Clinical Guidance on the Use of Next Generation Sequencing

(NGS) Tests for Infectious Diseases

Table of Contents
EXECUTIVE SUMMARY:...................................................................................................................................................................... 2
Clinical utility and limitations of NGS tests ................................................................................................................................ 3
Table 3a: Focal site(s) of infection ................................................................................................................................................. 5
Table 3c: Respiratory Infection....................................................................................................................................................... 8
Table 3d: Meningoencephalitis ....................................................................................................................................................... 9
Table 3e: Invasive Fungal Infection, Febrile Neutropenia, and Fever of Unknown Origin ................................. 10
Clinical scenarios in which Karius Testing may be considered ...................................................................................... 12
Clinical scenarios in which Karius Testing should usually be avoided ....................................................................... 12
Clinical scenarios in which Karius Testing should NOT be used.................................................................................... 13
How to order a Karius Test ............................................................................................................................................................ 14
How to read the Karius report ..................................................................................................................................................... 15
Karius Test classification of microorganisms ........................................................................................................................ 15
Karius Test results: Example scenarios .................................................................................................................................... 16
How to order University of Washington Broad-Range PCR ............................................................................................. 18
How to read the Broad-Range report ........................................................................................................................................ 19
How to order Delve Bio cell free DNA (previously University of California San Francisco Center for Next-
Gen precision diagnostics CSF cfDNA) ...................................................................................................................................... 20
How to read the Delve Bio report ............................................................................................................................................... 20
References............................................................................................................................................................................................. 21

Developed by: Cathy Cichon, MD - Jan 2024

Reviewed and approved by: Antimicrobial Stewardship Committee – Feb 2024
 Clinical Guidance on the Use of Next Generation Sequencing
(NGS) Tests for Infectious Diseases

EXECUTIVE SUMMARY:
Next-Generation Sequencing tests should ONLY be considered when the following
criteria are met:
1. Clearly identifiable focus on infection (do not use in undifferentiated clinical
conditions)
2. Anticipated prolonged course of antimicrobial therapy (i.e. weeks to months;
avoid NGS if anticipating short [≤14 day] course of empiric therapy or no plans to treat)
3. Negative conventional workup at >48 hours (or longer, depending on the clinical
scenario, suspected pathogen, and type of conventional testing sent)

An Infectious Diseases consult is required to order these tests.

Next-generation sequencing (NGS) is a culture-free method of analyzing the microbes within a sample. These
tests sequence all or part of the microbial genes in a patient specimen, such as serum, tissue, or CSF. This review
will focus on the clinical use of NGS tests, including the Karius Test, University of Washington Broad-Range
PCR, and Delve Bio (previously UCSF Center for Next-Gen precision diagnostics) CSF cfDNA. Separate
guidelines regarding the clinical use of multiplex molecular panels are available on the UNMC Clinical Microbiology
website: https://www.unmc.edu/intmed/divisions/id/asp/clinicalmicro.html

An Infectious Disease consult is required to obtain next-generation sequencing. Only ID clinicians are able
to order these tests in One Chart so they assist with interpretation of results and management decisions.

Abbreviations:
CAP = College of American Pathologists IFI = Invasive Fungal Infection MSSA = methicillin-sensitive
CLIA = Clinical Laboratory Improvement LDT = Laboratory Developed Test staphylococcus aureus
Amendments of 1988 LRTI = lower respiratory tract infection NGS = next generation sequencing
FDA = Food and Drug Administration MRSA = methicillin-resistant SOTR = solid organ transplant recipient
HSCT = hematopoietic stem cell staphylococcus aureus URTI = upper respiratory tract infection
transplant

Table 1: Next-generation sequencing technology1

Type of next- Mechanism Examples

generation
sequencing (NGS)

Whole genome Sequencing of the entire microbial genome from isolated Identification of microbial
sequencing (WGS) colonies pathogens for hospital and public
health epidemiological studies

Metagenomic NGS Sequencing of all the nucleic acids directly from patient Karius Test and
(mNGS) specimens (including pathogen and human DNA) without Delve Bio CSF test
culture

Targeted NGS Sequencing 16s rDNA (bacterial) or internal transcribed University of Washington Broad-
(tNGS) spacer (ITS; fungal) regions following amplification Range PCR
directly from patient specimens

2
Table 2: Available molecular diagnostic assays

Clinical specimen Available tests Turnaround time Estimated cost

Multiplex molecular panels (please see separate guidance documents for use and interpretation)

CSF BioFire Meningoencephalitis Panel 60 min $$

Respiratory BioFire Respiratory Pathogen Panel 45 min $$

(for URTI)

BioFire Pneumonia Pathogen Panel 60 min $$

(for LRTI)

Fungal plus PCR profile 1 and 2 (for 24 hours from $$$

LRTI) receipt of specimen
(send-out)

Stool BioFire Gastrointestinal Pathogen 60 min $$

Panel

Joint BioFire Joint Pathogen Panel 60 min $$

Next generation sequencing assays

Plasma* Karius Test (cell free DNA) 2-4 business days $$$$
from receipt of
specimen (send- List price (2024): $2,000
out)

Tissue University of Washington Broad-Range 5-7 business days $$ - $$$$ (depending on how
PCR (can include bacterial, fungal, from receipt of many target groups are
nontuberculous mycobacterial, and/or specimen (send- selected for testing, i.e.
MTB complex targets all of which must out) bacterial only, bacterial +
be ordered seperately) fungal, etc)

List prices (2024):

- Bacterial: $300
- Fungal: $490
- MTB: $220
- Non-MTB: $330

CSF Delve Bio cell free DNA (previously 1-2 weeks from $$$$
UCSF Center for Next-Gen precision receipt of specimen
diagnostics) (send-out) List price (2024): $3,100

*Plasma specimens can be stored at ambient temperature for 96 hours and at -20°C for up to 6 months. Karius
testing can be performed on specimens that are >96 hours old, if they were appropriately frozen.2

Clinical utility and limitations of NGS tests

Patient outcomes are typically not included in studies of NGS tests, making it difficult to determine the clinical
utility of these tests. Real-world studies have demonstrated limited meaningful clinical impact, even when these
tests are restricted to Infectious Diseases specialists.3,4,5 NGS testing may be advantageous for detecting
obscure or rare pathogens, pathogens that are difficult to grow using conventional methods, or in patients
previously treated with antimicrobials (where culture-based testing may be falsely negative6). Some NGS tests
(i.e. Karius Test) also offer a relatively rapid turnaround time, which can be advantageous for a limited number
of clinical syndromes described later in this document; however, the majority of NGS tests take several weeks
to result. The numerous NGS testing limitations must be taken into account including:

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Potential Limitations of NGS:
1. While additional pathogens may be detected via NGS, false negative results do occur when compared
to conventional testing. Thus a negative result does not rule out infection and must be put in context
of the pre-test probability of disease.
2. NGS tests do not distinguish commensal or colonizing organisms from pathogenic organisms, so
results must be interpreted based on the clinical picture.
3. NGS tests do not usually provide antimicrobial susceptibility data, although the Karius Test now
incorporates some common antimicrobial resistance markers (e.g. mecA, CTX-M, etc.). As with all
genotypic resistance testing, detection of the resistance markers does not always correlate with
phenotypic resistance, nor does the abscence of the markers guarantee susceptbility. As such, NGS-
identified resistance markers must be interpreted with caution and should be confirmed with standard
cultures as much as possible.
4. The Karius Test only detects pathogens that have DNA (e.g. bacteria, fungi, DNA viruses). Therefore
it is not able to detect RNA viruses such as HIV, hepatitis C, Zika, or coronaviruses.

All currently available NGS tests have not been approved by the FDA, although the FDA is actively exploring
regulatory approaches to this technology to ensure that NGS tests have adequate analytical and clinical
performance.7 Rather, these tests are developed by their respective parent laboratories as Laboratory
Developed Tests (LDT). As a result, each test is only commercially available from its own CLIA-certified, CAP-
accredited laboratory, leading to longer turnaround times due to shipping of the specimen.

Additional details regarding how to order and interpret each test are provided at the end of this document.

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 Recommended clinical use of NGS tests:
Table 3a: Focal site(s) of infection
Table 3(a-e) is loosely adapted from the American Society of Transplantation (AST) consensus
conference’s statements on best practice use of next generation sequencing assays in SOT recipients.8

Clinical Clinical scenario Testing order of preference Guidance on Use of NGS

syndrome

Focal site(s) of Suspected (1) Tissue cultures and Conventional testing is the preferred
infection infectious focus on histopathology initial step (i.e. biopsy with cultures
(Figure 1) radiographic (2) Broad-Range PCR if and histopathology, blood cultures,
evaluation that tissue is available* (ID antigen testing, and serology as
cannot be sampled consult required) appropriate). If conventional testing is
due to anatomical (3) Karius Test if tissue not negative, NGS testing from an infected
site or procedure available (ID consult sample source may be considered.
risk (ex: deep brain required) ● NGS testing of tissue
abscess) or has specimens (Broad-Range
negative PCR) is preferred over Karius
conventional testing Test. Karius Test can be
(ex: spinal considered if samples from
osteomyelitis) infected sites are negative by
standard testing or cannot be
obtained.
● Negative NGS testing does
not rule out infection.
* Note: The yield of Broad-Range PCR can vary, depending on the quality of the tissue specimen.

Figure 1: Algorithm for considering NGS test for patients with focal sites of infection
Algorithms modeled after University of Washington / Seattle Children’s infectious encephalitis algorithm.9

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Table 3b: Endovascular Infection
Clinical Clinical Testing order of preference Guidance on Use of NGS
syndrome scenario

Endovascular Culture-negative (1) Blood cultures Conventional testing is the preferred

Infection endocarditis (2) Tissue cultures and initial step (i.e. biopsy with cultures and
(Figure 2) histopathology histopathology, blood cultures, antigen
Mycotic (3) If blood cultures are testing, and serology as appropriate). Select
aneurysm or negative at 48 hours, populations (SOTR with severe illness,
vascular graft Infectious Disease should HSCT) may benefit from plasma cfDNA
infection be consulted and send testing earlier in the disease course due to
second line of testing (i.e. the diversity of pathogens encountered.
Q fever, Bartonella ● Conventional testing (including
quintana and henselae, testing for infections associated with
Brucella Antibodies, and culture-negative endocarditis such
Histoplasma serum and as Q fever, Bartonella, Brucella, and
urine Ag). Simultaneously, Histoplasma) should precede NGS
save a plasma sample for testing.
possible Karius Test. ● Other causes of culture-negative
(4) Broad-Range PCR if endocarditis including Legionella,
tissue is available (ID Mycoplasma and Tropheryma
consult required) whipplei should be considered
(5) Karius Test if tissue is not before or in paralell with Karius
available (ID consult testing
required) ● Plasma samples obtained early in
the course of the infection can be
saved and frozen for later NGS
testing in case conventional testing
is unrevealing.
● NGS testing of plasma cfDNA after
broad antimicrobial use may provide
a retrospective diagnosis, limiting
prolonged or unnecessary broad-
spectrum antimicrobial therapy,
although the likelihood of a positive
test decreases with each day of
therapy. Karius Test (either by
stored plasma or as initial testing)
can be preferred if bacterial
endocarditis is suspected but
patient recieved antibiotic therapy
prior to blood culture collection.

NOTE: Although the usefulness of NGS for

blood culture-negative endocarditis (BCNE) is
still being evaluated, the ISCVID Working
Group, in their updated 2023 Duke Criteria for
Infectious Endocarditis (IE), has proposed
that a positive result for C. burnetti, Bartonella
species, or T. whipplei from a NGS platform
should constitue a Major Criterion for IE.
Positive NGS testing for any other organisms
should be considered as a Minor Criterion,
although keeping in mind that data is limited
regarding differentiation of “true positive” from
“contamination” in NGS tests as well as that
these tests can have limited sensitivity.10

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Figure 2: Algorithm for considering NGS test in patients with endovascular infections

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Table 3c: Respiratory Infection
Clinical Clinical scenario Testing order of preference Guidance on Use of NGS
syndrome

Respiratory Radiographic (1) If sputum available, obtain Conventional testing is the preferred
Infection findings (interstitial pneumonia pathogen panel initial step (i.e. sputum cultures,
(Figure 3) infiltrates, tree-in- and sputum Gram stain and pneumonia pathogen panel,
bud opacities, culture bronchoscopy as appropriate). If
consolidation, (2) If sputum not available, obtain conventional testing is negative, NGS
nodules, cavities) respiratory viral panel, urine testing may be considered.
and negative/ streptococcal Ag, urine ● If a tissue specimen is
inconclusive Legionella Ag available NGS testing of
conventional (3) Serum Beta-D-Glucan if infected sites (Broad-Range
testing suspicion for Pneumocystis PCR) is preferred over Karius
pneumonia (PCP) Test. We recommend sending
Culture-negative (4) Bronchoscopy specimens for Broad-Range PCR from tissue
severe community- Gram stain, fungal stain, specimens only; it should not
acquired aerobic bacterial culture, be sent directly from BAL fluid.
pneumonia nocardia culture, fungal culture ● If a respiratory specimen is
and pneumonia PCR panel if available (i.e. BAL) and
Immuno- not previously obtained. invasive fungal infection is
compromised host Consider fungal studies if suspected, consider Fungal
with concern for concern for invasive fungal Plus PCR 1 or 2 prior to Karius
lower respiratory infection (see IFI below), or Test
opportunistic silver stain if concern for PCP; ● Non-pathogenic members of
infection Infectious Disease should be the respiratory microbiome
consulted in these cases. must be clinically differentiated
(5) Broad-Range PCR if tissue is from pathogens.
available (i.e. lung biopsy) (ID
consult required). This should We do not recommend NGS testing if
not be sent from BAL fluid. planning to treat with short empiric
(6) If all standard cultures/ course of therapy (<14 days).
serologies are negative,
infection is still suspected, and
Karius Test is being
considered, Infectious Disease
must be consulted for input.

Figure 3: Algorithm for considering NGS test in patients with respiratory infections

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Table 3d: Meningoencephalitis
Clinical Clinical scenario Testing order of preference Guidance on Use of NGS
syndrome

Meningo- Clinical syndrome (1) CSF cell count, protein, Conventional testing is the preferred
encephalitis of meningo- glucose initial step (i.e. CSF cultures,
(Figure 4) encephalitis, CSF (2) Meningoencephalitis panel meningoencephalitis panel). If
pleocytosis, or and CSF cultures conventional testing is negative, NGS
other abnormal (3) Consider studies for West testing may be considered although the
parameters Nile virus, toxoplasmosis, incremental diagnostic yield is low and it
suggestive of syphilis, endemic fungal may miss organisms detected using
infection, but infection, and tuberculosis traditional testing.
negative as appropriate. ● NGS testing of CSF (Delve Bio) is
conventional (4) If standard cultures are preferred over Karius Test due to
testing negative, CSF indices are the proximity of CSF to the
suggestive of infection, and source of infection.
Delve Bio CSF mNGS is ● CSF samples obtained early in
being considered, consult the course of the infection can be
Infectious Disease for input. saved and frozen for later NGS
testing in case conventional
testing is unrevealing.*
● In cases where conventional
testing is positive for a likely
pathogen, do not use NGS to
confirm a diagnosis.
*Samples can be stored <6 hours at room temperature, <7 days at 2-8°C, and up to 90 days at -70°C. If the CSF specimen
is frozen, it is acceptable for testing provided it has been stored at -70°C, and has undergone no more than 2 freeze/thaw
cycles. We would recommend discussing with Delve Bio / UCSF prior to sending frozen specimens, to ensure that the
specimen will be accepted.

Figure 4: Algorithm for considering NGS test in patients with meningoencephalitis

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Table 3e: Invasive Fungal Infection, Febrile Neutropenia, and Fever of Unknown Origin

Syndrome Clinical scenario Testing order of preference Guidance on Use of NGS

Invasive Clinical concern for The menu of diagnostic testing is Conventional testing is the
fungal invasive fungal extensive, and should be tailored to the preferred initial step (i.e.
infection infection based on individual patient based on risk factors, galactomannan, fungal
(IFI) clinical, laboratory, patient history, clinical presentation, antigens/serology, etc.).
and radiographic epidemiology, and radiologic NGS testing has not
evaluation appearance. consistently exceeded detection
(particularly lung (1) See Table 3c regarding initial workup of invasive fungal infections by
infiltrates or sinusitis) for suspected lower respiratory conventional testing and clinical
but negative infection. This should include criteria even in high-risk
conventional workup consideration of Pulmonology consult populations such as SOT or
and bronchoscopy. HSCT recipients.
Biopsy specimen with (2) See Table 3a and Table 3d regarding ● Note that NGS
fungal forms on workup for CNS lesions (either focal demonstrated decreased
pathologic evaluation site of infection or sensitivity in detecting
but negative meningoencephalitis). infection with Aspergillus
conventional workup (3) If there is concern for fungal infection, species as compared to
Infectious Diseases should be other molds.
consulted to help guide workup based
on the patient’s risk factors and the
radiographic appearance. The
following diagnostic tests can be
considered but will vary according to
patient’s immunocompromised
status:
- Blood fungal diagnostic tests:
(1,3)-β-d-glucan (Fungitell),
cryptococcal Ag, Aspergillus
galactomannan, and endemic fungi
serologies (i.e. Histoplasma,
Coccidioides, Blastomyces
depending on geographic location
or exposures)
- Urine fungal diagnostic tests:
Histoplasma Ag
- Bronchoscopy fungal studies:
Fungal stains, fungal cultures,
Aspergillus galactomannan,
nocardia culture. Consider Fungal
Plus PCR 1 or 2 panel if there are
focal consolidations, nodules,
and/or cavities on chest imaging; it
is less useful for evaluating diffuse
ground glass opacities.
(4) Biopsy of involved organ (lung, sinus,
skin, GI tract, etc.) for fungal culture
and histopathology with GMS stains
- Consider adding Fungal Broad-
Range PCR if biopsy is done
(5) If biopsy is not done, IFI is strongly
suspected, and Karius Test is being
considered, Infectious Disease must be
consulted for input

Febrile LOW RISK: MASCC (1) Blood cultures; additional testing based Conventional testing is the
neutropenia Risk-Index Score of on symptoms and physical exam preferred initial step (i.e.
(Fevers with ≥21 or CISNE score findings (CXR, urinalysis, GI pathogen blood cultures, other cultures,
ANC <500 or of <3 panel or C. difficile assay, viral antigen testing, and serology as

10
<1000 with diagnostics, etc.) appropriate). If conventional
predicted HIGH RISK: MASCC (2) Consider additional diagnostics based testing is negative, NGS testing
decline to Risk-Index Score of on initial clinical presentation (i.e. from an infected sample source
<500 over <21 or CISNE score abdominal CT, sinus CT, etc.), in a high-risk patient may be
the next 48 of ≥3 particularly if neutropenic fevers last >3- considered.
hours) 5 days. Early ID consultation of is ● NGS testing of
recommended. affected sites/tissues
(3) If lung infiltrates or sinusitis are present, (Broad-Range PCR) is
consider workup for IFI evaluation (see preferred over Karius
above). Test.
(4) If patient is high-risk (see criteria to the ● Karius Test could be
left), there is no obvious focal site of considered if samples
infection, all standard cultures/ from infected sites are
serologies are negative, fevers persist, negative by standard
an infectious cause is still suspected, testing or cannot be
and Karius Test is being considered, obtained (ex: patient
Infectious Disease must be consulted with severe
for input. thrombocytopenia).
● Negative NGS testing
does not rule out
infection.

Fever of Patient with We recommend a tiered evaluation based Conventional testing is the
unknown temperature >101F on clinical history and exam. Some preferred initial step (i.e.
origin for >5 days inpatient considerations, which should be tailored to blood cultures, antigen testing,
or >10 days the individual patient, include: and serology as appropriate). If
outpatient but (1) Blood cultures conventional testing is negative,
negative (2) Imaging (i.e. CT chest / abdomen / NGS testing from an infected
conventional workup pelvis, transthoracic echocardiogram, sample source may be
CT/PET scan) considered.
(3) Simultaneous evaluation for malignancy ● NGS testing of
and rheumatologic conditions as infected sites/tissues
appropriate (Broad-Range PCR) is
(4) Infectious Disease consult and preferred over Karius
consideration of targeted infectious Test. However Karius
workup based on exposure history and Test could be
clinical findings (i.e. endemic fungi, TB, considered if samples
rickettsial, etc). from infected sites are
(5) If all standard cultures/ serologies are negative by standard
negative, fevers persist, infection is still testing or cannot be
suspected, and Karius Test is being obtained.
considered, Infectious Disease must be ● Saving of plasma
consulted for input. specimen for Karius
Test is not needed as
patients with FUO
should have persistant
fevers and not be on
antimicrobials.
● Negative NGS testing
does not rule out
infection.

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 Additional Clinical Guidance on the Use of Karius Testing
Karius testing has the ability to detect approximately 1,250 different pathogens (including bacteria, fungi, viruses, and
parasites). The full pathogen list is available here: https://kariusdx.com/karius-test/pathogens. As noted at the top of this
document, it only detects DNA-based organisms, so it is unable to detect RNA-based pathogens such as HIV, hepatitis C,
Zika, or coronaviruses. It also does not distinguish commensal organisms from pathogenic organisms, so test results must
be interpreted with care. The sensitivity of the test is undefined and a negative test result does not necessarily rule out
infection unless the pre-test probably is already low. Although some antimicrobial resistance markers can be reported, the
detection of these markers does not always correlate with phenotype resistance and we generally recommend correlating
these results with standard cultures. The typical turnaround time for this test is 2-4 business days (includes shipping time).
Karius is expensive and often not covered by insurance, although it can be billed under diagnosis-related group codes.
Finally, the clinical actions which should occur based on test results are currently unclear. For these reasons we
recommend judicious use of this test and provide guidance regarding appropriate clinical scenarios below.

Clinical scenarios in which Karius Testing may be considered

Culture-negative endovascular infections Tiered evaluation:

(endocarditis, mycotic aneurysm, vascular graft ● Perform standard evaluation (see Table 3b)
infections) ● Day 3+: Karius Test (if no surgical
intervention) or
● If valve surgery performed consider Broad-
Range tissue PCR (University of Washington;
send out test)

Deep seated infection with contraindication to or See Table 3a. Examples:

desire to avoid an invasive procedure - Deep brain abscesses with no plan for
aspiration or surgery
- Spinal osteomyelitis/discitis with negative
blood and bone/disc cultures (if biopsy was
done, would recommend Broad-Range tissue
PCR prior to Karius Test)

Deep seated infection with nondiagnostic invasive If standard microbiological workup (including tissue
workup, but imaging or pathology remain highly cultures and pathology) negative but infection is still
suspicious for infection highly suspected, consider Broad-Range PCR prior to
Karius testing.

Clinical scenarios in which Karius Testing should usually be avoided

Severe pneumonia* Tiered evaluation:

For high risk patients (patients with severe CAP,
patients with CAP on expanded-spectrum therapy such
as vancomycin/cefepime, patients not responding to
typical therapy, or patients with hospital-acquired or
ventilator-associated pneumonia), we recommend:
(1) Sputum culture, pneumonia pathogen panel,
etc. (see Table 3c)
(2) Bronchoscopy (with Pulmonology input)
(3) Karius Test (with ID input) only if all testing is
unrevealing and infectious cause is still
strongly suspected and anticipating prolonged
(>14 days) of antimicrobial therapy

Fever of unknown origin (i.e. temperature >101°F for See Table 3e. Limited data (case reports/series only)
>5 days inpatient or >10 days outpatient, with suggests that Karius testing is unlikely to be beneficial
negative workup) in FUO cases. Could consider if microbiological workup
is negative and infectious etiology remains high on the
differential.

12
*Data is strongest for pediatric patients with complicated pneumonia, suggesting that Karius has a much higher yield than standard
testing and can significantly impact antimicrobial management.11,12 A prospective study among mechanically ventilated adult
patients demonstrated only marginally better yield than standard antimicrobial evaluation (60% vs 52%), and it was unclear if this
led to any meaningful outcomes.13 Karius testing may be useful in adult patients with complicated pneumonia if standard
microbiological workup is unrevealing, particularly in deescalating/targeting antibiotics.

Clinical scenarios in which Karius Testing should NOT be used

Sepsis or suspected bacteremia There is no data regarding Karius testing’s impact on

meaningful outcomes compared to standard testing (i.e.
duration of hospital stay, mortality, etc.).

Febrile neutropenia See Table 3e. There is no data regarding Karius

testing’s impact on clinical outcomes compared to
standard testing (i.e. duration of hospital stay, mortality,
etc.)

Advanced HIV/AIDS (CD4 <200 cell/mm3) with fevers There is no evidence that Karius provides utility over
standard microbiologic testing, and many fevers may
resolve with appropriate treatment of the HIV

Polyarthritis There is no data regarding Karius testing’s impact on

diagnostic yield or meaningful outcomes in patients with
polyarthritis.

Serial monitoring in asymptomatic transplant Prospective studies suggest that Karius testing has
recipients some ability to predict fungal infections and bacterial
bloodstream infections, however there is no data yet
regarding the test’s impact on meaningful clinical
outcomes and cost justification in order to rationalize
serial testing.

Outbreak investigation for rare or difficult-to-isolate Karius testing has been used as a diagnostic tool for
pathogens outbreaks caused by very difficult-to-grow pathogens
(i.e. NTMs), but in general we would not recommend its
use.

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How to order a Karius Test
Karius testing can be ordered in Epic under “Karius Test”:

Specimens can also be requested to be held for possible future testing. This can decrease the effect of empiric antimicrobial
therapy on Karius Test results while awaiting standard microbiological workup. This can be ordered in Epic under “Hold
for Karius”:

If Karius testing is needed you must place a Karius order and notify the Sendout Dept to let them know a sample is being
held and now needs to be sent out. Samples will only be held for one week then disposed.

Sendout Lab can be notified via email (SendoutLab@nebraskamed.com) or phone call (402.559.9353); email would be
the preferred method for documentation purposes.

Specific information about the tube top, serum volume, storage, etc. can be found here: https://kariusdx.com/karius-
test/karius-test-process. Plasma specimens can be stored at ambient temperature for up to 96 hours and at -20°C for up
to 6 months.1

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How to read the Karius report

(1) Microbe(s) detected: Must be part of Karius test’s database (see website for full list), and must have cell-free
DNA detected in statistically significant amounts
(2) Classification of microbes: Microbes will be classified by Karius into one of three categories: (1) Obligate &
Opportunistic Pathogens, (2) Commensal Pathogens & DNA Viruses, or (3) Microbes with Published Case
Reports.
(3) Quantification: The concentration of microbial DNA is measured in molecules of DNA fragments per
microliter of plasma (molecules per microliter, MPM). Many variables can affect the MPM value, including the
turnover rate of the organism and the genome size of the organism, so the MPM value should not be
compared across different microorganisms. The gradient visualizes the MPM based on 10,000 specimens
with positive Karius Test results.
(4) Antimicrobial resistance: The presence or absence of 7 AMR markers (SCCmec, mecA, mecC, vanA,
vanB, CTX-M, and KPC) are reported across 18 bacterial pathogens. These results are typically reported as
an amended report the following day.

Karius Test classification of microorganisms

15
Karius Test results: Example scenarios
What is detected Example of microorganism(s) Example of clinical Interpretation
detected scenario

No organisms N/A Adult with valvular Typically indicates no infection

detected vegetation and does not or source has been controlled,
meet any other Duke however it should be noted
Criteria for infective that negative NGS testing
endocarditis does not completely rule out
infection. Test results can be
affected by location of the
infection and antimicrobial
therapy.

Microorganism with >316,000 MPM Toxoplasma gondii Immunocompromised Typically associated with
very high patient with fevers after advanced or disseminated
quantification stem cell transplant disease
engraftment

Obligate pathogen 87 MPM MTB complex Patient post-liver Typically indicates infection
with low quantification transplant with nodular
pneumonia

Microorganism with 20 MPM Candida albicans Immunocompromised Clinical context required -

very low patient with febrile could represent early
quantification neutropenia infection, end of infection,
pre-treated infection, or
commensal/colonizer/
contaminant

Polymicrobial, with 4,764 MPM Histoplasma Adult with pneumonia One organism (Histoplasma in
one significant capsulatum and multiple pulmonary this case) is the possible
microorganism and nodules causative pathogen, and the
multiple non-specific 1,470 MPM Veillonella parvula other organisms are
microorganisms consistent with GI
408 MPM E Coli commensals (suggestive of
mucosal barrier disruption)
322 MPM Neisseria bacilliformis

NOTE: Cannot compare MPMs across

different microbes!

Polymicrobial, with 979 MPM Fusobacterium Adult with prolonged Multiple commensal
multiple non-specific nucleatum hospitalization in ICU and organisms consistent
microorganisms on pressor support for 3 with mucosal barrier
852 MPM Actinomyces oris weeks for septic shock injury and GI
translocation
580 MPM Streptococcus
salivarius Can be seen in critically ill
patients and
512 MPM Rothia dentocariosa immunocompromised
patients

Polymicrobial, with 4,764 MPM Histoplasma Immunocompromised Clinical context required - all
multiple pathogenic capsulatum patient with known organisms could potentially be
microorganisms candidemia and pathogenic
2,480 Trichosporon faecale disseminated
Histoplasmosis, now with
648 Candida albicans febrile neutropenia

16
Factors to consider when interpreting the Karius Test: Frequent incidental findings:

- Location of infection - H. pylori

- Recent infections - Low level DNA viruses
- Latent DNA viruses - Commensal microbes (i.e. staphylococcus
- Antimicrobial pre-treatment epidermidis, corynebacterium, streptocci,
- Mucosal barrier injury actinomyces, etc.)
- GVHD
- Poor dentition
- Aspiration

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How to order University of Washington Broad-Range PCR

Broad-Range PCR testing can be ordered in Epic as discussed below. See screenshot from Epic for ordering details.
You must be logged into an Infectious Disease context and have saved these labs as “preferences” to be able to
sign the order.

To order PCR testing for individual bacterial targets, fungal targets, or mycobacterial targets (non-TB mycobacteria
and MTB complex), order the desired individual test as follows:
• Bacterial Detect PCR (Code: LAB4531)
• Fungal Detection PCR (Code: LAB4532)
• Non TB Mycobact PCR (Code: LAB4533)
• M. tuberculosis PCR (Code: LAB4534)

To order PCR testing for both mycobacterial targets (non-TB and mycobacteria), use the following test order:

• AFB PCR (Code LAB4538)

To order PCR testing for all four targets (bacterial, fungal, NTM, and MTB) for a specimen, use the following test
order:
• Broadrange PCR (Code: LAB 4537)

Locating the specimen:

(1) If you place the order before the sample is collected, the sample for broad-range PCR generally gets split in
Specimen Receiving. Send-outs will receive a portion without further intervention. You can confirm that send-
outs has received the specimen by calling 402-559-9353.
(a) Note: If the patient discharges before Specimen Receiving receives the sample, the broad-range PCR
order will auto cancel. If you are requesting broad-range PCR on a sample obtained through an
outpatient procedure or on a patient that will be discharging, please contact send-outs to confirm that
the specimen has been received.
(2) If you are adding broad-range PCR on after cultures in micro have been set, please call the micro lab at 402-552-
2090 and request that tissue be sent to send-outs for broad-range PCR. If multiple samples were collected for
the patient, please indicate if you would like the specimens pooled. Tissue samples are held for 7 days. Please
note that the micro lab will not always have remaining tissue, depending on the quantity of specimen received
and the orders placed.
(3) If you are adding broad-range PCR on to a sample in pathology (such as a paraffin block), please call send-outs
at 402-559-9353 to coordinate with Pathology. Note that formalin-fixed specimens will have lower sensitivity than
fresh specimens, due to degradation of the DNA.

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How to read the Broad-Range report

Each PCR category (Bacterial, Fungal, NTM, and MTB complex) will list if any pathogens were **Detected**, followed by
the name of the pathogen that was identified.

If a pathogen is detected in one section, then one or more of the other sections might be labeled as **Indeterminate**
due to testing interference.

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How to order Delve Bio cell free DNA (previously University of California San Francisco
Center for Next-Gen precision diagnostics CSF cfDNA)

Delve Bio CSF testing can be ordered in Epic as a “Special Procedure”:

How to read the Delve Bio report

Each section (DNA viruses, RNA viruses, Bacterial, Fungi, and Parasites) will list if any pathogens were **Detected**,
followed by the name of the pathogen. Results are interpreted by laboratory physicians prior to release, so organisms that
are considered commensal or environmental are excluded from the report.

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American Association for Clinical Chemistry (AACC). Published online September 1, 2021. Accessed June 4,
2023. https://www.aacc.org/cln/articles/2021/september/next-generation-sequencing-for-infectious-diseases-
diagnostics-is-it-worth-the-hype
2. Karius. Karius Test Process. Accessed June 4, 2023.https://kariusdx.com
3. Hogan CA, Yang S, Garner OB, Green DA, Gomez CA, Dien Bard J, Pinsky BA, Banaei N. Clinical Impact of
Metagenomic Next-Generation Sequencing of Plasma Cell-Free DNA for the Diagnosis of Infectious Diseases:
A Multicenter Retrospective Cohort Study. Clin Infect Dis. 2021 Jan 27;72(2):239-245. doi: 10.1093/cid/ciaa035.
PMID: 31942944.
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10.1097/INF.0000000000003395. PMID: 34845152.
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print. PMID: 37815489.
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Hollemon DH, Blauwkamp TA, Ho C, Seng H, Shah P, Wanda L, Fowler VG, Ahmed AA. Microbial Cell-Free
DNA Identifies Etiology of Bloodstream Infections, Persists Longer Than Conventional Blood Cultures, and Its
Duration of Detection Is Associated With Metastatic Infection in Patients With Staphylococcus aureus and
Gram-Negative Bacteremia. Clin Infect Dis. 2022 Jun 10;74(11):2020-2027. doi: 10.1093/cid/ciab742. PMID:
34460909; PMCID: PMC9187311.
7. FDA. Optimizing FDA’s Regulatory Oversight of Next Generation Sequencing Diagnostic Tests—Preliminary
Discussion Paper. Published online December 2014. https://www.fda.gov/media/90403/download
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disease diagnostics in solid organ transplant recipients. American Journal of Transplantation. 2022;22(12):3150-
3169. doi:10.1111/ajt.17147
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Infectious Diseases. Clinical Laboratory News. Published online January 1, 2023.
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María Nazarena Pizzi, Stephen D Preston, Albert Roque, Francois Vandenesch, Jan T M van der Meer,
Thomas W van der Vaart, Jose M Miro, The 2023 Duke-International Society for Cardiovascular Infectious
Diseases Criteria for Infective Endocarditis: Updating the Modified Duke Criteria, Clinical Infectious Diseases,
Volume 77, Issue 4, 15 August 2023, Pages 518–526, https://doi.org/10.1093/cid/ciad271
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