UPIP, a precision metagenomics panel, detected uropathogens and antimicrobial resistance markers in urine samples with greater sensitivity than shotgun metagenomics. UPIP identified one or more of the eight most common uropathogenic bacteria in 86% of samples compared to 66% for shotgun metagenomics and 51% for urine culture. UPIP also showed improved detection of extended-spectrum beta-lactamase genes, methicillin-resistant Staphylococcus aureus, and vancomycin-resistant enterococci compared to shotgun metagenomics. The enhanced sensitivity of UPIP makes it a promising alternative to standard testing for urinary tract infections.

1. 22.EP.21.01
Marta Mangifesta1, Rita Stinnett1, Kate Broadbent1, Amy Hanson1, John Rossen1, Jamie Lemon1, Robert Schlaberg1
1IDbyDNA, Inc
Introduction
Methods
Discussions
References
Results
Conclusions
Precision Metagenomics Vs. Shotgun Metagenomics for Common Gram-Negative and
Gram-Positive Uropathogens For any questions, please
contact:
Research and
Development, IDbyDNA
research@idbydna.com
Acknowledgements
We are grateful to the entire Microbiology Unit at PathGroup Labs for coordination of sample selection, de-identification and shipment, and to Illumina for reagents for library preparation and enrichment in support of this study. We also thank the following
individuals for their key contributions to this work: the IDbyDNA Laboratory, MSA and Bioinformatics teams.
FEMS
Session Date 7/1/2022
• Urinary tract infections (UTIs) affect ~150 million (M)
people globally per year, including 2.6 M emergency
departments (ED) visits in US, for recurrent or difficult-to-
treat UTIs, in 2016 alone.1,2
• Antimicrobial resistance (AMR) in common uropathogens
is increasing. Production of ESBL and other mechanisms
of resistance are associated with clinical treatment failure
in patients with UTIs.3
• Precision Metagenomic (PM) next-generation sequencing
(NGS) offers a culture-independent alternative to rapidly
and accurately detect and quantify uropathogens and
AMR markers.
• The Explify® Urinary Pathogen ID/AMR Panel (UPIP) uses
target-enriched PM to detect and quantify 174
uropathogens (121 bacteria, 35 viruses, 14 fungi, 4
parasites), and >3,500 AMR markers associated with
resistance to 46 antimicrobials in 24h and includes fully
automated data analysis.
• We compared detection of the the 8 most common
bacterial causes of UTIs and relevant AMR markers by
UPIP and shotgun metagenomics (SM) using 399 urine
samples from patients with suspected UTI that were also
tested by culture.
• Samples selection, specifically flagged by conventional
urine culture and antimicrobial susceptibility testing
(AST), was performed by a commercial reference
laboratory. Growth of ≥10,000 colony-forming units was
interpreted as positive.
• DNA was extracted (Quick-DNA Urine Kit, Zymo) for UPIP
and SM, sequencing libraries were enriched in 3-plex for
UPIP (IDbyDNA), sequenced (1x147bp, NextSeq550,
Illumina) to a depth of ~1 M (UPIP) or ~10 M (SM) reads,
and automated data analysis performed with Explify
(IDbyDNA).
• Results were considered positive if any of the following
were detected: Escherichia coli, Klebsiella
pneumoniae, Proteus mirabilis, Pseudomonas
aeruginosa, Enterococcus faecalis, Enterococcus
faecium, Staphylococcus aureus and Staphylococcus
saprophyticus
SM-Pos. SM-Neg.
101
102
103
104
105
106
107
108
109
Pathogen
Load
(copies/mL)
UPIP SM Culture
0
50
100
150
200
250
300
350
400
Samples
with
Positive
Results
SM
UPIP Positive Negative
Positive 61 77
Negative 4 52
Table 2. Detection in culture-
positive samples
SM
UPIP Positive Negative
Positive 197 7
Negative 0 1
Table 3. Detection in culture-
negative samples
Organism Group UPIP SM UPIP Yield Increase
Escherichia coli
Gram-negative
114 76 33%
Klebsiella pneumoniae 80 40 50%
Proteus mirabilis 38 18 53%
Pseudomonas aeruginosa 28 16 43%
Enterococcus faecalis
Gram-positive
209 116 44%
Enterococcus faecium 19 10 47%
Staphylococcus aureus 26 21 19%
Staphylococcus saprophyticus 45 29 35%
UPIP Increases Diagnostic Yields for All 8 Top Bacterial Uropathogens
Table 4. Compared to SM, UPIP increased the yield for detection of Gram-negative
bacteria by 42% and in Gram-positive bacteria by 41%.
Improved Uropathogen Detection by UPIP
Higher Detection Rates for ESBL, MRSA, and VRE by UPIP
SM
UPIP Positive Negative
Positive 258 84
Negative 4 53
Table 1. Detection of top 8
uropathogens by UPIP and SM
SM
UPIP Positive Negative
Positive 9 1
Negative 0 0
Table 5. Prediction of ESBL
phenotype by UPIP and SM.
Table 6. Detection of S. aureus and
prediction of MRSA phenotype.
Table 7. Detection of Enterococcus sp.
and prediction of VRE phenotype.
Figure 1. (A) One or more of the 8 uropathogens were detected in
342/399 (86%) urines by UPIP, in 262/399 (66%) by SM, and in 205
patients (51%) by urine culture. (B) Bacterial load by UPIP was higher in
SM-positive than in SM-negative samples (p<0.0001).
Higher Detection Rates for Top 8 Uropathogens by UPIP
SM
UPIP Positive Negative
Positive 3 2
Negative 0 0
SM
UPIP Positive Negative
Positive 3 1
Negative 0 0
A B
UPIP was more sensitive for
detection of the top 8 bacterial
uropathogens than SM. The
difference was most pronounced
in culture-negative samples
where UPIP was positive in 138
(71%) samples compared to 65
(34%) by SM.
All (n=399) Culture-Pos. (n=205)
Culture-Neg. (n=194)
ESBL (n=10)
MRSA (n=5) VRE (n=4)
Sensitivity (UPIP & SM)
• Although shotgun metagenomics can detect hundreds of
microorganisms in the urine, targeted-enrichment with
UPIP offers higher sensitivity for detection and
quantification of the most common uropathogens.
• UPIP provides comprehensive and more sensitive
detection of genomic markers for antimicrobial resistance
than SM with fully automated analysis from sequencing
data to result. Rapid profiling of AMR markers has the
potential to inform antimicrobial decision making earlier
than current standard of care testing.
• Common empiric antimicrobial therapy for UTI in
outpatients (cephalexin, trimethoprim-sulfamethoxazole,
nitrofurantoin) and hospitalized patients (ceftriaxone)
often provide insufficient coverage for ESBL-producing
gram-negative uropathogen. Early detection of an ESBL-
producing bacteria allows for appropriate escalation of
antimicrobial therapy.
• UPIP provides improved detection of the most common
bacterial causes of UTI and AMR markers associated with
ESBL/MSRA phenotype when compared to a validated
shotgun metagenomic workflow while reducing the
required sequencing depth by 10-fold to ~1 M reads per
sample.
• UPIP, a comprehensive NGS test with fully automated data
analysis, provides a culture-independent alternative for
detection and quantification of 174 bacterial, viral, fungal,
and parasitic uropathogens and >3,500 AMR markers.
• In the future, prospective clinical studies will be
conducted to assess the clinical impact of UPIP on patient
care and clinical outcomes in patients with UTI.
1. Stamm WE, Norrby SR. J Infect Dis. 2001;183(Suppl
1):S1-S4.
2. Goebel MC, Trautner BW, Grigoryan L. 2021 Clin Micro
Rev 34(4): e00003-20
3. Anesi JA, Lautentech E, Nachamkin E, et al. 2018. Infect
Control Hosp Epidemiol 39:1431-1435
ESBL MRSA VRE
0
20
40
60
80
100
90
Sensitivity
(%)