Update on Molecular Diagnostics How does new molecular technology change the field of clinical microbiology ? Stefan Riedel, MD, PhD, D(ABMM) FASCP, FCAP Assistant Professor, Pathology The Johns Hopkins University, School of Medicine Director, Clinical Pathology Laboratories Johns Hopkins Bayview Medical Center
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Update on Molecular Diagnostics
How does new molecular technology change the field of clinical microbiology ?
Stefan Riedel, MD, PhD, D(ABMM) FASCP, FCAP
Assistant Professor, Pathology
The Johns Hopkins University, School of Medicine
Director, Clinical Pathology Laboratories Johns Hopkins Bayview Medical Center
Conflict of Interest / Disclosures
Microbiology Scientific Advisory Board: • Iris Diagnostics, Chatsworth, CA
Speakers’ Bureau • BD Diagnostics, Franklin Lakes, NJ, ª • Iris Diagnostics, Chatsworth, CA, ª • Siemens Healthcare Diagnostics, Tarrytown, NY ª
Research support: • BRAHMs Diagnostics, Annapolis, MD* • Thermo-Fisher, Scientific, Middletown, VA • BD Diagnostics, Franklin Lakes, NJ • Iris Diagnostics, Chatsworth, CA • Cubist Pharmaceuticals, Lexington, MA • Meridian Bioscience, Cincinnati, OH • AdvanDx, Woburn, MA • Siemens Healthcare Diagnostics, Tarrytown, NY
*Funding and materials used in the study described in this presentation were provided by BRAHMS Diagnostics, Annapolis, MD. Dr. Riedel has continued financial and material support for procalcitonin research, provided by Thermo Fisher and BRAHMS. The terms of this agreement are being managed by the Johns Hopkins University in accordance with its conflict of interest policies.
ªDr. Riedel’s participation in various speakers’ bureaus is managed by the Johns Hopkins University in accordance with its conflict of interest policies. All opinions expressed and/or implied in this presentation are solely those of Dr. Riedel. The content of this presentation does not represent or reflect the views of the Johns Hopkins University or the Johns Hopkins Health System.
Objectives – Describe the available molecular diagnostic tests that hold
promise for use in clinical microbiology
– Understand the limitations of molecular diagnostic methods, and how those limitations impact diagnosis, treatment, and surveillance in clinical settings
– Understand the path of implementation and quality assurance issues related to molecular diagnostics
– Understand and discuss potential for new technologies and applications in the rapidly changing diagnostic environment of clinical microbiology
Conventional vs. Molecular Diagnostic Methods
Even 10 years ago … conventional culture-based methods were routine methods for pathogen detection
• require viable organisms • require lengthy time of incubation
• result in longer TAT (> 24 h) for results
Christian Gram (1853 – 1938)
Robert Koch (1843 – 1910)
Friedrich Loeffler (1852 – 1915)
Standard Microbiology References
• Bergey’s Manual of Systematic Bacteriology • ASM – Manual of Clinical Microbiology
Clinical Microbiology Laboratories
• match results for their unknown clinical isolates to references • not infrequently: IMPERFECTED MATCHES FOR IDs
1980s – a new standard is approaching….
phylogenetic relationships of bacteria (and all life forms) by analysis of 5S, 16S, 23S rRNA
Woese CR 1987; Microbiol Rev 51: 221-271 Woese CR, Stackebrandt E, Macke TJ, Fox GE 1985; Syst Appl Microbiol 6: 143-151
Universal phylogenetic tree based on the 16S rRNA gene sequence comparisons.
Clarridge J E. Clin. Microbiol. Rev. 2004;17:840-862 Pace N R. Science 1997;276:734-740
The Clinical Continuum
Intervention !
Exposure
Incubation Period
Disease manifestations
Seek provider care
Clinical evaluation
Care plan
Implementation Specimen ordering
Specimen collection
Specimen transport
Specimen processing
Organism identification
Performance of AST
AST results reporting
Clinician processing information
To treat or not to treat….
Today’s Concerns for Antimicrobial Utilization
Who truly benefits from antibiotic therapy ?
What is the optimal duration of therapy ?
What factors drive inappropriate prescribing of antibiotics ?
• diagnostic uncertainty • lack of knowledge
• unavailability of microbiology test results / services • unavailability of infectious disease specialty consultation
• pharmaceutical marketing pressure • fear of missing a life-threatening infection
Length of Time to Detection Analysis
Earlier is better !
• 17% decrease in mortality when Gram stains from positive blood cultures are reported within < 1 hours Bauer K, et al. Clin Infect Dis 2010; 51: 1074
• More rapid detection of bacteremia will improve antimicrobial treatment/ switching and therefore clinical outcome of sepsis Kerremans J, et al. J Clin Microbiol 2009; 47 (11): 3520
• Timely reporting of AST results greatly improved patient outcomes Barenfanger J et al. J Clin Microbiol 1999; 37: 1415 Doern GV, et al. J Clin Microbiol 1994; 32: 1757
Laboratory interventions that decrease TAT can be effective
The Evolution of Molecular Diagnostic Methods
“If you build it they will come…”
Technological developments and
improvements
Commercial profit
The Evolution of Technology in Clinical Microbiology
Can and will molecular tests replace traditional microbiology methods?
Can and will molecular tests replace traditional microbiology methods?
Can molecular tests replace traditional methods?
Can current laboratory professionals be effectively trained to routinely perform these highly complex assays ?
Will physicians understand & accept the results and change their practice ?
The questions & concepts to consider
Are the decreased TAT and improved sensitivity worth the additional cost ?
What pre-analytical and analytical changes need to be implemented ?
Baron EJ 2011; J Clin Microbiol 49: S43
And there are more questions….
How does the emerging molecular technology affect smaller clinical microbiology laboratories ?
Is ribosomal RNA gene sequencing the new or current “gold standard” in microbial identification?
• 10% to 20% of clinical isolates are novel organisms and escape phenotypic identification methods • molecular methods have greatly advanced in recent years • molecular methods have (very) short TAT to results reporting • 16S rRNA analysis is new basis for taxonomy
BD-GeneOhm StaphSR • Multiplex real time PCR assay run on the SmartCycler®
• Amplifies specific target sequence of S. aureus and a specific target near the SCCmec insertion site (orfX junction in MRSA)
• Contains internal control to detect inhibition • Results are reported as negative or positive for S. aureus and/or MRSA
Stamper, PS et al 2007. J Clin Microbiol 45: 2191-2196 • 300 positive blood cultures from 295 patients containing gpc
• 89 grew S. aureus (29.7%); 211 grew species other than S. aureus (mostly CoNS) • Overall results: 96.7% agreement between culture and PCR assay • MRSA detection
Freezing of reagent master mix (up to 6 months) will decrease reagent waste and cost without compromising accuracy of test results
Cepheid Xpert MRSA/SA assay Rapid Detection of Staphylococcus aureus and MRSA in
Wound Specimens and Blood Cultures Wolk DM, et al. J Clin Microbiol 2009; 47: 823-826
Source and organism
% (no. of positive samples/total no.) Sensitivity Specificity PPV NPV
SSTI MRSA
97.1 (34/35)
96.2 (76/79)
91.9 (34/37)
98.7 (76/77)
S. aureus
100 (55/55)
96.6 (57/59)
96.5 (55/57)
100 (57/57)
BC MRSA
98.3 (57/58)
99.4 (346/348)
96.6 (57/59)
99.7 (346/347)
S. aureus
100 (120/120)
98.6 (282/286)
96.7 (120/124)
100 (282/282)
Cepheid Xpert MRSA/SA assay Rapid Detection of Staphylococcus aureus and MRSA in
Wound Specimens and Blood Cultures Wolk DM, et al. J Clin Microbiol 2009; 47: 823-826
Primers and probes detect sequences in:
• staphylococcal protein A (spa) gene, • the SCCmec inserted into the S. aureus chromosomal attB insertion site
• mecA gene
Sensitivity & specificity 100% and 98.6% for BC/SA isolates
Sensitivity & specificity 98.3% and 99.4% for BC/MRSA isolates
No issues with revertant strains
False positives due to MR-CoNS and isolates with SCCmec empty cassette
Broad-based Assays
• SepsiTest (Molzym) – performed directly on whole blood – targets conserved regions of 16S rRNA – broad range PCR combined with sequencing – detects > 300 different pathogens – TAT 8-12 h
• LightCycler SeptiFast (Roche) – performed directly on whole blood – multiplex real-time PCR – detects 25 different pathogens – TAT 3-30 h
Mancini N, et al. Clin Microbiol Rev 2010; 23: 235-251
Performance of the LCSeptiFast and the SepsiTest Leitner E, et al. J Microbiol Methods 2013; 92: 253-255
• samples were tested in parallel with BC, LCSF, and ST
• organisms considered true positive when growth in at least one BC bottle • potential skin contaminants considered true positives when present in two BC bottles • 33.3% (25/75) specimens were positive for 1 or more pathogens by any method used • 8 samples positive by LCSF but not by BC • 10 samples positive by ST but not by BC • “special gold standard”: BC plus reports of BC positivity within 7 days prior to specimen collection
Performance of the LCSeptiFast and the SepsiTest Leitner E, et al. J Microbiol Methods 2013; 92: 253-255
Comparison of LCSF and ST against BC/”gold standard”
Broad-based Assays and Direct Pathogen Detection Compared to BC, some assays have sufficient
diagnostic sensitivity & specificity. Combination of LCSF with procalcitonin may increase sensitivity.
Assays have improved TAT: LCSF 6 h ; ST 4-5 h
Positive ST results have TAT of 8-9 h due to sequencing
Automated DNA extraction is essential when implementing Assays in routine clinical laboratories.
Wolk DM, et al. J Clin Microbiol 2011; 49: S62-S67 Mauro MV, et al. Diagn Microbiol Infect Dis 2012; 73: 308-311 Leitner E, et al. J Microbiol Methods 2013; 92: 253-255
Molecular Technology for HAI screening
MRSA
Clostridium difficile
• culture remains common method for MRSA screening in most European countries
• recent Clinmicronet survey (70 laboratories) found that 54% adopted molecular methods for MRSA detection
• chromogenic agar media have shown increased sensitivities of 93% t0 99% compared to standard media
Marlowe EM, Bankowski MJ. J Clin Microbiol 2011; 49: S53-S56
Methods for MRSA screening
Method Sensitivity Specificity TAT Cost Technologist skill level
Culture Low* 100 % 18-48 h low moderate
Molecular high <100% < 24 h high moderate to high
*improved sensitivity when using chromogenic agar media
Marlowe EM, Bankowski MJ. J Clin Microbiol 2011; 49: S53-S56
Chromogenic agar media • MRSASelect Bio-Rad nares, wounds • Spectra MRSA Remel nares, positive BC • ChromID MRSA bioMérieux nares • BBL CHROMagar MRSA Becton Dickinson nares
Points to consider for method selection for MRSA Screening
Impact of MRSA on hospitals remains high!
laboratory – infection control – pharmacy – AMT
Screening of high-risk patients upon admission
Culture method has longer TAT but amenable to optimization of sensitivity
Culture method has longer TAT but amenable to optimization of sensitivity
Standardization of MRSA screening for both methods
Surveillance of test performance (emerging new variant strains?)
Points to consider for method selection for MRSA Screening
Hospital / Institution will need to determine the best fit for their setting
Collaboration between
Laboratory – Infection Control – Pharmacy – AMT
More evidence based studies needed to determine
• cost effectiveness of chosen methods (molecular vs. cultue) • studies focused on patient outcomes • continued method accuracy evaluation
Clostridium difficile
Clostridium difficile
• Formidable nosocomial pathogen
• responsible for up to 25% of antibiotic associated diarrhea • severe CDI results in extensive morbidity and mortality (6%)
• recently increased CDI frequency both in hospital and community settings
• emergence of hypervirulent strains (ribotype 027; NAP1)
High sensitivity & specificity
Rapid TAT, in order to timely implement therapy & isolation
General Principles
• Enterotoxin (Toxin A) – TcdA : cdtA
• Cytotoxin (Toxin B) – TcdB : cdtB
• additional toxins: cdtC, cdtD, cdtE
• Clostridium difficile – first described 1978
• Significant increase in incidence over the past decade
• NAP-1 (North American pulsed-field gel electrophoresis type 1 strain) – increase of more severe cases ?
TcdA+/TcdB+ : all cytotoxic; majority of strains causing disease in animals and humans TcdA-/TcdB- : nontoxigenic strains; not cytotoxic or virulent; may be PCR positive due to having parts of the toxin gene TcdA-/TcdB+ : produce only cytotoxin; may not be detected by commercial EIA method (10% of clinical cases; >40% in pediatric patients)
C. difficile – Laboratory Diagnosis
Test Target detected
TAT Sensitivity (%)
Specificity (%)
Cytotoxin Toxin B 1-3 days 90-95 95
Toxin Culture Toxigenic C. difficile
3-5 days 80-90 >95
EIA Toxin A or A/B
Toxin A or Toxin A&B
hours 97-98 75-80
EIA GDH C. difficile
hours 70-80 95-100
EIA GDH and Toxin A/B
C. difficile and Toxin A/B
hours 97-98 95-100
RT-PCR Toxigenic C. difficile
hours 80-99 >98
Bartlett J. ICHE 2010; 31: S35 Stamper P, et al. J. Clin. Microbiol. 2009; 47: 373
Performance Characteristics Molecular C. difficile Tests
Viala C, et al. J Microbiol Methods 2012; 90: 83-85
Performance Characteristics Molecular C. difficile Tests
BD GeneOhm , Cepheid Xpert, Illumigene
• relatively rapid and easy-to-perform tests
• similar performance regarding sensitivity/specificity
• Xpert and Illumigene are not time-consuming
• Xpert will detect ribotype 027
Additional studies will need to evaluate the role of the Quidel AmpliVue test
Promising new technologies
PCR/ESI-MS PCR combined with electrospray ionization mass spectrosmetry
MALDI-TOF MS Matrix assisted laser desoprtion ionization-time of flight mass spectrometry
DNA-Pyrosequencing-based Pathogen detection
FilmArray real-time PCR assays
MALDI-TOF MS • Identification of protein profiles derived from highly
conserved proteins
• Currently requires subculture before identification
• Low consumable cost
• Rapid organism identification
• Evidence of accurate bacterial organism ID – contaminant bacterial organisms – Whole colony needed for analysis – feasibility in clinical laboratory? – Cost for instrumentation?
Wolk DM et al. J Clin Microbiol 2011; 49: S62-S67 Maier T, et al. Chem Today 2007; 25: 68-71 Moussaoui W, et al. Clin Microbiol Infect 2010; 16: 1631-1638
FilmArray Technology (Idaho technology, Inc.)
Highly multiplexed automated PCR assay
• integrates specimen processing, nucleic acid amplification, and detection into a pouch
• premarket version detects 17 respiratory viruses plus three bacteria
• mechanical cell lysis using zirconium beads; nucleic acid capture and purification using metallic beads
• nested PCR; first stage is highly multiplexed PCR, second stage individual PCR mixtures (real-time PCR)
Adenovirus, bocavirus, hMPnV, influenza, parainfluenza 1-4, rhinovirus, RSV, Enterovirus, coronavirus, B. pertussis, Ch. Pneumoniae, M. pneumoniae)
FilmArray has excellent performance characteristics and allows for detection of a large number of pathogens.
Loeffelholz MJ, et al. J Clin Microbiol 2011; 49: 4083-4088
Specific Pathogen Identification, when conventional methods fail to achieve high level confidence for organism identification,
incl. pathogen discovery
Specific Pathogen Identification, for organisms associated with nosocomial transmission (infection control):
e.g. MRSA , C. difficile
Applications for Molecular Diagnostics
Specific Pathogen Identification, for organisms associated with disease outbreak or emerging pathogens:
e.g. H1N1 influenza , E. coli O104:H4
Specific Pathogen Identification for disease surveillance and other epidemiologic purposes (e.g. microbiome project)
“Advanced diagnostic technology will continually rely on the basic principles and practice of culture and identification.”
Dunne W.M., Pinckard J.K., Hooper L.V. Clinical Microbiology in the year 2025. J. Clin. Microbiol. 2002; 40 (11): 3889-3893
TRUE FALSE
If the new, molecular technology can be adapted to all hospitals, and
Assuming that accuracy and performance characteristics remain high ….
Molecular- and protein-based testing methods will replace traditional biochemical test methods, but will interface with current/traditional
antimicrobial susceptibility testing.
However: Culture methods are NOT obsolete !
Future Trends in Laboratory Diagnostics
Continued development of new technologies, and greater awareness & use of molecular test methods.
• need for expert groups to combine and assess data from multicenter studies
to meet regulatory requirements (e.g. CLIA, CAP, FDA)
• establish expert groups to develop new technologies and diagnostic algorithms through multicenter, randomized, clinical studies
• integrate existing and novel technologies for diagnostic algorithms through
collaborative networks between clinicians and the laboratory
Conclusions • Considering cost, complexity, and throughput newly developed
technologies may only be available to university-based diagnostic laboratories
• Need for further development of rapid diagnostic methods, including simple molecular diagnostics; however, culture based methods will NOT obsolete in the near future
• Need to develop middleware products that allow for interfacing of HIS/LIS users from multiple healthcare institutions with new technologies such as mass spectrometry
• Infectious Disease Physicians, Infection Control Practitioners, and Hospital Epidemiologists will need to assist Microbiologists / Laboratory Directors in clinical validation of new technologies