New Laboratory Technologies · New Laboratory Technologies Karissa Culbreath, PhD D(ABMM)...
Transcript of New Laboratory Technologies · New Laboratory Technologies Karissa Culbreath, PhD D(ABMM)...
New Laboratory Technologies
Karissa Culbreath, PhD D(ABMM)Scientific Director – Infectious Disease, TriCore
Reference LaboratoriesAssistant Professor – Department of Pathology,
University of New Mexico
Objectives
• Describe the current state of routine and molecular infectious disease testing
• Provide case examples of the benefits and challenges of molecular diagnostics
• Explore further advances in molecular and conventional testing to improve infection control
• Hospital Laboratory for 2 major health systems in New Mexico– Presbyterian Health System
• 8 Hospitals
– University of New Mexico Health System
• 2 Hospitals (Level 1 Trauma Center, Cancer Center)
• Commercial Physician and Hospital Clients throughout the state– Samples are delivered by courier
24/7
• Infectious Disease Laboratory Test Volume– >850,000 Infectious Disease tests
per year
TriCore Reference Laboratories
• Infectious Disease Laboratory Test Volume– >850,000 Infectious
Disease tests per year
• Esoteric and routine testing
• 18,000 molecular tests per month
• 37,000 culture-based tests per month
TriCore Clinical Microbiology Laboratory
Diagnostic Tools for Pathogens
Serology Antigen Detection
Culture Molecular Detection
How does it work? Detection of antibodiesDeveloped against pathogen
Detection of antigens produced by the pathogen
Isolation of pathogen through growth
Nucleic acidamplification detection of specific genetic target
Advantages Can detect acuteand past infection
Rapid, remains positive after treatment
Inexpensive High sensitivity,Usually rapid
Disadvantages Delay due to time to develop antibodies
Lower sensitivity, may be strain limitations
Time consuming,dependent on appropriate growth conditions
Expensive,Must know the target of interest
When to use With pathogens unable or difficult to culture
Specific diseasescenarios
Emergence of new pathogens, susceptibility testing, strain typing
When rapid/accurate diagnosis is necessary
You decide… PCR vs. Culture
• Patient with liver abscess secondary to cirrhosis
• Patient with 3-week history of cough consistent with pertussis
• Patient with diarrhea following chemotherapy
• Identification of patients colonized with MRSA for infection control
• Patient presents to the emergency room with symptoms suspicious for bacterial meningitis
From There to Here
“An individual who gets used to hard work can thereafter never live without it. Work is the foundation of everything in this world.” ~Louis Pasteur
From There to Here
“An individual who gets used to hard work can thereafter never live without it. Work is the foundation of everything in this world.” ~Louis Pasteur
PCR PCR
A Microbiologist or Physician
~ 2017 Microbiologist or Physician
10 Years Ago
Viral Loads HIV, HCV
Molecular Methods
Bacterial CultureC. difficile testingRespiratory VirusesHerpes VirusesHPVAFB and Fungal Cultures
Culture/Antigen/ Microscopy
MRSA Screening
Current State
Viral Loads HIV, HCV, CMV, etcGenotypingHPV DetectionHSV, VZVRespiratory VirusesStool Pathogens
Molecular Methods
Bacterial CultureAFB and Fungal Cultures
Culture/Antigen/ Microscopy
MRSA ScreeningHPVC. difficilePositive Blood Culture
The Next 3-5 Years
Viral Loads HIV, HCV, CMV, etcGenotypingHPV DetectionHSV, VZVRespiratory VirusesStool PathogensWhole Genome SequencingDirect Sequencing from Specimens
Molecular Methods
Bacterial Culture but fewer
Culture/Antigen/ Microscopy
MRSA ScreeningHPVC. difficilePositive Blood CulturePrimary Sterile Fluid SampleAFB and Fungal Cultures
Does MRSA Still Matter?
• 86% of invasive MRSA infections are hospital-acquired infections (HAI)
– 20% result in death
• Risk of surgical site infections is 30-fold higher in MRSA colonized patients
• Several states have legislative requirements for MRSA screening
• New impact from Affordable Care Act (ACA)
http://www.cdc.gov/hai/pdfs/toolkits/MRSA_toolkit_white_020910_v2.pdf
Natural History of MRSA Colonization
• Clearance of MRSA colonization: 12%-79%
• 50% of patients clear colonization at 88 weeks after initial documentation of colonization
• Risk factors for persistent colonization
– Comorbidities (decubitus ulcers)
– Residence at health care facility
Shenoy et al, BMC Infect Dis. 2014, 14:177
Community MRSA Rates
Community MRSA Rates 1.3 – 2.0%
MRSA Rate in ICU
ICU MRSA Rates – 4%-8%
MRSA Rate in ICU
ICU MRSA Rates – 4%-8%Nosocomial Acquisition – 2%
MRSA Rate in ICU
30% of MRSA positive will develop invasive disease
• Resistance to β-lactams in MRSA is conferred by the genetic element SCCmeccarrying the mecA gene
• mecA encodes an altered PBP2a which has a reduced affinity for β-lactam antibiotics resulting in resistance
• Gold standard for confirming MRSA is either detection of mecA or PBP2a
Genetics of MRSA
Performance and Utility of Culture
• Performance– Sensitivity 80%-89%
• Broth enrichment can increase sensitivity to near equivalence with NAAT, but increase TAT
– Specificity 92%-99%– Less expensive
• Benefits– Organism available for strain typing– Phenotypic test not limited by changes in molecular
characteristics
• Limitations– Longer TAT (24 hours)
Performance and Utility of Molecular Screening Method
• Performance– Sensitivity 82%-100%– Specificity 64%-100%
• Benefits– Rapid turn-around-time (1-3 hours)– Value is most evident when patients are not
preemptively isolated
• Limitations– May miss mecA drops outs– Emerging genetic resistant characteristics– Significantly more costly than culture
Emergence of mecC-MRSA
mecA PCR PBP2a Agglutination
Not detected Negative
FOX
OX
Emergence of mecC-MRSA
• mecC – emerged as a novel gene distinct from mecAthat confers resistance– Phenotypic analysis: Often cefoxitin resistant – oxacillin
susceptible– Molecular analysis: Distinct sequences won’t be detected
by molecular methods specifically targeted for mecA• Many assays have incorporated universal mec gene primers or add
mecC-specific primers
– Protein analysis: PBP2a encoded by mecC is distinct, resulting in false negative PBP2a results
• Further investigation for Cefoxitin resistant, PBP2a-negative S. aureus isolates
– Overall prevalence in humans is low but mecC must be recognized and tests must be able to detect
MRSA Screening Strategies
• Universal Screening– All patients upon admission, periodically thereafter
(i.e. weekly)
– More expensive
– Limited demonstration of impact
• Targeted Screening– Specific units or procedures (i.e. ICU)
– Less expensive
– Focuses on patients at highest risk for invasive infection
When and How to Screen?
Situation Need Possible Method
Limitations Benefits
Pre-SurgicalScreening
Decolonization and prophylaxis in elective procedures
Culture –MRSA/MSSA
May miss some low level colonized patients, not transferrable to emergency surgery
Low cost, faster TAT generally not needed
Universal Screening
Facilities without preemptive isolation or double beds
NAAT CostlyFast TAT necessary to cohort patients appropriately
Universal Screening (single bed facilities or with preemptive isolation)
Facilities with preemptive isolation, single beds
CultureMay miss some low level colonized patients
Less expensive, fast TAT not required for room assignment
Targeted Screening (i.e. ICU, ED)
Focus on high riskpopulations NAAT Cost
Focus cost on highest risk for greatest benefit
DecolonizationScreening
Identify patients to remove from isolation
CultureMay miss some low level colonized patients
Only detects viable organisms
• All NAAT screening methods are FDA-cleared for Nares/Nasal swab
• 30-40% of patients are exclusively colonized with MRSA at a different site
• Molecular testing is only as good as the specimen that is collected
All this screening, still MRSA infections, what are we missing?
Diagnostic Strategies for C. difficile Infections
GDH – Glutamate DehydrogenaseToxin A/B
DNA Targets
Current Testing Options
Two-Step Algorithm
• Screen for GDH and Toxin Production by EIA
• Reflex to molecular for discordant results
• Benefits– Detects toxin as a surrogate
of active disease only
• Limitation– Lower sensitivity could miss
C. difficile in low abundance
Molecular Testing Only
• Perform molecular testing on all sample submitted
• Benefits– Most sensitive method to
detect C. difficile
• Limitations– Could potentially pick up
colonized patients
Which method should be used?
2018 IDSA/SHEA Recommendation for C. difficile
• Pre and post analytical understanding of the patient, specimen and result are included in the guideline for test selection
• Well coordinated algorithms and response between laboratory and providers is required for optimal test interpretation
So… is it really CRE? Well, there’s
CRE and there’s
CRE…
Carbapenemase Detection
Understanding molecular terminology will save many headaches and heartaches
Carbapnemase Producing Organism• Carbapenemase Production
– Type and expression of carbapenemase enzyme (KPC, NDM, OXA)
– More likely to be plasmid mediated (transferable)
Carbapenem Resistant Organism• Carbapenem Resistance
– Bacterial species and presence of other resistance mechanisms (ESBL, AmpC)
– Reduced permeability/efflux pumps
– Presence of porin alterations
– Intrinsic resistance to carbapenems
• Proteus, Providencia, Morganella species have elevated imipenem MICs
Factors Influencing Carbapenem MIC
Ambler Class A Ambler Class B Ambler Class D
Serine Carbapenemase
Metallo-Carbapenemase
OXA Carbapenemase
KPCSMENMC-AIMIGESSFOSFCIBC
VIMGIMSIMNDMIMPSPM
OXAPSE
Detection of Carbapenemase
• Carbapenemase production usually initiates more aggressive infection control interventions
• CP-CRE possess a more stable and transferable for of resistance
• Understanding specific resistance mechanism is important for therapeutic decisions for novel antibiotics with activity against certain resistance genes
Carbapenemase Detection Methods
Modified HodgeTest (MHT)
CarbapenemaseInactivation Method (CIM)
Molecular (PCR) Whole Genome Sequencing
Accuracy Moderate High High High
Turn AroundTime
Next Day Next Day Same Day Several Days
Information Provided
Detection of carbapenemaseactivity
Detection of carbapenemaseactivity
Detection of specificcarbapenemasegenes
Detection of carbapenemresistance mechanisms
Limitations Poor sensitivity for NDM producers; poor specificity when AmpC present
None known Unable to detect novel carbapenemase
Quantity of bioinformatics data to analyze, especially in the setting of novel carbapenemase
Targeted Sequencing
• Targeted Amplicon Sequencing– 16s rRNA common bacterial target
– Broad coverage for most organisms• Loss of species resolution for some
genera
– Most common for clinical application from direct specimens
• Contamination from reagents and environment may be detected
– Application for microbiome analysis
Lefterova, MI et al. (2015) Next-Generation Sequencing for Infectious Disease Diagnosis and Management. J Mol Diagn 17:623-634
Broad Sequencing Sequencing
Lefterova, MI et al. (2015) Next-Generation Sequencing for Infectious Disease Diagnosis and Management. J Mol Diagn 17:623-634
• Whole Genome Sequencing– Unbiased sequencing of nucleic acid
(RNA and DNA)• Requires human sequences and
sequencing errors to the filtered
– Useful in cases when all other culture and molecular options have been exhausted
– Useful in characterization of organisms for strain typing in epidemiologic investigations
– May identify resistance markers that can help to predict susceptibility
Increased Detection of Pathogens: Impact of Direct Specimen Sequencing
Primary Pathogen Detected
Different Primary Pathogen Detected
Culture NegativeSequencing Positive
12 2 1
Source Culture Result Sequencing Result
Cerebellar Abscess S. intermediusA. aphrophilus
F. nucleatumCapnocytophaga spp.S. intermediusA. aphrophilus
Bile P. aeruginosa K. pneumoniaeC. braakiiP. aeruginosa
CSF Negative S. intermedius
Identification by sequencing can identify the cause of the infection, but there is no organism available for susceptibility testing
Challenges of Molecular Testing
• Increased detection of organisms, are they causing disease or innocent by-standers?
• Molecular testing will only answer the question you ask
• Will the test provide clinically actionable information?
• How do you treat a bug you’ve never heard of?
• Identification by sequencing but no organism available for susceptibility testing
• Routine Surveillance
– Active surveillance• Detection of Colonized
Patients
– Chart Review• Manual review of
previous culture results
• Informatics-Driven Surveillance
– Prior positive microbiology results with (EMR)
– System Alerts for risk factors
Informatics Driven Surveillance
Is the answer always more testing?
Informatics-Driven Surveillance
Maaike S. M. van Mourik et al. Clin Infect Dis. 2013;57:85-93
• Large sparsely populated geographical areas
• Remote locations
• Socioeconomic conditions
• Specimen collection and quality control
TriCore Reference Laboratory in New Mexico
Real-Time MDRO Database
Patient is admitted to healthcare facility
Database of MDRO Isolates
Au
tom
ated
IT Q
ue
ry History of MDRO
No History of MDRO
Preliminary Results
• In a 1 week query
– 2 facilities
– Approximately 8,897 patients screened
MRSA ESBL MDRO CP-CRE TOTAL
73 (0.82%) 31 (0.34%) 69 (0.77%) 1 (0.0001%) 174 (2.0%)
Screening Results
• 9 Patients routinely screened for MRSA (targeted screening practices)
• 4 patients potentially carriers of MRSA not on contact precautions
Facility A (culture) Facility B (NAAT)
MRSA Positive 2 3
MRSA Negative 2 2
Preliminary Results
• In a 1 week query
– Approximately 8,897 patients screened
MRSA ESBL MDRO CP-CRE TOTAL
73 (0.82%) 31 (0.34%) 69 (0.77%) 1 (0.0001%) 174 (2.0%)
• Isolated from urine• Isolated in outpatient setting• 5 months between CP-CRE isolate and
hospitalization• Admitted for suspected congestive heart failure• No cultures collected upon admission
Questions and Limitations
• Risk Stratification
– Increase Impact / Decrease Burden
– Refine risk
– Number of positive cultures in 365-day period
• Recent positive cultures (<30 days)
– Change notification/reporting timing for possible CRE
• Notification
– Notification to Infection Control?
– Notification to Bed Management System?
ARE ROUTINE CULTURES A THING OF THE PAST?
Automation in Microbiology
47
BEFORE AUTOMATION
A New Look for the Lab
AFTER AUTOMATION
Increased Pathogen Recovery with Automated Incubation
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Aerococcus Isolates Per Month from Urine Cultures
Automation
Implementation
PreAutomaton PostAutomation
Average isolates per month
3.4 17.6
Isolates per 1,000 cultures
0.22 1.22
Age 77 76
Gender 25% M, 75% F 23.7% M, 77.1% F
WBC 69.0 60.4
Positive UA 48.1% 37.4%
Culbreath, K, unpublished data 2017
Increased Pathogen Recovery with Automated Incubation
January 2015 – March 2016
April 2016 –December 2016
N. gonorrhoeae
1 4
LeukocyteEsterase
Nitrate WBC Other GC Test Performed
Patient 1 Trace Negative 15-20 No
Patient 2 Large Negative 150 Yes
Patient 3 Large Negative >150 No
Patient 4 Not performed
Not performed
Not performed
No
Patient 5 Moderate Negative TNTC No
Culbreath, K, unpublished data 2017
Neisseria gonorrhoeae from WASPLab
Transformation of a Culture Plate to a Digital Image
1 mm
1 mm
1 mm
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l
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High Resolution
Accurate coloration
Wide field depth
Quality Optics
What’s the value of a digital image?
• Competency assessment
• Training and review
• Clinical communication
• Colony recognition and identification
Algorithm-Based Colony Counting
• Separate Algorithm for Each Plate Side– Software identifies bi-plate and reads growth on each side using
different light/algorithm. Image is presented to users with the plastic separator oriented vertically.
Quantitation of Colonies Regardless of Morphology
y = 0.9539x + 2.1719R² = 0.9152
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Image Analysis Count
Colony Count
Colony Count
Linear (Colony Count)
Automated MRSA Detection
57,690 Sample Multi-Center Study• Automatic Detection and Segregation of
Positive from Negative MRSA Samples
Using Different Manufacturers’ Chromogenic agar
• Sensitivity of 100%• Specificity of 90-96% (varied by location)
• Detection of 153 Positive Plates that were
Missed Manually
Automated VRE Detection
104,730 Sample Multi-Center Study• Automatic Detection and Segregation of
Positive from Negative VRE Samples
Using Colorex VRE or Oxoid VRE
• Sensitivity of 100%• Specificity of 89.5% (varied by location)
• Detection of 498 Positive Plates that were
Missed Manually
Image Analysis on Chromogenic Media
Faron et al, J. Clin. Microbiol. March 2016 vol. 54 no. 3 620-624
Faron et al, J. Clin. Microbiol. October 2016 vol. 54 no. 10 2464-2469
Phenotypic Colony Recognition
Size
Morphology
Growth Characteristics
It’s E. coli!
Performance of Colony Recognition
Organism Classifications nCorrect
ClassificationPercent Unclassified Percent Misclassified Percent
Correct Gram Classification
Percent
Staphylococci 40 36 90% 4 10% 0 0% 40 100%
Candida species 38 37 97% 0 0% 1 3% 38 100%
Streptococci 40 27 68% 5 13% 8 20% 40 100%
Enterobacteriacae 72 64 89% 6 8% 1 1% 72 100%
Pseudomonas 37 31 84% 6 16% 0 0% 37 100%
Enterococci 40 40 100% 0 0% 0 0% 40 100%
• 267 Isolates were challenged against the Image Analysis Library
• Organisms were considered correct if within the appropriate classification
• Unclassified: No single classification provided
• Misclassified: Organism classified into the incorrect group
Image Analysis Concordance with Manual Interpretation
Value of Kappa
Level of Agreement
% of Data that are Reliable
0-0.20 None 0-4%
0.21-0.39 Minimal 4-15%
0.40-0.59 Weak 15-35%
0.60-0.79 Moderate 35-63%
0.80 -0.90 Strong 64-81%
Above 0.90 Almost Perfect
82-100%
Manual Interpretation Analysis
No GrowthNormal Flora/Contaminated
Culture Reviewfor ID/AST
% Agreement Kappa Value
Soft
war
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terp
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n No Growth 2044 5 4 99.6% 0.98 (0.98-0.99)
NGUF/Contaminated (3 or
more organisms)7 360 8 96.0% 0.61 (0.59 -0.67)
Culture Review for ID/AST
15 194 2414 92.0% 0.92 (0.90- 0.93)
McHugh ML, Interrater reliability: the kappa statistic. Biochem Med. 2012 22(3):276-282
Summary
• Molecular testing is a valuable tool in the diagnostic arsenal
• Understanding the benefits and limitation of the testing is important for pre- and post- analytical decision making
• Laboratory informatics may be able to supplement unknown variables in infection control surveillance
• Routine culture and staining powered by artificial intelligence is being incorporated into routine laboratory workflow