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Transforming infectious disease diagnostics with genomics

STUDY SUMMARY

Karius Test for Pediatric Patients with Invasive Fungal Infections

CLIA #: 05D2121236CAP #: 9497749

Plasma Next-Generation Sequencing for Pathogen Detection in Pediatric Patients at Risk for Invasive Fungal Infection

The Karius Test can detect invasive fungal infections (IFI) directly from blood in high-risk pediatric immunocompromised patients and can identify non-fungal pathogens in individuals that have prolonged febrile neutropenia but do not meet criteria for proven or probable IFI.

Amy Armstrong, MD1, Jenna Rosso�, MD1, Romielle Aquino, BA2, Desiree Hollemon, MSN, MPH2, David Hong, MD2, Sonali Chaudhury, MD1 and William Muller, MD, PhD3 (1) Pediatric Hematology, Oncology, and Stem Cell Transplantation, Ann & Robert H. Lurie Children's Hospital of Chicago, Chicago, IL, (2 )Karius, Inc., Redwood City, CA, (3 )Pediatric Infectious Diseases, Ann & Robert H. Lurie Children's Hospital of Chicago, Chicago, IL

PATIENT POPULATION

RESULTS

STUDY DESIGN

This study enrolled 40 evaluable patients with underlying oncologic diagnoses who were determined to be at risk for IFI.

There were 6 patients who met established EORT-C/MSG criteria for proven IFI, one for probable IFI, and 13 for possible IFI.

Immunocompromised pediatric patients with suspected IFI were enrolled.

A plasma sample was obtained at the time of enrollment for Karius testing. Results were not available to clinicians in real-time.

The Karius Test identified the same pathogen as tissue or blood culture in four out of the six proven IFI cases. The test identified Pneumocystis jirovecii in one patient with probable IFI based on positive β-D-glucan test.

Among the 33 patients without proven or probable IFI, the Karius Test detected a fungal pathogen (Candida glabrata) in one patient.

In patients with prolonged fevers without proven IFI, the Karius Test identified viral pathogens that fit the clinical symptoms.

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Plasma Next-Generation Sequencing for Pathogen Detection in Pediatric Patientsat Risk for Invasive Fungal Infection

Amy E. Armstrong1, Jenna Rossoff1, Romielle Aquino,2 Desiree Hollemon2, David K. Hong2, Sonali Chaudhury1, William J. Muller3

1. Division of Hematology, Oncology and Stem Cell Transplantation, Ann & Robert H. Lurie Children’s Hospital of Chicago, Northwestern University Feinberg School of Medicine, Chicago, IL; 2. Karius, Inc., Redwood City, CA ; 3. Division of Infectious Diseases, Ann & Robert H. Lurie Children’s Hospital of Chicago, Northwestern University Feinberg School of Medicine, Chicago, IL

• Invasive fungal disease (IFD) is a major cause of mortality and morbidity among pediatric immunocompromised patients such as those who receive chemotherapy or hematopoietic stem cell transplantation

• The current diagnostic ‘gold standard’ of IFD remains culture of infected tissue obtained by biopsy

• Noninvasive biomarker testing for galactomannan or 1,3-β-D-glucan (BG) can have low sensitivity and does not provide species-level identification

• Next-generation sequencing (NGS) of cell-free plasma is a promising noninvasive approach to providing species-level identification of IFD via a blood test and can further guide specific treatment

Describe the incidence of positivity for fungal specific pathogens on NGS analysis in a high-risk immunocompromised pediatric population and correlate results with other ‘standard’ infectious studies if performed

Patient Demographics (n=40)

OBJECTIVES

BACKGROUND

METHODS

RESULTS

CONCLUSIONS

REFERENCE1. De Pauw, et al. Revised Definitions of Invasive Fungal Disease from the

EORTC/MSG Consensus Group. Clinical Infectious Diseases. 2008; 1813-21.

RESULTS

• In this single-institution study, we demonstrate the first successful use of NGS to detect infectious pathogens in immunocompromised pediatric patients

• NGS identified fungal pathogens (including PJP) in 7 of 40 high-risk patients for IFD• Antifungal therapy was administered to 32 patients without proven fungal disease or NGS detection of a

fungal pathogen (80%); of these, 23 (72%) received prolonged therapy• NGS identified non-fungal pathogens, mainly DNA viruses, in a majority of patients providing potential

alternatives for prolonged FN or abnormal imaging findings in patients at risk for IFD• We propose NGS as a non-invasive diagnostic tool that if positive for fungal pathogens in patients with

possible and probable IFD can preclude the need for invasive measures and guide treatment for systemic infection

• If results of NGS are negative for fungal pathogens and patient does not meet other diagnostic criteria or only has concerning findings on chest CT, consideration may be given to discontinuing fungal therapy

Criteria for Enrollment

None(21) Possible

(11)

Proven (6)

Probable (1)

Patient Fungal Classification Based on Clinical Testing

Fungus Isolated

SpecimenSampled

Detected by NGS?

Aspergillus fumigatus

Lung Yes

Candida albicans

Blood Yes

Rhizopus species

Scalp lesion (skin biopsy)

Yes

Candida albicans

Pancreaticpseudocyst

Yes

Aspergillus fumigatus*

Lung No

Rhizopusoryzae

Cheek lesion (skin biopsy)

No

*Aspergillus grew on day 27 of 28 of culture; 18s rDNA was negative as were fungal stains• IRB approved prospective trial in which eligible patients were approached for

consent, assent and/or parental permission to have 6 cc of blood drawn with routine lab draws for NGS and to undergo electronic chart review

• Inclusion criteria: hematology, oncology, and stem cell transplant patients at our institution with prolonged fever and neutropenia (FN) (≥96 hours) despite broad-spectrum antibiotics or any findings that triggered consideration of a new fungal infection (i.e. abnormal imaging, recrudescent fevers in setting of neutropenia, characteristic skin rashes)

• Exclusion criteria: patients without concern for fungal infection or those currently being treated for a presumed or documented fungal infection (on >3 days of treatment antifungal dosing)

• Plasma layer extracted by centrifugation (1500 rpm for 10 minutes at room temperature) and aliquots frozen at -80°C until shipment to Karius, Inc.

• Cell-free DNA extracted from plasma, libraries prepared, and agnostic NGS applied (human sequences removed, remaining reads aligned to a curated database including >1200 pathogens where organisms present at a significance-level above a predefined threshold were reported)

• Clinical data from all infectious evaluations performed around time of enrollment and patient and disease information obtained via electronic chart review

• Toxoplasma gondii detected in a patient with prolonged FN and CT chest suggestive of fungal infection

• Candida glabrata isolated in a patient with prolonged FN but no other criteria of IFD

• NGS detected Pneumocystis jirovecii in 2 patients with abnormal CT chests and elevated BG (369 and >500 pg/mL); in 1 of these patients, lung biopsy was negative for Pneumocystis jirovecii but eventually grew Aspergillus fumigatus

• Numerous pathogens also identified on NGS that could explain the clinical parameters for enrollment, including HSV1, CMV, VZV, HHV6, EBV and BK polyoma virus. NGS accurately identified:

• 4/4 patients with detectable CMV (range >137-3647 IU/mL)

• 2/2 patients with BK viremia (20-22 x103 copies/mL)

Fungal Classification

Received > 1 Week

AntifungalTreatment

None 13/21 (62%)

*NGS identified Candida glabrata

in 1 patient

Possible 9/11 (82%)

Probable 0/1 (0%)

*NGS identified Pneumocystis

jirovecii

Use of Antifungals for Presumed IFD

Definitions of Invasive Fungal Disease1

Possible At risk host + meets 1 clinical criterion: i.e. abnormal imaging (CT/MRI), bronchoscopic or ophthalmologic analysis

Probable Above criteria and meets mycological criterion: i.e. direct detection of mold in sputum, BAL, sinus aspirate or positive Galactomannan or BG

Proven Recovery of a mold or yeast by microscopic analysis or culture of normally sterile site(biopsy, blood, CSF)

Other Findings

11/11 patients had abnormal chest CTs

Age - year

Median 11.0 years

Range 1.2-24.2 years

Sex – no (%)

Male 26 (65%)

Female 14 (35%)

Race – no (%)

White 15 (38%)

Non-white 25 (63%)

Disease Classification – no (%)

Leukemia/Lymphoma 22 (55%)

Solid/CNS Tumor 7 (18%)

Stem Cell Transplant 9 (23%) (6 Allo,3 Auto)

Other 2 (5%)