Download Shigella - First Coast ID/CM Symposium

Molecular Based Enteric
Bacterial Panels in
Clinical Microbiology
Joel E. Mortensen, PhD
Director
Diagnostic Infectious Disease Testing Laboratory
Cincinnati Children’s Hospital
Cincinnati, Ohio
1
The presenter has professional relationships
with the following companies:
Becton Dickinson, bioMerieux, Check-points
Ssytems, Eli Lilly, JMI, Meridian Bioscience,
Nanosphere, ThermoFisher
The Golden Age of Microbiology
• Louis Pasteur – 1822- 1895
• Silkworms, beer, wine, anthrax, spontaneous
generation, rabies
• Pasteur Institute
• Roux, Duclaux,
• and others
The Golden Age of Microbiology
• Robert Koch – 1843 – 1910
• 1905 Nobel Prize for Medicine
• Berlin Institute for Infectious Diseases
• Löffler, Gaffky, von Hess, Petri, Klebs
• Koch’s postulates
The Golden Age of Microbiology
• Christian Gram - 1853 - 1938
• Developed a stain for bacteria in tissue
• Fundamental differences between Gram-positive
and Gram-negative bacteria
1900 to 1940’s
• Serological differentiation based on expression of
surface markers
• Phenotypic differentiation based on fermentation
products
• Other differentiating characteristics
• Coagulase
• Lipase
• hemolysis
1950 to 2000
• Phenotypic differentiation formalized
• Use of fermentation products commercialized
• Other differentiating characteristics also
commercialized
1970’s
Enterotube II – Roche Diagnsotics
Itinvented in the 1970s in the United States by Pierre Janin of Analytab Products, Inc
Computers meet miniaturization
• Vitek System
• Microscan
• MicroMedia
• Abbott Diagnostics
• Roche Diagnostics
• Many others
“We are on the cusp of the
biggest change in
microbiology since Louis
Pasteur declared that life
does not arise
spontaneously”
16
State of the stool world today
• Diarrhea is a big deal world wide
• In the developed world, two basic populations
• Louis would be happy with most of the methods
used as recently as yesterday
• What are the opportunities and challenges to our
patients and our laboratories
GI pathogens in community
• Bacterial –
• outbreaks or clusters of Salmonella, Shigella and
Campylobacter
• Clostridium difficile has been growing in the
community
• Norovirus is number one cause of acute diarrhea
• Parasitic – sporadic, low incidence, specific populations
• Immunocompromised a special case
Why even talk about molecular
methods?
Bacteria
• Stool cultures are the most common test ordered
• Positivity is only ~2- 6 %
• Molecular test may double the positivity rate
• Culture
• Variable sensitivity
• Good specificity, but some limitations
• Turn around time 48 hours (mean) for a negative,
range up to 96 hours
• Wide variety of media and methods
Why even talk about molecular
methods?
Viruses
• Viruses represent the highest prevalence – 25% in
most populations
• Norovirus
• Rotavirus has declined in US
• Nucleic acid amplification has replace nearly all
conventional culture
Why even talk about molecular
methods?
Parasites
• Highly variable sensitivity
• Tech dependent
• High expertise
• Standard O&P does NOT readily detect:
• Cryptosporidium spp.
• Cyclospora spp.
• Microsporidia spp.
• Single sample for molecular shown to be sensitive
FDA Cleared Platforms
• Prodesse® - Progastro SSCS
• Nanosphere - Verigene® Enteric Pathogen
• Biofire Diagnostics - FilmArray® GI panel
• Luminex® - xTAG Gastrointestinal Pathogen Panel
• BD MAX® Enteric Bacterial Panel
Prodesse
• Salmonella spp.
• Shigella spp.
• Campylobacter spp. (C. jejuni and C. coli only,
undifferentiated)
• Shiga Toxin 1 and 2 genes
Prodesse - performance
Target
%Sensitive
% specific
C. coli/jejuni
84 – 100
99 - 100
Salmonella spp.
86 - 100
99 - 100
Shigella spp.
81 - 100
99 – 100
stx1/stx2
78 - 100
99 - 100
Buchan et al. J Clin Microbiol. 2013 Dec;51(12):4001-7.
Nanosphere
• Bacteria
• Campylobacter Group
• Salmonella spp.
• Shigella spp.
• Vibrio Group
• Yersinia enterocolitica
• Shiga Toxin 1and 2
• Viruses
• Norovirus
• Rotavirus
Nanosphere performance
Combined Results after Resolution of Discrepants
Target
% Sens
% Spec
93.0
99.7
96.5
99.7
Shigella
100
100
Vibrio
97.0
100
Y. enterocolitica
100
100
stx1
100
99.9
stx2
97.4
99.9
Norovirus
90.9*
98.9
Rotavirus
66.7*
99.9
 611 prospective stool
samples in Cary Blair. Campylobacter
 228 contrived
Salmonella
samples.
*Discrepant analysis not complete
Novak, S Abstract 1317 CVS 2014
BioFire - Film Array
• Bacteria
• Campylobacter (jejuni, coli and upsaliensis)
• Clostridium difficile (Toxin A/B)
• Plesiomonas shigelloides
• Salmonella
• Yersinia enterocolitica
• Vibrio (parahaemolyticus, vulnificus and cholerae)
• Vibrio cholerae
• Diarrheagenic E.coli/Shigella
• Enteroaggregative E. coli (EAEC)
• Enteropathogenic E. coli (EPEC)
• Enterotoxigenic E. coli (ETEC) lt/st
• Shiga-like toxin-producing E. coli (STEC)
stx1/stx2
• Shigella/Enteroinvasive E. coli (EIEC)
• Parasites
•
•
•
•
Cryptosporidium
Cyclospora cayetanensis
Entamoeba histolytica
Giardia lamblia
• Viruses
•
•
•
•
•
Adenovirus F40/41
Astrovirus
Norovirus GI/GII
Rotavirus A
Sapovirus (I, II, IV and V)
Film Array performance
Prospective Results – positive in >10 samples
Target
% Sens
% Spec
Bacteria
Target
% Sens
% Spec
Viruses
Campylobacter
97.1
98.4
Adenovirus
95.5
99.1
C. difficile
98.8
97.1
Norovirus
94.5
98.8
Salmonella
100
99.6
Sapovirus
100
99.1
Shigella/EIEC
95.9
99.9
STEC
100
99.7
Cryptosporidium
100
99.6
EPEC
99.1
97.2
Cyclospora
100
100
ETEC
100
99.4
Giardia
100
99.5
EAEC
98.8
98.2
Buss JCM 53:915 2015
Parasites
Luminex
• Bacteria and Bacterial Toxins
• Campylobacter
• Clostridium difficile, Toxin A/B
• Escherichia coli O157
• Enterotoxigenic E.coli (ETEC)
LT/ST
• Shiga-like Toxin producing E.coli
(STEC) stx1/stx2
• Salmonella
• Shigella
• Vibrio cholerae
• Yersinia enterocolitica
•
•
•
•
•
•
•
•
Viruses
Adenovirus 40/41
Norovirus GI/GII
Rotavirus A
Parasites
Cryptosporidium
Entamoeba histolytica
Giardia
Luminex Performance
BD MAX
• Salmonella spp.
• Campylobacter spp. (jejuni / coli)
• Shigellosis disease causing agents
• Shigella spp.
• Enteroinvasive E. coli (EIEC) as well as Shiga-toxin
producing E. coli
BD MAX
• Parasite panel submitted to FDA for review
• Viral and additional bacteria in clinical trials
BD MAX - Performance
PPA
NPA
Salmonella spp.
97.3
99.8
Shigella spp.
99.2
100
Campylobacter spp.
97.5
99.0
Shiga-toxin
100
99.7
PPA = positive percent agreement
NPA = negative percent agreement
Molecular testing considerations
• Most molecular platforms are as sensitive or more
sensitive than conventional methods
• Use in appropriate patient populations
• Will results influence patient outcome
• Positive signal in the absence of disease
• Most viral/bacterial infections are self-limiting
Molecular testing considerations
• Cost will be the most significant factor
• Disease may not warrant testing
• High risk of adverse outcome in some patients
• Billing questions and CPT codes
• *What analytes should be on the test panel*
• Traditional approach - panels focused on bacteria,
viruses and parasites
• Very broad/all inclusive panels
• Guideline directed testing
IDSA Clinical Testing Guidelines
Enteric bacteria Multicenter Clinical Trial
• 4,240 preserved or unpreserved stool specimens, including
3,457 collected prospectively, and 783 frozen, retrospective
• Collected from sites in U.S. (10), Canada (1) and Mexico (2)
• BD MAX compared to standard culture methods for
Salmonella, Shigella, and Campylobacter and a commercial
EIA (Meridian Bioscience) for Shiga-toxins 1 and 2
• Alternate target PCR with bi-directional sequencing was used
to confirm results of retrospective samples and for discrepant
analysis of prospective sample
Results
PPA
NPA
Salmonella spp.
97.3
99.8
Shigella spp.
99.2
100
Campylobacter spp.
97.5
99.0
Shiga-toxin
100
99.7
PPA = positive percent agreement
NPA = negative percent agreement
Multicenter Clinical Trial
• BD MAX detected additional organisms compared
to conventional culture
• 22 Campylobacter spp.
• 19 Salmonella spp.
• 9 Shigella spp.
• 9 samples with shiga toxins
BD MAXTM EBP Time Motion Studies
• Look at the impact of BD MAXTM on workflow
• Compare BD MAXTM to conventional culture
• Examine measurable parameters
• Time to reportable result
• Time and effort spent performing tests
• Cost comparison
TAT Comparison Typical Negative Culture
44 hr 37 mm
7 hrs 6 min
85% average reduction of specimen TAT
*Assumes MAX processing occurs at 7:00 am and 8:00 pm daily
Process Steps for Negative Culture
Events / Decisions
Culture Activity
per specimen
BD MAX
Activity
Events /
Decisions per
Specimen
Receipt
3
Receipt
3
Accession
7
Accession
1
EHEC
4
BAP
43
Sample Prep
8
MacConkey
26
13
Hektoen
26
System
Operation
MAC
14
Total
25
Campy
18
Total
141
82% reduction of processing steps per specimen (141 Culture events / decisions
vs. 25 BD MAX events / decisions)
Conventional Process
Legend:
= Day 1
= Day 2
= Day 3
BD MAXTM EBP Process
Legend:
= Day 1 =
Cost Comparison – Negative test
Conventional
Culture $ (min)
BD MAX
Basic Test
Labor (minutes)
Supplies
$6.75 – 7.65 (15-17)
17.31
$0.67 (1.4 min)
33.62**
Workup*
Labor
Supplies
Total cost in $
15.75-18.00 (35-40)
6.84 – 19.34
$26.00 – 64.00
N/A
N/A
$32.00 – 37.00**
*10-20% require significant work up of suspected pathogens
** Cost dependent on contract pricing
Meeting the needs of CCHMC
• Move forward with implementation and integration
of BD MAX
• Enteric stool pathogens
• Stool parasite screening
• MRSA/MSSA screening
• VRE screening
Additional considerations – All Platforms
• Space and platform menu
• QC/QA
• Education
• Training
• Interfaces
• Validation and reproducibility
• Reporting
• Billing
Space for instrumentation
• Common molecular specimen set up area
• Open vs closed amplification systems
• Foot print of instrumentation
• BD MAX – Approximately 8 feet of bench space
• Storage for supplies – dirty vs clean
• Flow from dirty to clean areas
Space for instrumentation
• Storage requirements
• Room temperature
• Refrigerator
• Freezer
• Packaging of reagents
Space for instrumentation
Menu – all platforms
• Balance between strength of an individual assay
and efficiencies of a single platform
• Scientific - positive and negative percent
agreement
• Operational issues including labor, QC, QA, etc
QC/QA – All systems
• External QC requirements
• CAP requirements for molecular platforms is
viewed as onerous by many
• Positive and negative control for each analyte,
new lot and shipment
• Positive and negative control for each analyte
each run unless alternate method utilized (20
replicates then rotating system of targets)
• Commercially available stains or samples
QC/QA
• Combined external controls, pooled
organisms/targets
• 20 day QC and then weekly or other routine
• Rotation of targets?
Education
• laboratory staff - general education of Lab rounds
and demos
• “Lunch and learn”
• Specific education in the laboratory
• Early introduction and preparation
• Overlap with training
Education
• Clinician education - Key user groups
• Physician
• Nurses/nurse practitioners
• Collection supplies and test ordering
• Resulting
• Clinician education – other user groups
• Other in house groups
• Outside clinics and offices
Training
• Core users
• Additional users
• Set ups - processing samples
• Entire laboratory staff to answer questions, etc
• Detailed check list for training and competency
• Quizzes to document procedure review
• Online systems work well (Compliance 360)
Training
• Resources after training
• Technical service – 24/7 ?
• Online resources and training
• Online or paper manuals
• List serves
• Instrument problem log (CCHMC)
Training topics - example
• System overview
• Reagents and consumables
• Specimen collection and transport
• Software navigation
• System workflow
• System setup – hands on
• Results review
• Trouble shooting
Interfaces – All systems
• Non- interfaced
• Bi – directional interfaces
• Costs
• Vendor side often free
• LIS side in the range of $5,000
• Available/proven interfaces are critical
• Middleware solutions
• Vendor and non-vendor
Verification, validation and reproducibility
• Vendor data
• Published studies
• Abstracts and posters
• Peer reviewed journal articles (JCM)
• Internal studies
Verification
• A one time process completed before a test system
or instrument is used for patient testing.
• Verification studies should be performed by the
technologists that will perform patient testing.
Verification
• Studies – ASM Cumitech 31A
• Stool samples
• How many positive samples needed - 20
• Challenge sets
• Patient isolates (freeze positive samples now!)
• Number of organisms
• In test set
• Out of test set (cross reaction)
Assay Verification Checklist
Test Name: _______________________________________________________
TASK
1. Create plan for verification with dept Leadership team
a. Design study with approval of director and manager
b. Write up study design
c. Study design reviewed and approved by director and manager
2. Electrical check & KN # issued for new equipment/instrument by CE
3. Create verification data sheet
4. Perform verification testing including repeat on discrepant samples
5. Write up QA of verification including statistical analysis of data:
a. Precision
b. Accuracy
c. Reportable range
d. Sensitivity & Interferences
6. Submit QA of verification for review by manager and director
7. Write procedures
a. Testing
b. Preventive Maintenance (PM)
8. Create following logs
a. QC sheet
b. Calibration log, if applicable
c. PM log
d. Inventory sheet
e. Training checklist
f. Verified Results Review sheet, if applicable
9. Clinical Lab Index: submit modifications on Issue Tracking
10. LIS: interface with instrumentation, if applicable
11. Cerner Millenium: CERT and PROD (follow LIS.DBA.131 procedure-Test
validation checklist for Lab Staff)
a. Build test procedure, results and interpretive data if applicable
b. Labels
c. Reflexive orders, if applicable
d. Testing with a “test patient”
12. Order CAP proficiency or establish alternative if not available
13. Complete Chemical Inventory Product Form for new or deleted products
14. Communicate changes to clinicians, if applicable
15. Communicate changes to other lab departments, if applicable
16. Complete cost analysis with manager
17. Submit billing information (CPT code and cost) for compendium
18. Submit information on Issue Tracking to LIS for Cerner Millenium & EPIC
DATE
TECH
Validation
• Once an instrument or system has been verified,
validation demonstrates that it repeatedly
continues to give the expected results as performed
over time and continues to meet the
manufacturer’s claims.
Validation
• QC tracking over time
• Proficiency testing
• Maintenance and calibration records
• Parallel testing of duplicate instruments
• Correlation with clinical findings
Reproducibility
• Reproducibility testing
• Number of replicates - 20
• Number of users – at least 4
• Day-to-day variance
• Technologist variability
Reporting
• Adapt and integrate - CCHMC
• Insert MAX EBP results into present culture reporting
format
• Preliminary result for primary pathogens
• Final at 24 hours for extended
• Modify as new pathogens become available
• Add method note as required by CAP
• Develop routine viral stool panel test and report
CPT and billing
• Phase 1 - BD MAX with Culture
• 87505 x 1 (3-5 targets) Includes, Campylobacter, Salmonella,
Shigella, Shiga Toxin
• 87046 x 2 (BAP& MAC) for Aeromonas, Plesiomonas, Vibrio,
Yersinia
• Additional ID and susceptibility may be added
• Phase 2 - no culture
• 87506 x 1 (6-11 targets)
• Includes all of the above when FDA approved for remaining
enteric bacterial pathogens
Adapt and integrate
• New technology must be integrated into a new
process, not just dropped into a lab
• New digital cell phone with an analog network
• Old processes must evolve and adapt to the new
analytic and diagnostic paradigms
Aknowledge
• Cindi Ventrola – Manager of Diagnostic Infectious
Diseases Testing Laboratory
• Barb DeBurger and Sarah Hanna – Technical
Specialist