 
20150413].  

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4. Chandra Deb L, McGrath BM, Schlosser L, et al. Antibiotic Prescribing Practices for Upper 
Respiratory Tract Infections Among Primary Care Providers: A Descriptive Study. Open Forum Infect 
Dis 2022;9(7):ofac302 doi: 10.1093/ofid/ofac302 [published Online First: 20220617].  
5. Shapiro DJ, King LM, Fleming-Dutra KE, Hicks LA, Hersh AL. Association between use of diagnostic 
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Epidemiology 2020;41(4):479-81 doi: 10.1017/ice.2020.29 [published Online First: 2020/02/24].  
6. Snyder RL, King LM, Hersh AL, Fleming-Dutra KE. Unnecessary antibiotic prescribing in pediatric 
ambulatory care visits for bronchitis and bronchiolitis in the United States, 2006–2015. Infection 
Control & Hospital Epidemiology 2021;42(5):612-15 doi: 10.1017/ice.2020.1231 [published Online 
First: 2020/10/16].  
7. Björkman I, Erntell M, Röing M, Lundborg CS. Infectious disease management in primary care: 
perceptions of GPs. BMC Fam Pract 2011;12:1 doi: 10.1186/1471-2296-12-1 [published Online First: 
20110111].  
8. Clark TW, Lindsley K, Wigmosta TB, Bhagat A, Hemmert RB, Uyei J, Timbrook TT. Rapid multiplex 
PCR for respiratory viruses reduces time to result and improves clinical care: Results of a systematic 
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9. Andrews D, Chetty Y, Cooper BS, et al. Multiplex PCR point of care testing versus routine, 
laboratory-based testing in the treatment of adults with respiratory tract infections: a quasi-
randomised study assessing impact on length of stay and antimicrobial use. BMC Infect Dis 
2017;17(1):671 doi: 10.1186/s12879-017-2784-z [published Online First: 20171010].  
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Emergency Departments: A Systematic Review and Meta-Analysis. JAMA Internal Medicine 
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11. Egilmezer E, Walker GJ, Bakthavathsalam P, Peterson JR, Gooding JJ, Rawlinson W, Stelzer-Braid S. 
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with acute respiratory tract infection. Rev Med Virol 2018;28(5):e1995 doi: 10.1002/rmv.1995 
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13. Sanchez GV, Fleming-Dutra KE, Roberts RM, Hicks LA. Core Elements of Outpatient Antibiotic 
Stewardship. MMWR Recomm Rep 2016;65(6):1-12 doi: 10.15585/mmwr.rr6506a1 [published Online 
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14. Gerber JS, Prasad PA, Fiks AG, et al. Effect of an outpatient antimicrobial stewardship intervention 
on broad-spectrum antibiotic prescribing by primary care pediatricians: a randomized trial. Jama 
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15. Finkelstein JA, Huang SS, Kleinman K, et al. Impact of a 16-community trial to promote judicious 
antibiotic use in Massachusetts. Pediatrics 2008;121(1):e15-23 doi: 10.1542/peds.2007-0819.  
16. Stenehjem E, Wallin A, Willis P, et al. Implementation of an Antibiotic Stewardship Initiative in a 
Large Urgent Care Network. JAMA Network Open 2023;6(5):e2313011-e11 doi: 
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17. Roberts RM, Hicks LA, Bartoces M. Variation in US outpatient antibiotic prescribing quality 
measures according to health plan and geography. Am J Manag Care 2016;22(8):519-23.  
18. Melville BL, Musser T, Fishman E, Rainis D, Byron SC. Developing a quality measure to assess use 
of antibiotic medications for respiratory conditions. Antimicrob Steward Healthc Epidemiol 
2023;3(1):e13 doi: 10.1017/ash.2022.328 [published Online First: 20230117].  
19. Bauer MS, Kirchner J. Implementation science: What is it and why should I care? Psychiatry 
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20. Livorsi DJ, Drainoni ML, Reisinger HS, et al. Leveraging implementation science to advance 
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15. APPENDICES 
Appendix 1: SPOTFIRE RST IFU 
Appendix 2: SPOTFIRE RST Quick Guide 
Appendix 3: Study Flow Diagram 
Appendix 4: Study Schedule of Activities 
Appendix 5: Study Data Table 
Appendix 6: Provider Survey 
Appendix 7: Patient Survey  
Appendix 8: Diagnostic Treatment Algorithm 

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APPENDIX 1: BIOFIRE® SPOTFIRE® Respiratory/Sore Throat (R/ST) Panel IFU 
 
Document version: 02 
Document Date: 07/09/2024 
See attached for full document. 

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APPENDIX 2: BIOFIRE® SPOTFIRE® Respiratory/Sore Throat (R/ST) Panel Quick Guide 
 
Document version: 02 
Document Date: 07/09/2024 
See attached for full document 
 
 
 

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APPENDIX 3: Study Flow Diagram 
 
 
 
 
 

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APPENDIX 4: Study Schedule of Activities

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Appendix 5: Study Data Table 
bioMérieux/AFC SPOTFIRE Study Data Table 
Variable
Unit of Measurement
Definition 
Subject ID 
As assigned by NRCG
De-identified study subject ID as assigned by NRCG
Site
Gardendale, Grove 
Location patient was seen at as reported in EMR
Date of visit
DD/MM/YYYY 
Date patient was seen in clinic and enrolled in 
study as reported in EMR 
Patient Age
Years
Patient age at time of visit, recorded in whole 
years, as reported in EMR 
Patient Race
White, Black/African American, 
Native Hawaiian/Pacific Islander, 
Asian, American Indian/Alaska 
Native, Other
Patient race as reported in EMR at time of visit 
Patient Ethnicity
Hispanic/Latino, NOT 
Hispanic/Latino
Patient ethnicity as reported in EMR at time of 
visit
Patient biological 
sex 
M, F 
Patient 's biological sex as reported in EMR 
Time of arrival
hh:mm 
Patient check-in time 
SPOTFIRE test run SPOTFIRE R or SPOTFIRE ST 
SPOTFIRE test (R or ST) run for patient at time of 
study visit as reported in EMR. 
Adenovirus
Positive, Negative 
Result of SPOTFIRE test: Adenovirus target 
detected. Positive = detected. Negative = not 
detected 
Coronavirus SARS-
CoV-2 
Positive, Negative, Not Tested
Result of SPOTFIRE test: Coronavirus SARS-CoV-2 
target detected. Positive = detected. Negative = 
not detected. Not tested = target not offered on 
test used for patient.  
Coronavirus 
(seasonal)
Positive, Negative 
Result of SPOTFIRE test: Coronavirus (seasonal) 
target detected. Positive = detected. Negative = 
not detected 
Human 
metapneumovirus Positive, Negative 
Result of SPOTFIRE test: Human metapneumovirus 
target detected. Positive = detected. Negative = 
not detected

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Human 
rhinovirus/entero
virus
Positive, Negative 
Result of SPOTFIRE test: Human 
rhinovirus/enterovirus target detected. Positive = 
detected. Negative = not detected 
Influenza A Virus Positive, Negative 
Result of SPOTFIRE test: Influenza A Virus target 
detected. Positive = detected. Negative = not 
detected 
Influenza A virus/ 
H1-2009 
Positive, Negative 
Result of SPOTFIRE test: Influenza A virus/ H1-
2009 target detected. Positive = detected. 
Negative = not detected
Influenza A virus/ 
H3 
Positive, Negative 
Result of SPOTFIRE test: Influenza A virus/ H3 
target detected. Positive = detected. Negative = 
not detected 
Influenza B virus
Positive, Negative 
Result of SPOTFIRE test: Influenza B virus target 
detected. Positive = detected. Negative = not 
detected 
Parainfluenza 
virus
Positive, Negative 
Result of SPOTFIRE test: Parainfluenza virus target 
detected. Positive = detected. Negative = not 
detected 
Respiratory 
syncytial virus
Positive, Negative 
Result of SPOTFIRE test: Respiratory syncytial virus 
target detected. Positive = detected. Negative = 
not detected
Bordetella 
parapertussis 
Positive, Negative, Not Tested
Result of SPOTFIRE test: Bordetella parapertussis 
target detected. Positive = detected. Negative = 
not detected. Not tested = target not offered on 
test used for patient. 
Bordetella 
pertussis
Positive, Negative, Not Tested
Result of SPOTFIRE test: Bordetella pertussis target 
detected. Positive = detected. Negative = not 
detected. Not tested = target not offered on test 
used for patient.  
Chlamydia 
pneumoniae 
Positive, Negative 
Result of SPOTFIRE test: Chlamydia pneumoniae 
target detected. Positive = detected. Negative = 
not detected
Mycoplasma 
pneumoniae 
Positive, Negative 
Result of SPOTFIRE test: Mycoplasma pneumoniae 
virus target detected. Positive = detected. 
Negative = not detected 

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Streptococcus 
dysgalactiae
(Group C/G Strep) Positive, Negative, Not Tested
Result of SPOTFIRE test: Streptococcus 
dysgalactiae (Group C/G Strep) target detected. 
Positive = detected. Negative = not detected. Not 
tested = target not offered on test used for 
patient. 
Streptococcus 
pyogenes (Group 
A Strep)
Positive, Negative, Not Tested
Result of SPOTFIRE test: Streptococcus pyogenes 
(Group A Strep) target detected. Positive = 
detected. Negative = not detected. Not tested = 
target not offered on test used for patient. 
SOC test run 
Healgen COVID-19/Flu A&B Ag 
Combo Rapid Test, RSV Ag Rapid 
Test, Strep A Ag Rapid Test 
(follow-up throat culture for 
children and adolescents with 
negative RADT results)
SOC test run on control group or intervention 
group patient at time of visit as reported in EMR. 
SOC respiratory 
test result 
Organism detected
SOC respiratory test result for control group 
patient at study visit as reported in EMR. No 
organism detected = neg test result. Organism 
detected = positive test result. 
SOC test run on 
study patient at 
intervention site? Y/N 
Y/N on if patient seen at intervention site had SOC 
test run in addition to SPOTFIRE test. This is a 
variable that will not be directly collected via EMR, 
but will be a binary variable developed by the 
bioMérieux biomathematics team based on site of 
visit and type of testing performed as reported via 
patient EMR. 
SOC respiratory 
test result 
(intervention 
group)
Organism detected
If Y to SOC respiratory test being run on 
intervention group patient - SOC respiratory test 
result for intervention group patient at study visit 
as reported in EMR. No organism detected = neg 
test result. Organism detected = positive test 
result.  
Recorded ongoing 
diagnoses
Diagnosis ICD-10 code 
Any relevant persistent or ongoing patient 
diagnoses as reported in EMR for study subject at 
time of study visit.  

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Asthma at 
presentation
Y/N 
Y/N bioMérieux created binary variable on 
whether pt has ICD-10 code associated with 
asthma at time of visit. 
Study visit 
diagnosis 
Diagnosis ICD-10 code
Patient diagnosis as reported in EMR for study 
visit.  
ABX prescribed?
Y/N 
Y/N on if study subject was prescribed at least one 
antibiotic for diagnosis at time of study visit. , as 
reported in EMR (variable that bioMérieux creates 
based on what patient was prescribed)
ABX 1 type 
name, class
If Y to patient being prescribed ABX at time of 
study visit - name and class of antibiotic 1 
prescribed, as reported in EMR. Antibiotic class 
will be determined by trained pharmacists during 
the analysis phase. This information is not 
reported in the EMR.  
ABX 1 dose 
dosage
If Y to patient being prescribed ABX at time of 
study visit - dosage of antibiotic 1 prescribed, as 
reported in EMR 
ABX 1 duration 
days
If Y to patient being prescribed ABX at time of 
study visit - number of days patient is prescribed 
antibiotic 1 for, as reported in EMR 
ABX 2 type 
name, class
If Y to patient being prescribed ABX at time of 
study visit - name and class of antibiotic 2 
prescribed, as reported in EMR 
ABX 2 dose 
dosage
If Y to patient being prescribed ABX at time of 
study visit - dosage of antibiotic 2 prescribed, as 
reported in EMR 
ABX 2 duration
days
If Y to patient being prescribed ABX at time of 
study visit - number of days patient is prescribed 
antibiotic 2 for, as reported in EMR 
ABX 3 type 
name, class
If Y to patient being prescribed ABX at time of 
study visit - name and class of antibiotic 3 
prescribed, as reported in EMR 
ABX 3 dose 
dosage
If Y to patient being prescribed ABX at time of 
study visit - dosage of antibiotic 3prescribed, as 
reported in EMR 

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ABX 3 duration
days
If Y to patient being prescribed ABX at time of 
study visit - number of days patient is prescribed 
antibiotic 3 for, as reported in EMR 
Antiviral 
prescribed?
Y/N
Y/N on if study subject was prescribed at least one 
antiviral for diagnosis at time of study visit. , as 
reported in EMR (variable that bioMérieux creates 
based on what patient was prescribed)
Antiviral 1 type
name, class
If Y to patient being prescribed antiviral at time of 
study visit - name and class of antiviral 1 
prescribed, as reported in EMR 
Antiviral 1 dose
dosage
If Y to patient being prescribed antiviral at time of 
study visit - dosage of antiviral 1 prescribed, as 
reported in EMR 
Antiviral 1 
duration
days
If Y to patient being prescribed antivirals at time of 
study visit - number of days patient is prescribed 
antiviral 1 for, as reported in EMR 
Antiviral 2 type 
name, class
If Y to patient being prescribed antiviral at time of 
study visit - name and class of antiviral 2 
prescribed, as reported in EMR 
Antiviral 2 dose
dosage
If Y to patient being prescribed antiviral at time of 
study visit - dosage of antiviral 2 prescribed, as 
reported in EMR 
Antiviral 2 
duration
days
If Y to patient being prescribed antivirals at time of 
study visit - number of days patient is prescribed 
antiviral 2 for, as reported in EMR 
Antiviral 3 type
name, class
If Y to patient being prescribed antiviral at time of 
study visit - name and class of antiviral 3 
prescribed, as reported in EMR
Antiviral 3 dose
dosage
If Y to patient being prescribed antiviral at time of 
study visit - dosage of antiviral 3 prescribed, as 
reported in EMR 
Antiviral 3 
duration
days
If Y to patient being prescribed antivirals at time of 
study visit - number of days patient is prescribed 
antiviral 3 for, as reported in EMR 
Prescribing 
Provider Type 
PA, NP, MD, DO
Prescribing provider license type according to 
registered NPI number 

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Appropriateness 
of Treatment
Y/N 
Was treatment prescribed appropriate? Y = 
Antibiotic prescribed for bacterial infection OR 
antiviral prescribed for viral infection. N = 
Antibiotic prescribed for anything other than 
bacterial infection OR antiviral prescribed for 
anything other than viral infection. 
Did patient 
receive test result 
prior to 
discharge? 
Y/N 
Y/N on if patient received test result prior to 
discharge from clinic.  
Time of discharge hh:mm 
Time patient discharged from clinic on the day of 
study visit, as reported in EMR 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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APPENDIX 6: Provider Survey 
 
 

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APPENDIX 7: Patient Survey 
 
 

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APPENDIX 8: Diagnostic Treatment Algorithm