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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