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Transforming infectious disease diagnostics with genomics

STUDY SUMMARY

Karius Test for the Detection and Monitoring of CMV Viremia

CLIA #: 05D2121236CAP #: 9497749

#558: Use of the Quantitative Karius® Plasma Next Generation Sequencing Cell-Free Pathogen DNA Test to Detect and Monitor Cytomegalovirus Infection in Allogeneic Stem-Cell Transplant Recipients

Monica Fung, MD1, Justin Teraoka1, Kathy Lien1, Hon Seng2, Adama Parham, MBA2, Desiree Hollemon, MSN, MPH2, David K. Hong, MD2, Lily Blair, PhD2, Simona Zompì, PhD, MD2, Aaron C. Logan, MD, PhD1, Joseph D. Yao, MD3 and Peter Chin-Hong, MD1

(1)University of California, San Francisco, San Francisco, CA(2)Karius, Inc., Redwood City, CA(3)Mayo Clinic, Rochester, MN

POSTER SESSION II - Infectious Diseases and Cytotoxic T LymphocytesSaturday, February 23rd: 6:45 - 7:45 PM

The Karius Test may be used to monitor CMV viral load in patients while also being able to detect and quantitate 1,000+ pathogens from a single blood draw.

PATIENT POPULATION / STUDY DESIGNAnalytical comparison and validation of the Karius Test included 125 residual plasma samples that were negative or positive for CMV using two separate FDA approved assays. Quantitative CMV results from the Karius Test were compared and correlated with results from the two quantitative PCR tests for CMV monitoring.

To determine clinical utility of the Karius Test for monitoring CMV infection levels, plasma samples from 70 allo-SCT recipients with CMV viremia were tested.

RESULTSThe Karius Test showed high correlation with two FDA-approved CMV viral load tests. It had 94% agreement with the cobas CMV test using its cut-off for negative samples (<37.5 IU/ml) and 95% using a clinical utility cut-off (<2000 IU/ml). There is 95% agreement between the CAP/CTM and cobas CMV tests for both cut-offs.

Among 70 allo-SCT patients with known CMV viremia, the Karius Test showed potential clinical utility as a CMV monitoring assay with significant mean differences in CMV quantities (MPM) when compared between Day 0 and ≥ Day-7 as well as between Day 0 and ≥D14 of antiviral treatment.

Background: Allogeneic stem-cell transplant (allo-SCT) recipients are at risk of developing severe CMV infection. The pre-emptive approach to CMV prevention, weekly monitoring with CMV PCR with treatment for positive viral load (VL), is standard of care. The quantitative Karius plasma Next Generation Sequencing test (KT) can identify DNA from >1,000 human pathogens. We report on the clinical validation and utility of KT for detecting and monitoring CMV viremia in allo-SCT patients.

Methods: Plasma samples negative or positive by CMV PCR (COBAS®

AmpliPrep/COBAS® TaqMan® CMV Test [CAP/CTM] or cobas 6800/8800 [cobas® CMV]) were tested with KT for analytical comparison. For KT, after cell-free DNA (cfDNA) was sequenced, human-derived reads were excluded bioinformatically, and remaining DNA sequences were aligned to a curated microbial pathogen database. KT results were reported as molecules per microliter (MPM). Linear regression was used to correlate log10 transformed CMV VL results within the overlapping quantifiable range of comparison assays. Using the regression equation to convert KT results from MPM to IU/mL, percent agreement was calculated and assay result agreement was analyzed with Bland-Altman plots. Allo-SCT recipients with CMV viremia were selected from the prospective observational DISCOVER trial (NCT02804464), and their plasma specimens were tested with KT. Mean differences in MPM between ≥7 days before start of antiviral treatment (D-7) and start of treatment (D0), and between D0 and ≥14 days after start of treatment (≥D14) were examined with Wilcoxon rank-sum tests.

Results: Among 125 residual samples tested by all 3 assays, 1 sample failed KT sequencing and was excluded from the analysis. Percent agreement between KT and cobas CMV (n = 124) was 94% using the cobas CMV cut-off for negative samples (VL<34.5 IU/mL) and 95% using a clinical utility cut-off (VL <2,000 IU/mL). Comparatively, the percent agreement between CAP/CTM and cobas CMV was 95% for both cut-offs. Correlation coefficients were 0.921 for qKT vs CAP/CTM (n = 74) and 0.951 for KT vs cobas CMV (n = 87), with result differences ranging -0.90 to 0.90 log10 and -0.81 to 0.81 log10, respectively. Among 70 DISCOVER trial patients, those with detectable KT CMV showed significant mean differences in MPM between ≥D-7 and D0 of antiviral treatment (n = 11; p = 0.042) and between D0 and ≥D14 of antiviral treatment (n = 10; p = 0.009).

Conclusions: High correlation was observed between KT and two FDA-approved CMV VL assays. KT identified significant increases in CMV clinical utility of VL at start of treatment and significant decreases at ≥14 post-treatment in allo-SCT recipients, showing potential KT as a monitoring assay, with the added value of detecting >1,000 pathogens in the same blood draw.

RESULTS

BACKGROUND AND METHODS

Use of the Quantitative Karius® Plasma Next Generation Sequencing Cell-free Pathogen DNA Test to Detect and Monitor Cytomegalovirus Infection in Allogeneic Stem-Cell Transplant Recipients

Monica Fung1, Justin Teraoka1, Kathy Lien1, Hon Seng2, Adama Parham2, Desiree Hollemon2, David K. Hong2, Lily Blair2, Simona Zompì2, Aaron C. Logan1, Joseph D. Yao3 and Peter Chin-Hong1

1University of California, San Francisco, CA; 2Karius, Inc., Redwood City, CA; 3Mayo Clinic, Rochester, MN, U.S.A.

CONCLUSION

Sample Processing and Workflow

Figure 4: Case Examples of CMV Viremia and and Early Detection of Bacterial Co-Infections Identified by Conventional Testing and the Karius Test

1. A strong correlation was observed between the Karius Test and two FDA-approved CMV Viral load assays.

2. The Karius Test is a novel quantitative method for monitoring CMV viremia in patients with potential utility for monitoring CMV treatment response.

3. The Karius Test may be used to monitor for CMV viremia and other significant infections simultaneously

Figure 2: Correlation and Concordance of Karius Test versus cobas® CAP/CTM and cobas ® CMV qPCR Testing

Figure 3: Response to Treatment for Symptomatic and Asymptomatic DISCOVER Subjects with CMV Viremia

CAP/CTM CMV VL and Karius® Test MPM kinetics of two DISCOVER subjects with symptomatic CMV infection (A, B) and two DISCOVER subjects with asymptomatic CMV viremia (C, D) and their response to treatment.

Conflict of Interest Disclosures:

This study was funded by Karius, Inc. MF and PCH received a research grant from Karius to conduct the DISCOVER Study. AL and JY have no conflict of interest. JY has no conflict of interest. HS, AP, DH, DKH, LB, SZ are employees of Karius, Inc.

Abstract #558

Karius® Test is compared with CAP/CTM CMV assay (n=74) (A, C) and with cobas® 6800/8800 CMV assay (n=87) (B, D). Regression equations from each comparison were used to log transform MPM Karius Test results into IU/mL. Log transformed mean VL (x axis) are compared to the VL differences (y axis) in the Bland-Altman plots (C, D).

(A) (B)

(C) (D)

Figure 1: Performance of Karius® Test versus cobas ® CMV qPCR Testing

ABSTRACT

87 3

3 31

41 5

3 75

cobas CMV/8800 cobas CMV/8800Pos Neg

(≥titer min*) (<titer min)Pos Neg

(≥2,000 IU/mL) (<2,000 IU/mL)

Neg

Po

s(≥

titer

min

)

(<tit

er m

in)

Neg

Pos

(≤20

00 IU

/mL)

(

≥200

0 IU

/mL)

Kar

ius

Kar

ius

N= 124Sensitivity 97%Specificity 91%% Agreement 95%

N= 124Sensitivity 93%Specificity 94%% Agreement 94%

Contingency table assessing agreement between Karius Test and cobas CMV/8800 using the titer min cutoff of 34.5 IU/mL (A) and a clinical cutoff of ≥2,000 IU/mL (B). Regression equations from each assay comparison were used to convert MPM Karius Test results into IU/mL.

*Titer min = 34.5 IU/mL

(A) (B)

Subject #1 Case Details:

21yo M with AML s/p allogeneic stem cell transplant. Patient was admitted on HD #60 with urethral pain and acute kidney injury. Underwent ureteroscopy on HD#63 which showed diffuse bladder inflammation. R ureter dilated and stent placed. HD#70 urine Chlamydia testing positive

Plasma NGS positive for Chlamydia trachomatis on HD#39 and HD#50 (31 days before clinical testing)

Subject #2 Case Details:

69yo F with myelodysplastic syndrome s/p allogeneic stem cell transplant.Transplant complicated by VOD, renal failure, respiratory failure.MRSA bacteremia on HD#26 which was treated and cleared. MRSA bacteremia again on HD#53.

Plasma NGS positive for Staphylococcus aureus on HD#25 and HD#46(1 day and 7 days before clinical testing)

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