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ASM Microbe 2019 Abstract Summary

Karius® Test for Diagnosis of Nocardia Infections

Genomic insights for infectious diseases

Poster Session CPHM-102—Molecular Diagnostic Microbiology Friday | June 21 | 11:00 AM–12:00 PM, 4:00–5:00 PM

Poster CPHM-957

Next-generation Sequencing of Pathogen Cell-free DNA in Plasma (Karius Test) Reveals Nocardia Species Diversity in Clinical InfectionsAsim A. Ahmed1, Michael Rosen1, David K. Hong1, Sudeb C. Dalai1, Ann MacIntyre1, Lily Blair1, Martin Lindner1, Jeev Bala1, Sivan Bercovici1

(1) Karius, Inc., Redwood City, CA

Results The Karius Test detected the following N. asteroides complex species: N. farcinica (N=3), N. abscessus (N=3), N. arizonensis (N=1), and N. niwae (N=1).

In the remaining two samples, the exact Nocardia species could not be resolved. In one of the samples, sequencing reads were shared between three closely-related species of N. asteroides complex type III. Additionally, data suggested the presence of a yet un-sequenced genome belonging to the Nocardia nova/elegans/africana cluster. In the second sample, sequencing reads were shared between two closely-related species with data suggesting divergence and evolution of the strain from those two.

Study Design Results from all Karius Tests reported during 2018 were reviewed for cases of Nocardia. Ten such results were analyzed at the sequence level for the specific Nocardia species identified by the Karius Test.

Abstract

Background Analysis of indeterminate Nocardia species

Next-Generation Sequencing of Microbial Cell-Free DNA in Plasma (The Karius® Test)Reveals Nocardia Species Diversity in Clinical Infections

Asim A. Ahmed MD, Michael Rosen PhD, David K. Hong MD, Sudeb C. Dalai MD, PhD, Ann MacIntyre DO, Lily Blair PhD, Martin Lindner PhD, Jeev Bala PhD, Sivan Bercovici PhD

Karius, Inc., Redwood City, CA, USAkariusdx.com

BackgroundNocardia can cause pulmonary, brain, skin or disseminated infections in immunocompromised and immunocompetent hosts. Definitive diagnosis can be challenging, requiring direct testing of respiratory, brain or skin samples. The gold standard for Nocardia diagnosis involves culture with buffered charcoal yeast extract that is not available in all microbiology laboratories. Modified acid-fast staining can also be performed but has limited yield. Molecular methods have led to reclassification of Nocardia and identification of new species. N. asteroides complex – the cause of the majority of invasive disease – has been reclassified into six phenotypic groups based on antibiotic sensitivity profile.

MethodsThe Karius Test (KT) is a CLIA certified/CAP-accredited next-generation sequencing (NGS) plasma test that detects microbial cell free DNA (mcfDNA)1. After mcfDNA is extracted and NGS performed, human reads are removed and remaining sequences are aligned to a curated database of > 1000 organisms. Organisms present above a statistical threshold are reported. KT results from the prior year (Redwood City, CA) were reviewed for detections of Nocardia. Clinical information was obtained from medical consultations with Karius medical directors. For unresolved Nocardia species (cases 2, 5 and 11) Venn diagrams were generated showing the intersection between the strain detected and the three closest Nocardia reference strains in the database. Identity plots were generated showing varying similarity of the strain detected against a larger set of genome references. A heat map demonstrating the diversity and phylogenetic relationships within the genus was generated to provide context for the Nocardia species detected.

ResultsThe KT detected Nocardia species in thirteen patients including nine adults and four children with a male predominance (N=10). Ten patients were immunocompromised; two patients were heart transplant recipients. Nine patients had a pulmonary focus with a presumptive lower respiratory tract infection, five had brain lesions and four had evidence of multifocal, disseminated disease. Most Nocardia detected were in the N. asteroidescomplex, including N. farcinica (N=3) which belongs to type V, N. abscessus(N=3) belonging to type I, N. cyriaciegoergica (N=3) belonging to type VI, N. arizonensis (N=1) and N. niwae (N=1). In three samples the exact Nocardia species could not be resolved. In one sample reads were shared between N. elegans, N. nova and N. africana (all closely-related species in N. asteroidescomplex type III). Sequences suggest the presence of a yet un-sequenced genome belonging to the Nocardia nova/elegans/africana cluster. In two other samples reads were shared between N. gamkensis and N. exalbida (two closely-related species), with sequence data suggesting the strain diverged and evolved from the two reference strains.

ConclusionOpen-ended unbiased sequencing reveals a diverse spectrum of Nocardiaspecies including members of the former N. asteroides complex. NGS for mcfDNA offers a non-invasive means of diagnosing Nocardia infections and capturing species diversity, potentially uncovering new insights on genus complexity not resolved by current taxonomic classification.

References1. Blauwkamp TA, Thair S, Rosen MJ, Blair L, Lindner MS, Vilfan ID et al. Analytical and Clinical Validation of a Microbial Cell-Free DNA Sequencing Test for Infectious Disease. Nature Microbiology 2019 April, 4(4):663-674.

Contact: asim.ahmed@kariusdx.com(617) 780-8899

Nocardia detections using The Karius® Test

MPM=molecules/microliter, RR=reference range (97.5%ile MPM among self-reported normal individuals), F=female, M=male, A=adult, C=child, IC=immunocompromised, Y=yes, N=no, Cx=culture, I-VI superscript denotes former Nocardia asteroides complex phenotypic sub-type

Case 2

Case 5

Case 11

Percent Identity Plots Alignment Overlap Venn DiagramNumbers = aligned reads

Friday June 21, 2019CPHM – 957

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ilarit

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Reference Similarity and Phylogenetic Heat Map