value of genetic testing and a. lenore ackerman, md phd
TRANSCRIPT
A. Lenore Ackerman, MD PhDDivision of Pelvic Medicine and Reconstructive Surgery
Value of Genetic Testing and
Next-Generation Sequencing
in Diagnosis of UTI
Bacteria in the Urinary Tract…
…always means infection, right?
Urinary Tract InfectionsNo universally accepted definitions of UTI-associated symptoms, cut-offs for culture results, or definitive accepted uropathogens that can guide us objectively in the management of UTI.
DEFINITIONS
“Microbial infiltration of the normally sterile urinary tract”1
“Significant bacteriuria in a patient with symptoms or signs attributable to the urinary tract and no alternate source”2
1Barber et al. Clin Infect Dis 2013. 57(5):719-7242Hooton et al. Clin Infect Dis 2010. 50(5):625-663
Even in healthy, immunocompetent hosts Even in subjects without urinary symptoms
Microbes reside within the urinary tract
“Urine is sterile”
Nope, not anymore1
1Hannan and Hultgren. ASM Microbes 2014
The Human Microbiome
Commensal, symbiotic polymicrobial communities
At least as many bacteria as there are cells in our bodiesHighly diverseEnvironment-specificCarefully balanced
Rosen et al. PLOS Medicine 2007 4(12): e329.
Normal functions of bacteria in the GU tract1
Tell your audience what needs to happen first to achieve your outcome.
Normal Functions of Bacteria in the GU Tract1. Production of neurotransmitters
that interact with the nervous system
2. Competition with pathogens for resources
3. Regulation/maintenance of epithelial junctions
4. Production of antimicrobial compounds
5. Priming of epithelial and immune defenses
6. Degradation of harmful compounds
7. Development of the urinary tract8. Creation/maintenance of a
barrier at the uroepithelium
1Whiteside, et al. Nat Rev Urol 2015
DYSBIOSISMicrobial imbalance or maladaptation on or inside the body
Implicated in diseases of every organ system, from depression to diabetes to cancer
Kinross et al. Genome Medicine 2011. 3:14
MECHANISMS OF DYSBIOSIS IN DISEASE
• Loss of the normal bacteria that maintain the gut barrier
• Overgrowth of pathobionts/pathogenic bacteria
• Generation of a local inflammatory response
• Loss of gut barrier• Local organ dysfunction,
systemic inflammation, even cancer promotion
1Kamada et al. Nat Rev Immunol 2013 13:321–335
Two (not so) similar patients
2-3 episodes a yearSudden onset dysuria with severe urgency
Always has >100,000 E. coli on cultureResponds to antibiotics within a 1-2 days
No symptoms between infections
Case 1
Let’s take…
34 yo F5-6 episodes a year
Worsening pressure, frequency, dysuriaCultures typically negative
Needs 2-3 weeks of antibiotics to feel better Still has milder symptoms between infections
Case 2
34 yo F
Follow the AUA Recurrent UTI Guidelines…
I’m not so sure WHAT to do…
• Recurrent uncomfortable urinary tract symptoms perceived to be similar to UTI
• No microbiological evidence of infection on standard clinical urine culture
• Failure to improve with standard UTI-type therapies
UTILS(Yoo-tîls)
UTI-like Symptoms
Lynn Stouthers, MD
A MICROBIOME-INSPIRED THEORY
Symptoms in patients with UTILs must be due to some infection that we have been unable to detect due to limitations in culture sensitivity
Characterizing the Urinary Microbiome and “Dysbiosis”
What is in there?Is it a living community? Are we sure it’s coming from the urinary tract?
Pros ConsCulture-dependent:Microbes must grow!
• Ensure viable organisms• Cheaper, Faster
• Underestimates Diversity• <1% organisms thought to be
cultivable Culture-independentDetects other markers of microbes (DNA, proteins, RNA)
• More sensitive• Can detect difficult to grow
organisms • Can detect minor species
• Overestimates Diversity• Contamination can be a
problem• Biases of analytic methods• Some methods more costly,
time consuming
CULTURE-DEPENDENT METHODS
STANDARD CLINICAL URINE CULTURENO universally accepted definition of positive
ENHANCED QUANTITATIVE URINE CULTURELonger incubationsLarger amount of urine culturedMultiple culture conditions
Wolfe, A. ASM Microbes 2016.
EQUC provides improved detection of urinary bacteria
Many of the bacteria detected by sequencing are in fact cultivable from urine samplesBacteria can be grown using EQUC from subjects with AND without symptoms
• Several studies detected bacteria in ~90% of subjects considered controls (no LUTS)
• Equivalent detection of E. coli in control and UTI groups
• Bacterial counts alone could not distinguish UTI from control groups
Wolfe, A. ASM Microbes 2016Hilt et al. J Clin Microbiol 2014. 52(3): 871-876Price et al. J Clin Microbiol 2016. 54(5): 1216-1222
CLINICAL UTILITY OF EQUC
Price et al. J Clin Microbiol 2016. 54(5): 1216-1222
For +EQUC and Negative standard culture, no difference in improvement +/-antibiotics
Use of Culture-Dependent Assays for the Diagnosis and Treatment of UTI?
• Standard culture remains the usual clinical gold standard, but has many shortcomings
• EQUC provides better identification of uropathogens, as well as commensal bacteria (asymptomatic bacteriuria)
• But EQUC has yet to demonstrate a clinical benefit
• Combination of EQUC with other urine biomarkers may provide the best information to transform UTI care
NOT YET READY FOR PRIME TIME
CULTURE-INDEPENDENT METHODS
Ilhan, Z.E. ASU PhD Dissertation. 2016.
NEXT GENERATION SEQUENCING• Detects microbes through the isolation of microbial DNA fragments • Amplification of a sufficiently variable DNA region allows identification of microbial
taxa • Most commonly uses the ribosomal DNA locus
16S NGS for “UTI”• No big differences in16S
NGS between UTI and controls
• Enterobacteriaceae (which contains E. coli) detected in all samples
• NGS cannot measure levels of bacteria, only relative composition of the population (which looked pretty similar)
Sathiananthamoorthy et al. J Clin Microbiol 2019. 57 (3) e01452-18;UTI Control
Quantitative PCR (qPCR)FOR UROPATHOGENS
In a population of over 200 patients revealed 98% concordance between rapid PCR-based detection and standard culture.1
PCR-based detection of E coli was positive in 80% of UTI patients with symptoms who were culture-negative. However, the test also detected E. coli in 12% of controls using these cut-offs.2
1 Van der Zee et al. PLOS One 2016. 11(3):e01507552 Heytens et al. Clin Microbiol Infect 2017. 23(9):647-652
“16S PCR is more expensive than targeted PCR, with both assisting in microbiological diagnosis but uncommonly enabling antimicrobial change”
IS MORE REALLY BETTER?• Identify bacteria in more patients
• Identify fastidious bacteria• Provide faster assessment of
antibiotic susceptibility• Identification of drug resistance• Recognition of community
effects that affect treatment responses
MOLECULAR DIAGNOSTIC APPROACHES:
Aggarwal, D. et al. Sci Rep 10, 7965 (2020).
“Return on investment on the program happens swiftly (within the first month), with the ability to earn 7 figures or more in net income for practices with 5 providers or more on an annual basis.”
-Marketing Materials
???????
-Alan Wolfe, PhD
Loyola Univeristy
You can actually identify bacteria in almost everybody with this test. So what we don't
want to do is to have clinicians over-treat, try to sterilize the bladder, right?
You want to get rid of the symptoms.”
So, how do we NOW define UTI vs. UTILs vs. Asymptomatic Bacteriuria?
EQUC, NGS, qPCR and Metagenomics all aim to give a more complete characterization of GU microbes
BUT in gaining the ability to identify bacterial communities from everyone, regardless of symptoms, we now must redefine the questions of what is a UTI?
-Debbie Downer
Urinary bacterial communities are diverse and complex: There is not as yet a simple way to define microbiologically what is healthy and what needs treatment or intervention
Senior VP of Buzzkilling
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Should ALL Urinary Tract Infections Should be Treated with Antibiotics?
Primary goal of treatment should be the amelioration of symptoms and prevention of complications, such as pyelonephritis/sepsis
Progression to Pyelonephritis is infrequent1: <0.4% in untreated AND antibiotic-treated patients2
Duration of symptoms is only longer in untreated patients by 1-2 days
Management of symptoms, recovery and progression to pyelonephritis in patients given ibuprofen similar to those given antibiotics3
Actual Benefit of Antibiotics is Limited:
1Foxman, B. Nat Rev Urol 2010. 7:653–6602Christiaens et al. Br J Gen Pract 2002. 52, 729–734
3Bleidorn et al. BMC Med. 2010. 8: 30
*For Research Purposes Only…
…at least for now
While increasingly sensitive and specific methods* to detect urinary bacteria are
increasing our understanding of genitourinary microbial communities, it is not yet clear how to integrate this knowledge into clinical care
CONCLUSIONS
• Prognostic biomarkers • Assays for bacterial
virulence factors (uropathogenic strains)
• Microbial Manipulation
Healthy genitourinary commensal bacteria
play an important role in preventing UTI
Bacteria aren’tall bad
“Which patients have bacteria in their urine?” Future Studies
“Which patients would benefit from intervention and what kind?”
