Plasma-Based Next-Generation Sequencing for Pathogen Detection and Quantification in Children With Musculoskeletal Infections
Phase: NA
Conditions: Musculoskeletal Infection, Acute Hematogenous Osteomyelitis, Septic Arthritis, Osteomyelitis, Pyomyositis
Sponsor: Indiana University

 
Clinical Study Protocol 
Title:  Plasma-Based Next-Generation Sequencing for Pathogen Detection 
and Quantification in Children with Musculoskeletal Infections 
 
Supersedes: 0.8 
Document Number: KDG-002 
Clinicaltrials.gov #: NCT03846804 
Effective Date: 19Oct2021 
Page: 1 of 30 
 
 
 
Clinical Study Protocol  
Protocol Title:  
Plasma-Based Next-Generation Sequencing for 
Pathogen Detection and Quantification in Children 
with Musculoskeletal Infections 
Protocol Number: 
KDG-002 
Study Sponsor: 
Karius Inc. 
Protocol Version  
Version 0.9 
 
 
 
CONFIDENTIALITY STATEMENT 
This document contains confidential information.  By accepting this document, you agree to maintain the 
information as confidential and to use it only for the purpose of conducting the study. 

 
Clinical Study Protocol 
Title:  Plasma-Based Next-Generation Sequencing for Pathogen Detection 
and Quantification in Children with Musculoskeletal Infections 
 
Supersedes: 0.8 
Document Number: KDG-002 
Clinicaltrials.gov #: NCT03846804 
Effective Date: 19Oct2021 
Page: 2 of 30 
 
 
CLINICAL STUDY PROTOCOL  
 
 
Protocol Title: Plasma-Based Next-Generation Sequencing for Pathogen Detection and 
Quantification in Children with Musculoskeletal Infections 
 
 
Protocol Number: 
KDG-002 
 
 
Protocol Version: 
 0.9 
 
 
 
Karius Approvals: 
 
 
____________________________________                          __________________ 
Asim Ahmed, MD 
Sr. Medical Director   
 
 
 
 
Date 
 
_____________________________________                       __________________ 
Radha Duttagupta, PhD 
Head of Clinical Operations  
 
 
  
Date 
 
_____________________________________                 
 _________________ 
Carine Ho  
Sr. Clinical Research Associate 
 
 
 
 Date 
 
 
 
 
 
 
 
 
 
 
 
 

 
Clinical Study Protocol 
Title:  Plasma-Based Next-Generation Sequencing for Pathogen Detection 
and Quantification in Children with Musculoskeletal Infections 
 
Supersedes: 0.8 
Document Number: KDG-002 
Clinicaltrials.gov #: NCT03846804 
Effective Date: 19Oct2021 
Page: 3 of 30 
 
 
 
Principal Investigator Protocol Acceptance Page 
 
Protocol Title: Plasma-Based Next-Generation Sequencing for Pathogen Detection and 
Quantification in Children with Musculoskeletal Infections 
 
Protocol Version: 
Version:  0.9 
By signing this protocol acceptance page, I confirm I have read, understood, and agree to the 
following:    
 
The conduct of the study will be in accordance with the current protocol and in accordance with the 
ethical principles that have their origin in the Declaration of Helsinki, Title 21 of the Code of Federal 
Regulations§§50,56, the International Conference of Harmonization E6, and any applicable 
regulatory requirements.   
 
The study protocol and any amendments are to be approved by the sponsor and reviewed and 
approved by an Institutional Review Board (IRB) before implementation. 
 
Written informed consent is to be obtained from each study subject prior to the conduct of any 
study procedures that exceed or differ from standard practice at the study site. 
 
Confidential information in this document will not be used for any purpose other than the 
evaluation or conduct of the clinical investigation without prior written consent of Karius.  
 
 
  
Principal Investigator (Print Name) 
 
  
 
  
Principal Investigator (Signature) 
          Date 
 
 
Please return original form to Karius at the address provided below. 
 
Attn:          Clinical Operations 
Karius Inc. 
975 Island Drive  
Redwood Shores, CA 94065  
 
After signing, please place a copy of this protocol and signature page in your study files. 

 
Clinical Study Protocol 
Title:  Plasma-Based Next-Generation Sequencing for Pathogen Detection 
and Quantification in Children with Musculoskeletal Infections 
 
Supersedes: 0.8 
Document Number: KDG-002 
Clinicaltrials.gov #: NCT03846804 
Effective Date: 19Oct2021 
Page: 4 of 30 
 
 
TABLE OF CONTENTS 
ACRONYMS .......................................................................................................................................................... 6 
STUDY SYNOPSIS .................................................................................................................................................. 8 
1 
BACKGROUND INFORMATION AND RATIONALE .......................................................................................... 9 
1.1 
BACKGROUND INFORMATION ................................................................................................................................ 9 
1.2 
RATIONALE FOR THE STUDY ................................................................................................................................... 9 
2 
STUDY OBJECTIVES AND ENDPOINTS ........................................................................................................ 10 
3 
STUDY DESIGN .......................................................................................................................................... 11 
4 
STUDY METHODS ...................................................................................................................................... 13 
5 
SELECTION AND WITHDRAWAL OF SUBJECTS ............................................................................................ 13 
5.1 
STUDY POPULATION .......................................................................................................................................... 13 
5.2 
MEDICATIONS .................................................................................................................................................. 13 
5.3 
INCLUSION CRITERIA .......................................................................................................................................... 14 
5.4 
EXCLUSION CRITERIA .......................................................................................................................................... 14 
5.5  SUBJECT SCREENING AND ENROLLMENT ................................................................................................................. 14 
5.6 
 SUBJECT WITHDRAWAL ..................................................................................................................................... 14 
5.6.1 
Withdrawal Criteria ............................................................................................................................ 14 
5.7 
STUDY TERMINATION ......................................................................................................................................... 15 
6 
TREATMENT OF SUBJECTS ......................................................................................................................... 15 
6.1 
TREATMENT REGIMEN(S) .................................................................................................................................... 15 
7 
STUDY PROCEDURES ................................................................................................................................. 15 
7.1 
SCREENING ...................................................................................................................................................... 15 
PATIENTS WILL BE SCREENED FOR ELIGIBILITY (SECTION 5.3) BY THE STUDY TEAM AT EVALUATION AT RHC. .................................. 15 
7.2 
STUDY VISITS .................................................................................................................................................... 15 
STUDY VISITS COINCIDE WITH A PATIENT’S COURSE OF TREATMENT FOR MSKI BY RILEY PEDIATRIC INFECTIOUS DISEASE AT RHC.  UP TO 
4 VISITS MAY OCCUR PER INPATIENT ADMISSION. ............................................................................................................... 15 
7.3 
SAMPLE HANDLING ........................................................................................................................................... 15 
7.4 
SAMPLE BANKING ............................................................................................................................................. 16 
8 
SAFETY MONITORING AND REPORTING .................................................................................................... 17 
9 
CLINICAL ADJUDICATION ........................................................................................................................... 17 
9.2 
ADJUDICATION PROCESS ..................................................................................................................................... 17 
9.3 
17 
10 
STATISTICAL CONSIDERATIONS ................................................................................................................. 17 
10.1 GENERAL CONDITIONS FOR STATISTICAL ANALYSIS ................................................................................................... 17 
10.2 ANALYSIS DATA SETS ......................................................................................................................................... 17 
MINIMAL CLINICAL DATA SET ............................................................................................................................. 17 
10.3 STUDY ENDPOINTS FOR CLINICAL SAMPLES AND DATA COLLECTION ............................................................................ 19 

 
Clinical Study Protocol 
Title:  Plasma-Based Next-Generation Sequencing for Pathogen Detection 
and Quantification in Children with Musculoskeletal Infections 
 
Supersedes: 0.8 
Document Number: KDG-002 
Clinicaltrials.gov #: NCT03846804 
Effective Date: 19Oct2021 
Page: 5 of 30 
 
 
10.4 DETERMINATION OF SAMPLE SIZE ......................................................................................................................... 19 
10.5 ANALYSIS POPULATION(S) ................................................................................................................................... 19 
Subject Disposition ............................................................................................................................................ 19 
Replacement of Subjects ................................................................................................................................... 19 
Procedures for Handling Missing, Unused, or Spurious Data ............................................................................ 19 
10.6 DEMOGRAPHICS AND BASELINE CHARACTERISTICS ................................................................................................... 19 
10.7 ANALYSIS ......................................................................................................................................................... 19 
11 
ADMINISTRATIVE CONSIDERATIONS ......................................................................................................... 20 
11.1 STUDY COMPLIANCE .......................................................................................................................................... 20 
11.2 INFORMED CONSENT AND PROTECTED SUBJECT HEALTH INFORMATION AUTHORIZATION ................................................ 20 
11.3 SUBJECT SCREENING LOG .................................................................................................................................... 20 
CASE REPORT FORMS .................................................................................................................................................. 21 
11.4 STUDY MONITORING ......................................................................................................................................... 21 
11.5 RETENTION OF RECORDS ..................................................................................................................................... 21 
11.6 CONFIDENTIALITY POLICY .................................................................................................................................... 22 
11.7 PRINCIPAL INVESTIGATORS OBLIGATIONS IN CONDUCT OF STUDY AND PROTECTION OF HUMAN SUBJECTS ......................... 22 
12 
APPENDIX A:  SCHEDULE OF EVENTS ......................................................................................................... 23 
13 
APPENDIX B:  ID-DX SAMPLE COLLECTION INSTRUCTIONS ......................................................................... 24 
14 
APPENDIX C. CASE REPORT FORM ............................................................................................................. 25 
15 
APPENDIX D: REFERENCES ......................................................................................................................... 30 
  

 
Clinical Study Protocol 
Title:  Plasma-Based Next-Generation Sequencing for Pathogen Detection 
and Quantification in Children with Musculoskeletal Infections 
 
Supersedes: 0.8 
Document Number: KDG-002 
Clinicaltrials.gov #: NCT03846804 
Effective Date: 19Oct2021 
Page: 6 of 30 
 
 
ACRONYMS 
 
AHO 
Acute hematogenous osteomyelitis 
CRP 
C-reactive protein 
cfDNA 
Cell-free DNA 
CFR 
Code of Federal Regulations 
CRF 
Case Report Forms 
DNA 
Deoxyribonucleic Acid 
DVT 
Deep vein thrombosis  
ESR 
Erythrocyte sedimentation rate 
GCP 
Good Clinical Practice 
ICF 
Informed Consent Form 
ICU 
Intensive care unit 
ID 
Identification  
IRB 
Institutional Review Board 
IU 
Indiana University 
LOT 
Length of therapy 
MPM 
Molecules per microliter 
MSKI 
Musculoskeletal infection  
NGS 
Next-generation sequencing 
PCT 
Procalcitonin 
PHI 
Protected Health Information  

 
Clinical Study Protocol 
Title:  Plasma-Based Next-Generation Sequencing for Pathogen Detection 
and Quantification in Children with Musculoskeletal Infections 
 
Supersedes: 0.8 
Document Number: KDG-002 
Clinicaltrials.gov #: NCT03846804 
Effective Date: 19Oct2021 
Page: 7 of 30 
 
 
RHC 
Riley Hospital for Children 
WBC 
White blood cell count 

 
Clinical Study Protocol 
Title:  Plasma-Based Next-Generation Sequencing for Pathogen Detection 
and Quantification in Children with Musculoskeletal Infections 
 
Supersedes: 0.8 
Document Number: KDG-002 
Clinicaltrials.gov #: NCT03846804 
Effective Date: 19Oct2021 
Page: 8 of 30 
 
 
STUDY SYNOPSIS 
 
Protocol Number: 
KDG-002  
Protocol Title: 
Plasma-Based Next-Generation Sequencing for Pathogen Detection and 
Quantification in Children with Musculoskeletal Infections 
 
 
Study Objectives: 
1. To evaluate and compare pathogen identification of children with 
musculoskeletal infections (MSKI) using Karius® plasma-based 
quantitative Next-Generation Sequencing Test (Karius Test) and standard 
culture methods. 
 
2. To examine the kinetics of pathogen cell-free DNA (cfDNA) in the 
plasma of children with MSKI and compare the quantity of cfDNA to 
clinical symptoms and inflammatory markers, C-reactive protein (CRP), 
Erythrocyte sedimentation rate (ESR), and procalcitonin (PCT), a 
biomarker thought to be more specific for bacterial infections.  
 
3. To evaluate whether samples quantitative cfDNA predicts severe disease 
in children with MSKI.  
 
Study Design: 
We will prospectively enroll children evaluated at Riley Hospital for 
Children (RHC) with musculoskeletal infections (osteomyelitis, septic 
arthritis, or pyomyositis) over a 12-24-month period. Eligible subjects will 
be identified by referral from the infectious diseases and orthopedic 
services at RHC. Subjects and their parent(s) or guardian(s) will be 
informed of the study and, upon providing informed consent, will be 
enrolled in the study.  Study samples will be collected at time points that 
closely parallel clinical testing as performed during routine, standard care 
for treatment of MSKI. Immediately after enrollment, up to two detection 
blood samples will be obtained for real-time NGS testing at Karius 
Laboratory (Redwood City, CA) to determine if additional study samples 
will be obtained. Detection of a pathogen by NGS will be interpreted as a 
positive result that qualifies the collection of subsequent study samples.  
Pathogen identification by NGS will be compared to standard cultures 
methods, and quantitative cfDNA will be evaluated over time.  
Duration of Study: 
12-Month Period 
Number of Sites: 
1:  Riley Hospital for Children 
Sample Size: 
38 Subjects 

 
Clinical Study Protocol 
Title:  Plasma-Based Next-Generation Sequencing for Pathogen Detection 
and Quantification in Children with Musculoskeletal Infections 
 
Supersedes: 0.8 
Document Number: KDG-002 
Clinicaltrials.gov #: NCT03846804 
Effective Date: 19Oct2021 
Page: 9 of 30 
 
 
1 
BACKGROUND INFORMATION AND RATIONALE  
1.1 
Background Information  
Musculoskeletal infections (MSKI; osteomyelitis, septic arthritis, and pyomyositis) are a 
common cause for hospitalization and, in children, among the most frequent type of invasive 
infection. Due to the risk of local tissue destruction and metastatic bacterial spread, MSKI 
require prompt diagnosis and targeted treatment. Current diagnostic modalities require isolation 
of a causative organism by culture from a sterile site (e.g., blood, bone or joint fluid); however, 
nearly half of all MSKI in children remain culture-negative [1,2,3]. Several reasons for this exist, 
including antibiotic use prior to obtaining a culture; lack of a surgical or drainage procedure; and 
the fastidious nature of certain organisms in culture [4]. Absence of a microbiologic diagnosis 
has significant ramifications on the management of children with MSKI, as patients are 
ultimately treated with broad, often multi-agent antimicrobial regimens to cover all likely 
causative pathogens.  
1.2 
Rationale for the Study 
A culture-independent molecular test, such as the Karius® plasma-based next-generation 
sequencing (Karius Test) test, would not be prone to the aforementioned barriers and has the 
potential to increase pathogen detection allowing for targeted antimicrobial therapy. 
 
Additionally, quantitative NSG has the potential to improve another component of the 
complex management of children with MSKI—length of therapy (LOT). Typically, length of 
therapy for MSKI in children is 2-6 weeks depending on the infection type and severity, 
however, variability in this practice exists [5,6]. Ultimately LOT is typically determined by 
clinical symptoms and inflammatory makers (e.g., C-reactive protein (CRP) and erythrocyte 
sedimentation rate (ESR)) at the end of the pre-determined timeframe. This approach, while 
often effective, is challenging in children given that the clinical exam can be unreliable and 
inflammatory markers are non-specific, often influenced by intercurrent illnesses or recent 
procedures. An objective assay with improved specificity, such as the Karius Test, which has the 
ability to quantitate cell-free pathogen DNA (cfDNA) and has been shown to 
correlate with clinical findings in other disease states [7], could allow for a tailored treatment 
approach and address an unmet need for children with MSKI. 
 
Finally, the ability of the Karius Test to quantitate cfDNA has the potential to inform 
disease severity and potentially predict complications in children with MSKI. While numerous 
proposed strategies have been published to develop a prediction algorithm for disease severity 
in children with MSKI, each of these methods has significant limitations [8], and none involve 
quantification of cfDNA. If the Karius Test could be used to predict severe disease, it could 
have a significant impact on early management of this complex disease. 
 

 
Clinical Study Protocol 
Title:  Plasma-Based Next-Generation Sequencing for Pathogen Detection 
and Quantification in Children with Musculoskeletal Infections 
 
Supersedes: 0.8 
Document Number: KDG-002 
Clinicaltrials.gov #: NCT03846804 
Effective Date: 19Oct2021 
Page: 10 of 30 
 
 
Taken together, the Karius Test has the potential to significantly impact antibiotic utilization and 
quality of care for children with MSKI. In this proposal, we seek to investigate the utility of this 
novel technology in children with MSKI. 
 
2 
STUDY OBJECTIVES AND ENDPOINTS 
2.1 PRIMARY OBJECTIVES 
 
1) To evaluate and compare pathogen identification of children with musculoskeletal 
infections using Karius plasma-based quantitative NGS test (Karius Test) and standard culture 
methods. We hypothesize that the Karius Test will have a high positive agreement for culture 
positive samples and increase pathogen detection in culture-negative infections. 
 
2) To examine the kinetics of pathogen cell-free DNA (cfDNA) in the plasma of children 
with MSKI and compare the quantity of cfDNA to clinical symptoms and inflammatory 
markers, CRP, ESR, and procalcitonin (PCT), a biomarker thought to be more specific for 
bacterial infections. We hypothesize that the cfDNA quantity will correlate strongly with 
clinical symptoms as well as inflammatory markers (CRP, ESR and PCT). 
 
3)  To evaluate whether initial detection samples quantitative cfDNA predicts severe 
disease1 in children with MSKI. We hypothesize that cfDNA quantity at admission will correlate 
with severe or complicated MSKI. 
1Severe disease defined by any of the following: need for intensive care unit (ICU) care, 
infection in two or more non-contiguous anatomic sites (disseminated disease), complicated 
disease (need for more than 1 debridement procedure, deep vein thrombosis [DVT] or 
thromboembolic disease, pathologic fracture)  
 
2.2 EXPLORATORY OBJECTIVE 
 
1) To evaluate and compare pathogen identification from synovial fluid of children with 
septic arthritis using Karius Test and standard culture methods.  
 
2.3 PRIMARY ENDPOINT MEASURES 
 
1) To evaluate and compare pathogen identification of children with musculoskeletal 
infections using Karius Test NGS and standard culture methods: 
a.  Describe pathogen identification by Karius Test vs. standard culture methods 
 
2) To examine the kinetics of pathogen cell-free DNA (cfDNA) in the plasma of children 
with MSKI and compare the quantity of cfDNA to clinical symptoms and inflammatory markers, 
CRP, ESR, and procalcitonin (PCT), a biomarker thought to be more specific for bacterial 
infections. 

 
Clinical Study Protocol 
Title:  Plasma-Based Next-Generation Sequencing for Pathogen Detection 
and Quantification in Children with Musculoskeletal Infections 
 
Supersedes: 0.8 
Document Number: KDG-002 
Clinicaltrials.gov #: NCT03846804 
Effective Date: 19Oct2021 
Page: 11 of 30 
 
 
a. Determine quantification of cf DNA in molecules per microliter (MPM) at each time 
point using the Karius Test.  
b. Determine CRP, ESR, PCT at each timepoint and compare to cfDNA MPM.  
 
3) To evaluate whether initial detection sample quantitative cfDNA predicts severe disease 
in 
children with MSKI. 
a. Determine the correlation of cfDNA level in MPM to severity of infection (defined in 
SECTION 2.1) 
 
2.4 EXPLORATORY ENDPOINT MEASURES 
 
1) To evaluate and compare pathogen identification from synovial fluid of children with 
septic arthritis using the Karius Test and standard culture methods.  
a.  Describe pathogen identification by NGS of the synovial fluid compared to standard 
culture methods.  
 
3 
STUDY DESIGN 
This is a prospective study of children 6 months to 18 years of age (inclusive) with the diagnosis 
of MSKI at Riley Hospital for Children (Indianapolis, IN) over a 12-month period. Eligible 
subjects will be identified by referral from the infectious diseases and orthopedic services at 
RHC. Subjects will be identified and screened for eligibility (Section 5.3) by the study team. 
Subjects and their parent(s) or guardian(s) will be informed of the study and, upon providing 
informed consent, will be enrolled in the study.  
Study samples will be collected to coincide with routine clinical care.  Samples will be collected 
when sufficient sample is available after routine care samples are obtained.  Additional 
venipunctures specific for collection of study samples will be performed only with participant’s 
consent. 
 
Collection of NGS samples for real-time testing: 
Detection samples will be collected prior to any surgical procedures and will be sent to Karius® 
Laboratory (Redwood City, CA) for real-time testing to determine if the pathogen is detected by 
NGS.  Karius will notify the study team of the result(s).  A positive result for any detection 
sample will qualify the collection of subsequent study samples.  Detection samples will be 
collected within 96 hours of enrollment, and if applicable, 12-72 hours after the first sample, 
upon any re-admission for MSKI, or when prior detection sample results are inconclusive.   
Collection of subsequent study samples 
If NGS testing identifies a pathogen in the detection sample(s), subsequent study samples will, 
will be collected ≥ 48 hours after previous sample collected.  When the Detection NGS result(s) 

 
Clinical Study Protocol 
Title:  Plasma-Based Next-Generation Sequencing for Pathogen Detection 
and Quantification in Children with Musculoskeletal Infections 
 
Supersedes: 0.8 
Document Number: KDG-002 
Clinicaltrials.gov #: NCT03846804 
Effective Date: 19Oct2021 
Page: 12 of 30 
 
 
is inconclusive or still pending, study samples may be collected at the appropriate timepoint(s). 
Collected sample(s) will be sent to Karius with qualifying result(s) from the Detection NGS 
sample(s).  Up to 4 samples, including detection samples, will be collected per inpatient 
admission.  (Figure 1)  
 
Collection of Synovial Fluid from patients with septic arthritis: 
When synovial fluid is obtained as part of routine medical care, excess or leftover synovial fluid 
may be collected and sent to Karius for potential NGS testing of the synovial fluid.  
Procalcitonin: 
A procalcitonin (PCT) level will be obtained at each study sample time point, along with routine 
clinical labs when sufficient sample can be collected. 
 
 If the Karius Test identifies an infectious agent in a clinically meaningful timeframe that may be 
of interest for the treating physician, Dr. Wood, at his discretion, can contact the attending 
Detection NGS Samples  
Sent for real-time NGS testing 
 
Collected within 96 hours of 
enrollment and/or hospital re-
admission for MSKI and prior to 
any surgical procedures.  Another 
sample may be collected 12-72h 
after previous sample. 
 
Subsequent NGS Samples* 
*Stored and sent for batch NGS testing if either detection sample is positive or 
result pending at collection time point 
 
Collected q >48 hours, after previous samples, while hospitalized (max 2) and at 
routine care outpatient visits (max 3)  
 
Figure 1. Study Design- Samples for NGS testing will be collected to coincide with routine clinical care testing. Detection 
sample(s) sent for real-time NGS testing to determine if the Karius test identifies a pathogen.  If a pathogen is detected by 
NGS, additional study samples will be collected throughout the treatment and follow up for MSKI.  
Routine Clinical Care Labs 
 
Blood & surgical cultures, ESR, CRP. 
 
PCT will be collected as a study lab with routine labs if available  
 
NGS samples will be paired with clinical care labs unless patient agrees to additional blood draw 
 
*Synovial fluid collected for NGS if leftover fluid is available (septic arthritis) 
 

 
Clinical Study Protocol 
Title:  Plasma-Based Next-Generation Sequencing for Pathogen Detection 
and Quantification in Children with Musculoskeletal Infections 
 
Supersedes: 0.8 
Document Number: KDG-002 
Clinicaltrials.gov #: NCT03846804 
Effective Date: 19Oct2021 
Page: 13 of 30 
 
 
physician and notify them. Dr. Wood will stress that these results are from a research-use only 
test but will guide the treating physician to confirm the diagnosis with a commercially available 
CLIA-approved test. 
   
 
4 
STUDY METHODS 
Plasma samples will be obtained at the time points outlined in Section 3.  Approx 2-4ml 
of whole blood will be collected in 2mL K2-EDTA tubes for NGS testing and centrifuged at 
1,600 RCF for 10 minutes at room temperature, within 24 hours of blood draw.  Plasma will be 
transferred into sterile polypropylene tubes. Detection samples will be sent at ambient 
temperature to Karius Laboratory within 96 hours of blood draw.  Subsequent NGS samples will 
be frozen at -20oC until shipping to Karius.  For patients with septic arthritis, in which synovial 
fluid is collected as part of standard of care, left-over synovial fluid will be frozen at -20oC until 
shipping to Karius. Blood and source specimen (bone biopsy, abscess aspiration, or synovial 
fluid) cultures for standard microbiologic diagnosis, will be collected as per the treating 
physicians.  Cultures will be performed per standard of care procedures by the Indiana 
University clinical laboratory. Standard of care laboratory testing (e.g., ESR, CRP) will be 
obtained per the treating physicians and run in the Indiana University (IU) clinical laboratory. 
Additionally, procalcitonin (PCT), which is not part of current standard of care laboratory 
testing, will be drawn with NGS samples and run in the IU clinical laboratory for research 
purposes only.  
 
5 
SELECTION AND WITHDRAWAL OF SUBJECTS  
5.1 
Study Population 
We will prospectively enroll children, 6 months to 18 years of age, evaluated at Riley Hospital 
for Children (RHC) with clinical presentation consistent with a musculoskeletal infection 
(osteomyelitis, septic arthritis, or pyomyositis; MSKI) over a 12-24-month period. Eligible 
subjects will be identified by referral from the infectious diseases and orthopedic services at 
RHC. 
 
5.2 
Medications 
There are no medication restrictions or medication requirements for this study.  
 
 

 
Clinical Study Protocol 
Title:  Plasma-Based Next-Generation Sequencing for Pathogen Detection 
and Quantification in Children with Musculoskeletal Infections 
 
Supersedes: 0.8 
Document Number: KDG-002 
Clinicaltrials.gov #: NCT03846804 
Effective Date: 19Oct2021 
Page: 14 of 30 
 
 
5.3 
Inclusion Criteria 
Eligible subjects will be identified by referral from the infectious diseases and orthopedic 
services at RHC. Subjects and their parent(s) or guardian(s) will be informed of the 
study and, upon providing informed consent, will be enrolled in the study if they meet the 
following inclusion criteria:  
1. 6 months (to ensure adequate blood volume drawn) to 18 years of age.  
2. Strong clinical suspicion of MSKI as evidenced by fever, osteoarticular pain (e.g. 
tenderness to palpation of a joint, bone pain, or refusal to bear weight); and elevated 
ESR or CRP. 
 
5.4 
Exclusion Criteria 
1. Subjects will be excluded if they have clinical evidence suggesting an alternative 
diagnosis; PI discretion; inability or unwillingness to consent for the study. 
2. Subjects will be excluded if they have a surgical procedure prior to obtaining NGS 
samples. 
 
 
5.5  
Subject Screening and Enrollment  
The study team will be notified by the pediatric infectious diseases and/or orthopedic team, at 
admission/evaluation, of any child presenting with possible MSKI. A study team member will 
screen the patient for eligibility (Section 5.3), and if eligible will obtain informed consent/assent 
for study participation. 
 
5.6 
 Subject Withdrawal  
5.6.1 
Withdrawal Criteria 
The primary consideration in any determination to discontinue a subject’s 
participation must be the health and welfare of the subject. Reasons for study 
withdrawal may include but are not limited to: 
a)  A serious medical complication or an unacceptable situation, whether 
attributed to study procedure or not.  
b)  Noncompliance with study procedures. 
c)  Subject’s right to withdraw their consent at any time during the study with or 
without stated reason. 
d)  The PI’s opinion is that it is not in the subject’s best interest to continue study 
participation. 
5.6.2 
Documentation of Withdrawal of Subjects 

 
Clinical Study Protocol 
Title:  Plasma-Based Next-Generation Sequencing for Pathogen Detection 
and Quantification in Children with Musculoskeletal Infections 
 
Supersedes: 0.8 
Document Number: KDG-002 
Clinicaltrials.gov #: NCT03846804 
Effective Date: 19Oct2021 
Page: 15 of 30 
 
 
Document the reasons for early withdrawal of any subject from the study on the 
appropriate Case Report Forms (CRF). If the reason for early withdrawal is an 
adverse event or an abnormal laboratory value, record the specific event or test 
result on the relevant CRF.  
5.7 
Study Termination 
Sponsor has the right to terminate this study at any time. Reasons for terminating the study 
may include, but are not limited to, the following: 
a) Subject enrollment is unsatisfactory. 
b) Data recording is inaccurate or incomplete. 
c) Investigators do not adhere to the protocol or applicable regulatory guidelines 
in conducting the study. 
6 
TREATMENT OF SUBJECTS 
6.1 Treatment Regimen(s) 
NA 
 
7 
STUDY PROCEDURES 
7.1 
Screening  
Patients will be screened for eligibility (Section 5.3) by the study team at evaluation at 
RHC.  
7.2 
Study Visits 
Study visits coincide with a patient’s course of treatment for MSKI by Riley Pediatric 
Infectious Disease at RHC.  Up to 4 visits may occur per inpatient admission and up to 
4 visits may occur per outpatient course. 
 
7.3 
Sample Handling  
Sample collection materials may be supplied by the sponsor. The main sample 
handling instructions are as below:  
 
All samples should be collected using the institutional guidelines for sterile sample 
collection.  The venous blood sample collection can be collected from either a 
peripheral vein or a central line catheter. 
 
A) Detection Samples: 
 

 
Clinical Study Protocol 
Title:  Plasma-Based Next-Generation Sequencing for Pathogen Detection 
and Quantification in Children with Musculoskeletal Infections 
 
Supersedes: 0.8 
Document Number: KDG-002 
Clinicaltrials.gov #: NCT03846804 
Effective Date: 19Oct2021 
Page: 16 of 30 
 
 
Specimen: Minimum 1.2 mL of plasma isolated from whole blood collected in K2-
EDTA tube within 24 hours of draw by centrifugation of the specimen at 1,600 RCF 
for 10 minutes and transferred to a sterile polypropylene tube.  Ship to Karius 
laboratory at ambient temperature. Must be received within 96 hours (4 days) of 
collection.   
 
B) Subsequent study samples: 
 
B1. Collect 2-4.0 mL of whole blood in a K2-EDTA 2.0 mL blood collection tube 
B2. CRITICAL STEP: Immediately after collection, gently invert the filled tube 180o  
       and back 8-10 times to adequately mix the blood and tube reagents together. 
B3. Place tube upright in rack at room temperature. The tube may be stored upright at 
        room temperature for no more than 24 hours prior to processing for plasma    
        collection. Do not freeze or refrigerate the tube. 
B4. For plasma collection, centrifuge the K2-EDTA blood tube at 1,600 rcf for 10 
minutes at 20oC. It is critical that tubes be centrifuged at the appropriate speed to       
ensure proper plasma separation. 
B5. Using a sterile micropipette, transfer blood plasma (top layer) into labeled 1.0 mL 
aliquot tubes. Take care not to disturb the pellet at the bottom of the tube by 
tilting the tube and placing the pipette tip along the lower side of the collection 
tube wall. 
B6. In order to ensure Karius receives sufficient sample for processing and to avoid 
tubes cracking prior to shipment, each aliquot tube should ideally be filled to 1.0 
mL with plasma after processing. If there is plasma that won’t fill a full tube, the 
remaining amount should still be sent to Karius. 
B7. Freeze the plasma samples immediately following processing by transferring to 
80°C freezer. Store all samples in -80oC until transport to Karius. 
 
7.4 
Sample Banking 
As agreed by the subject on the signed informed consent form (ICF), his or her blood 
(and blood products) will be kept in the Sponsor’s lab or designated facility equipped 
with freezers (at -80 ºC) and may be used by the sponsor for research. The ICF will 
include the subject’s right to release or restrict their sample for purpose of research by 
the sponsor.  
 
Each sample will be identified by a unique number on a bar-coded label (sample ID) 
which will not have any personal information in order to maintain subject 
confidentiality.  Only the study site staff will keep a matching list of sample barcode 
numbers, subject identifiers, subject names, and visit dates to fully identify these 
samples. 
 

 
Clinical Study Protocol 
Title:  Plasma-Based Next-Generation Sequencing for Pathogen Detection 
and Quantification in Children with Musculoskeletal Infections 
 
Supersedes: 0.8 
Document Number: KDG-002 
Clinicaltrials.gov #: NCT03846804 
Effective Date: 19Oct2021 
Page: 17 of 30 
 
 
8 
SAFETY MONITORING AND REPORTING 
No unexpected adverse events or side effects related to the conduct of the protocol are 
anticipated. All venous samples will be collected by routine techniques performed by 
qualified phlebotomists and/or licensed health care personnel.  All adverse events will be 
reported to the IRB and to the Medical Monitor.  
Medical Monitor: Jack Schneider , M.D., 317-944-7265; jgschnei@iu.edu.  
 
9 
CLINICAL ADJUDICATION  
9.2 
Adjudication Process 
NA 
9.3 
 
Data Review 
Upon the study completion, the comparison between Karius NGS testing and standard culture 
methods for pathogen identification will be reviewed by Dr. Wood and Karius.  The study results 
will be summarized in a peer-reviewed publication. 
 
10 STATISTICAL CONSIDERATIONS 
10.1 
General Conditions for Statistical Analysis  
Positive and negative agreement (percentage) will be used to compare culture and NGS for 
pathogen identification.  Correlation between inflammatory markers and cfDNA quantification 
will be compared using Spearman’s coefficient. Lastly, simple regression modeling will be used 
to assess the ability of cfDNA to predict severe disease.   
 
 
10.2 
Analysis Data Sets 
 
            MINIMAL CLINICAL DATA SET 
** All of these data will be striped of protected health information (PHI). The Investigator will 
maintain a log that can link these subject ID and sample IDs to medical record numbers and 
sample-collection dates. 
 
Variable 
 Acronym definition 
Type of data 
Subject ID 
Subject Identifier 
Numerical  
Sample ID 
Sample Identifier (i.e., IP1) 
Numerical  

 
Clinical Study Protocol 
Title:  Plasma-Based Next-Generation Sequencing for Pathogen Detection 
and Quantification in Children with Musculoskeletal Infections 
 
Supersedes: 0.8 
Document Number: KDG-002 
Clinicaltrials.gov #: NCT03846804 
Effective Date: 19Oct2021 
Page: 18 of 30 
 
 
 
 
 
Admission Time 
Date and time of hospital 
admission 
Date and time 
Discharge Time 
Date and time of hospital 
discharge 
Date and time  
Sample Time 
Date and time of NGS sample 
collection 
Date and time 
Age  
Age at the time of diagnosis of 
MSKI 
Years 
Sex 
 
F/M 
Race 
 
Nominal 
Infection Type 
Acute hematogenous 
osteomyelitis (AHO), Septic 
Arthritis, Pyomyositis 
Nominal 
Infection Location 
Name of bone/joint infected 
Nominal 
Days of Symptoms  
Days of symptoms (number) 
related to MSKI (fever, pain) 
prior to hospital admission 
Numerical 
Antibiotic Prior Admit 
Antibiotic given prior to 
admission (Y/N) 
Yes/No 
Antibiotic Name 
Name of initial antibiotic(s) 
given for MSKI 
Nominal 
Antibiotic Time 
Date and time initial 
antibiotic(s) given  
Date and time 
Surgical Procedure 
Was a surgical procedure 
performed 
Yes/No 
Culture Pathogen ID  
Name of pathogen by culture 
Nominal 
Culture source 
Source of culture (i.e., blood, 
bone, synovial fluid) 
Nominal 
NGS Pathogen ID 
Name of pathogen by NGS 
Nominal 
NGS Quantification 
Molecule per microliter 
(MPM) of cfDNA 
Numerical 
WBC 
White blood cell count 
Numerical 
WBC Timepoint 
Time and Date of WBC 
collection 
Date and time 
CRP 
C-reative protein 
Numerical 
CRP Timepoint 
Time and Date of CRP 
collection 
Date and time 

 
Clinical Study Protocol 
Title:  Plasma-Based Next-Generation Sequencing for Pathogen Detection 
and Quantification in Children with Musculoskeletal Infections 
 
Supersedes: 0.8 
Document Number: KDG-002 
Clinicaltrials.gov #: NCT03846804 
Effective Date: 19Oct2021 
Page: 19 of 30 
 
 
ESR 
Erythrocyte sedimentation rate 
Numerical 
ESR Timepoint 
Time and Date of ESR 
collection 
Date and time 
PCT 
Procalcitonin 
Numerical 
PCT Timepoint 
Time and Date of PCT 
collection 
Date and time 
 
 
 
10.3 
Study Endpoints for Clinical Samples and Data Collection 
Study objectives and endpoint measures are listed in Protocol Section 2.  
10.4 
Determination of Sample Size  
Based on current rates, it is estimated that at least 50 subjects will be admitted for MSKI at Riley 
Hospital for Children over a 12-month period. Based on an estimated >75% participation 
(consistent with similar previous studies) the target sample size of this study is 38.   
10.5 
Analysis Population(s)  
Subject Disposition 
Summary tables will be provided containing frequency counts for subject 
disposition (all enrolled subjects, all subjects with uncontaminated samples, all 
subjects who discontinued from study and reason for discontinuation).  
Disposition in terms of number of subjects excluded from the ITT analysis set will 
also be provided for the study. 
Replacement of Subjects 
No replacements for subjects withdrawing from the study are planned. 
Procedures for Handling Missing, Unused, or Spurious Data 
10.6 
Demographics and Baseline Characteristics 
As per above, baseline demographic information including age, race/ethnicity, and gender will 
be collected via review of the patient’s medical record after enrollment.   
10.7 
Analysis 
Statistical analysis is outlined in Section 10.1 
 

 
Clinical Study Protocol 
Title:  Plasma-Based Next-Generation Sequencing for Pathogen Detection 
and Quantification in Children with Musculoskeletal Infections 
 
Supersedes: 0.8 
Document Number: KDG-002 
Clinicaltrials.gov #: NCT03846804 
Effective Date: 19Oct2021 
Page: 20 of 30 
 
 
11 ADMINISTRATIVE CONSIDERATIONS 
This study will be conducted at one site in the United States.  The investigator will submit 
this protocol, the informed consent, and any other relevant supporting information to the 
appropriate IRB for review and approval prior to study initiation.  A letter confirming IRB 
approval of the protocol and informed consent, and a statement that the IRB is organized 
and operates according to Good Clinical Practice (GCP) and the applicable laws and 
regulations, and financial disclosures must be forwarded to the Sponsor prior to the 
enrollment of subjects into the study. Amendments to the protocol must also be approved by 
the IRB prior to the implementation of changes in this study. 
11.1 
Study Compliance 
The study will be conducted in compliance with this protocol, principles of ICH-6, 
GCP and the Declaration of Helsinki, and all applicable national regulations 
governing clinical trials.  
11.2 
Informed Consent and Protected Subject Health Information Authorization 
A copy of the IRB approved informed consent may be audited or requested by the 
Sponsor for regulatory purposes. The investigator or designee must explain to each 
subject the purpose and nature of the study, the study procedures, the possible 
adverse effects, and all other elements of consent as defined in 21CFR §50. Each 
subject must provide a signed and dated informed consent prior to enrollment into 
this study. Signed consent forms must remain in each subject’s study file and be 
available for verification by study monitors at any time. 
In accordance to individual local and national subject privacy regulations, the 
investigator or designee must explain to each subject prior to screening that for the 
evaluation of study results, the subject’s protected health information obtained 
during the study may be shared with the Sponsor and its designees, regulatory 
agencies, and IRBs. The study sponsor will not use the subject’s protected health 
information or disclose it to a third party without applicable subject authorization. It 
is the investigator’s or designee’s responsibility to obtain a written permission to 
use protected health information from each subject. If a subject withdraws 
permission to use protected health information, it is the investigator’s responsibility 
to obtain a written request from the subject or subject’s legal guardian and to ensure 
that no further data will be collected from the subject. Any data collected on the 
subject prior to withdrawal will be used in the analysis of study results. 
11.3 
Subject Screening Log 
The Principal Investigators must keep a record that lists all subjects considered for 
screening and enrollment in the study.  For those subjects subsequently excluded, 
record the reason(s) for exclusion. 

 
Clinical Study Protocol 
Title:  Plasma-Based Next-Generation Sequencing for Pathogen Detection 
and Quantification in Children with Musculoskeletal Infections 
 
Supersedes: 0.8 
Document Number: KDG-002 
Clinicaltrials.gov #: NCT03846804 
Effective Date: 19Oct2021 
Page: 21 of 30 
 
 
 
Case Report Forms 
Study site personnel will collect and record data on paper and/or electronically. If 
electronic data collection is not feasible, other forms of source document will be 
used to capture the data.  A single set of standardized case report forms (CRFs), 
which primarily include the Inclusion/Exclusion Criteria, Medical History, Height 
and Weight, Concomitant Medications, will be required for every study subject who 
has undergone any amount of study procedures.  Standardized CRFs will be 
completed for each study subject and monitored. 
11.4 
Study Monitoring  
Representatives of the Sponsor or its designee will monitor for proper completion 
of study procedures until completion.  Monitoring will be conducted through 
personal visits with the Principal Investigators and site staff as well as any 
appropriate communications by mail, fax, E-mail, or telephone.  The purpose of 
monitoring is to ensure compliance with the protocol and the quality and integrity 
of the data.  This study is also subject to Quality Assurance reviews and/or audits 
under the Sponsor’s practices or procedures. 
Every effort will be made to maintain the anonymity and confidentiality of all 
subjects during this clinical study.  However, the Principal Investigators agrees to 
allow the IRB, representatives of the Sponsor or its designated agent, and 
authorized employees of the appropriate regulatory agencies to inspect the facilities 
used in this study and have direct access to the hospital or clinic record of all 
enrolled into the study, for purposes of verification.  A statement to this effect will 
be included in the informed consent form authorizing the use of protected health 
information. 
11.5 
Retention of Records 
The Principal Investigators must retain a copy of all documents for 5 years unless 
the Sponsor notifies the Principal Investigators in writing that the documents no 
longer need to be retained.  The Principal Investigators must retain the documents 
for a longer period where so required by other applicable requirements. Essential 
documents shall be archived in a way that ensures that they are readily available, 
upon request, to the competent authorities.  The medical files of study subjects shall 
be retained in accordance with national legislation and the maximum period of time 
permitted by the hospital, institution, or private practice.  The Principal 
Investigators is responsible for contacting the Sponsor before the destruction of any 
study related documents and must wait for a written approval from the Sponsor 
before destruction proceeds. 

 
Clinical Study Protocol 
Title:  Plasma-Based Next-Generation Sequencing for Pathogen Detection 
and Quantification in Children with Musculoskeletal Infections 
 
Supersedes: 0.8 
Document Number: KDG-002 
Clinicaltrials.gov #: NCT03846804 
Effective Date: 19Oct2021 
Page: 22 of 30 
 
 
11.6 
Confidentiality Policy 
By conducting this study, the Principal Investigators affirms to the Sponsor that all 
study results and information furnished by the Sponsor will be maintained in strict 
confidence. Such information will be communicated to the Principal Investigator’s 
IRB under an appropriate understanding of confidentiality. 
 
11.7 
Principal Investigators Obligations in Conduct of Study and Protection of 
Human Subjects  
The Principal Investigators must ensure that: 
1. He or she will personally conduct or supervise the study. 
2. His or her staff and all persons who assist in the conduct of the study clearly 
understand their responsibilities and have their names included in the Study 
Staff Signature and Delegation of Authority log.  The Principal Investigators 
will sign the authorization log whenever it is updated with new responsibilities 
or staff membership. 
3. The study is conducted according to the protocol and all applicable regulations. 
4. The protection of each subject’s rights and welfare is maintained. 
5. Signed and dated informed consent and permission to use protected health 
information are obtained from each subject prior to conducting study 
procedures. If a subject withdraws permission to use protected health 
information, the investigators will obtain a written request from the subject and 
will ensure that no further data be collected from the subject. 
6. The consent process is conducted in compliance with all applicable regulations 
and privacy acts. 
7. The IRB complies with applicable regulations and conducts initial and ongoing 
reviews and approvals of the study. 
8. Any amendment to the protocol is submitted promptly to the IRB. 
9.   Any significant protocol deviations are reported to the Sponsor and the IRB 
according to the guidelines at the study site. 

 
Clinical Study Protocol 
Title:  Plasma-Based Next-Generation Sequencing for Pathogen Detection 
and Quantification in Children with Musculoskeletal Infections 
 
Supersedes: 0.8 
Document Number: KDG-002 
Clinicaltrials.gov #: NCT03846804 
Effective Date: 19Oct2021 
Page: 23 of 30 
 
 
12 APPENDIX A:  SCHEDULE OF EVENTS 
 
 
 
 
 
 
 
 
 
 
Procedure 
Screening  
Visit 
SHC ED  
Enrollment Visit 
(V1) 
 
 
 
 
  
 
  
  
     
  
 
     
  
 
     
 
 
 
  
 
 
 
 

 
Clinical Study Protocol 
Title:  Plasma-Based Next-Generation Sequencing for Pathogen Detection 
and Quantification in Children with Musculoskeletal Infections 
 
Supersedes: 0.8 
Document Number: KDG-002 
Clinicaltrials.gov #: NCT03846804 
Effective Date: 19Oct2021 
Page: 24 of 30 
 
 
13 APPENDIX B:  ID-DX SAMPLE COLLECTION INSTRUCTIONS  
 
 
 
 
 
 

 
Clinical Study Protocol 
Title:  Plasma-Based Next-Generation Sequencing for Pathogen Detection 
and Quantification in Children with Musculoskeletal Infections 
 
Supersedes: 0.8 
Document Number: KDG-002 
Clinicaltrials.gov #: NCT03846804 
Effective Date: 19Oct2021 
Page: 25 of 30 
 
 
14 APPENDIX C. CASE REPORT FORM 
Eligibility Evaluation 
 
In order to qualify for the study, the following inclusion criteria must be met: 
Inclusion criteria questions 1-3, 4 must be marked "yes". 
Exclusion criteria question 4 and 5 must be marked “no” 
Does the patient qualify for the study?    
 YES 
 NO  
 
 
 
 
 
Inclusion Criteria 
Yes 
No 
1. Is the patient 6 months to < 18 years old?  
 
 
 
2. Is there a strong clinical suspicion of MSKI? 
  Fever (temperature >38 degrees C 
 
  Osteoarticular pain 
 
  Elevated inflammatory markers CRP ≥ 3 mg/dL or ESR ≥ 40 mm/hr  
 
 
 
3. Is patient, or legal representative, capable and willing to give written 
informed consent prior to any screening procedures?  The Informed 
Consent form must be signed before any study procedures are 
performed.   
 
Date of Informed Consent:   _ _/ _ _/ _ _ _ _          (mm/dd/year) 
Time of Informed Consent:  _________________ (24-hr clock) 
 
 
4. Is there clinical evidence of an alternative diagnosis? 
 
 
 
5. Will the patient have a surgical procedure prior to obtaining NGS 
sample? 
 
 

 
Clinical Study Protocol 
Title:  Plasma-Based Next-Generation Sequencing for Pathogen Detection 
and Quantification in Children with Musculoskeletal Infections 
 
Supersedes: 0.8 
Document Number: KDG-002 
Clinicaltrials.gov #: NCT03846804 
Effective Date: 19Oct2021 
Page: 26 of 30 
 
 
 
 
 
Date of Enrollment  
 
(MM-DD-YYYY) 
 
Study Number 
 
 
Admission Date and Time 
 
 
(MM-DD-YYYY); 00:00 
Discharge Date and Time 
 
 
(MM-DD-YYYY); 00:00 
Sex: 
 Male 
 Female 
Age 
 
 
Years 
Sex 
F/M 
Race/Ethnicity (circle one) 
White Non-Hispanic, White Hispanic, 
Black Non-Hispanic, Black-Hispanic, 
Asian, Other _____ 
Infection Type (circle one) 
Acute hematogenous osteomyelitis 
(AHO), Septic Arthritis, Pyomyositis 
Infection Location 
 
 
Name of bone/joint infected 
Days of Symptoms  
 
 
Days of symptoms (number) related to 
MSKI (fever, pain) prior to hospital 
admission 
Antibiotic Prior Admit 
Antibiotic given prior to admission 
(Y/N) 
Antibiotic (Initial) Name 
 
 
Name of initial antibiotic(s) given for 
MSKI 

 
Clinical Study Protocol 
Title:  Plasma-Based Next-Generation Sequencing for Pathogen Detection 
and Quantification in Children with Musculoskeletal Infections 
 
Supersedes: 0.8 
Document Number: KDG-002 
Clinicaltrials.gov #: NCT03846804 
Effective Date: 19Oct2021 
Page: 27 of 30 
 
 
Antibiotic (Initial) Time  
 
(MM-DD-YYYY); 00:00 
Surgical Procedure 
Y/N 
Culture Pathogen ID  
 
 
Name of pathogen by culture 
Culture source 
 
 
Blood, bone/synovial fluid/tissue, both 
NGS Pathogen ID- (IP1) 
 
 
Name of pathogen by NGS 
NGS Quantification- (IP1) 
 
 
Molecule per microliter (MPM) of 
cfDNA 
NGS Pathogen ID- (IP2) 
 
 
Name of pathogen by NGS 
NGS Quantification- (IP2) 
 
 
Molecule per microliter (MPM) of 
cfDNA 
NGS Pathogen ID- (IP3) 
 
 
Name of pathogen by NGS 
NGS Quantification- (IP3) 
 
 
Molecule per microliter (MPM) of 
cfDNA 
NGS Pathogen ID- (IP4) 
 
 
Name of pathogen by NGS 
NGS Quantification- (IP4) 
 
 
Molecule per microliter (MPM) of 
cfDNA 
NGS Pathogen ID- (OP1) 
 
 
Name of pathogen by NGS 
NGS Quantification- (OP1) 
 

 
Clinical Study Protocol 
Title:  Plasma-Based Next-Generation Sequencing for Pathogen Detection 
and Quantification in Children with Musculoskeletal Infections 
 
Supersedes: 0.8 
Document Number: KDG-002 
Clinicaltrials.gov #: NCT03846804 
Effective Date: 19Oct2021 
Page: 28 of 30 
 
 
 
Molecule per microliter (MPM) of 
cfDNA 
NGS Pathogen ID- (OP2) 
 
 
Name of pathogen by NGS 
NGS Quantification- (OP2) 
 
 
Molecule per microliter (MPM) of 
cfDNA 
NGS Pathogen ID- (OP3) 
 
 
Name of pathogen by NGS 
NGS Quantification- (OP3) 
 
 
Molecule per microliter (MPM) of 
cfDNA 
WBC x 10^3/uL 
 
 
White blood cell count 
WBC Timepoint 
 
 
(MM-DD-YYYY); 00:00 
CRP  
 
 
mg/dL 
CRP Timepoint 
 
 
(MM-DD-YYYY); 00:00 
ESR  
 
mm/hr 
ESR Timepoint 
 
 
(MM-DD-YYYY); 00:00 
PCT 
 
 
ng/mL 
PCT Timepoint 
 
 
(MM-DD-YYYY); 00:00 
 

 
Clinical Study Protocol 
Title:  Plasma-Based Next-Generation Sequencing for Pathogen Detection 
and Quantification in Children with Musculoskeletal Infections 
 
Supersedes: 0.8 
Document Number: KDG-002 
Clinicaltrials.gov #: NCT03846804 
Effective Date: 19Oct2021 
Page: 29 of 30 
 
 
 
 
 
 
 
 
 

 
Clinical Study Protocol 
Title:  Plasma-Based Next-Generation Sequencing for Pathogen Detection 
and Quantification in Children with Musculoskeletal Infections 
 
Supersedes: 0.8 
Document Number: KDG-002 
Clinicaltrials.gov #: NCT03846804 
Effective Date: 19Oct2021 
Page: 30 of 30 
 
 
15 APPENDIX D: REFERENCES 
1. Williams DJ, Deis JN, Tardy J, et al. Culture-Negative Osteoarticular Infections in the Era of 
Community-Associated Methicillin-Resistant Staphylococcus aureus. Pediatr Infect Dis J. 
2010;30: 523-5. 
 
2. Saavedra-Lozano J, Mejías A, Ahmad N, et al. Changing trends in acute osteomyelitis in 
children: impact 
of methicillin-resistant Staphylococcus aureus infections. J Pediatr Orthop. 2008;28: 569-75. 
 
3. Thomsen I, Creech CB. Advances in the diagnosis and management of pediatric osteomyelitis. 
Curr Infect Dis Rep. 2011;13: 451-60. 
 
4. Verdier I, Gayet-Ageron A, Ploton C, et al. Contribution of a broad range polymerase chain 
reaction to the diagnosis of osteoarticular infections caused by Kingella kingae: description of 
twenty-four recent pediatric diagnoses. Pediatr Infect Dis J. 2005;24: 692-6. 
 
5. Peltola H, Pääkkönen M, Kallio P, et al. Short- versus long-term antimicrobial treatment for 
acute hematogenous osteomyelitis of childhood. Pediatr Infect Dis J 2010; 29: 1123–28. 
 
6. McMullan BJ, Andresen D, Blyth CC, et al. Antibiotic duration and timing of the switch from 
intravenous to oral route for bacterial infections in children: systematic review and guidelines. 
Lancet Infect Dis. 2016;16: e139-52. 
 
7. Wanda L, Ruffin F, Hill-Rorie J, et al. Direct Detection and Quantification of Bacterial Cell-
free DNA in Patients with Bloodstream Infection (BSI) Using the Karius Plasma Next 
Generation Sequencing (NGS) Test [abstract 2083]. In: ID Week 2017. San Diego, CA: 
Infectious Diseases Society of America, 2017. 
 
8. Benvenuti MA1, An TJ, Mignemi ME, et al A Clinical Prediction Algorithm to Stratify 
Pediatric Musculoskeletal Infection by Severity. J Pediatr Orthop. 2016 Sep 27.