Siemens Healthcare Diagnostics, Tarrytown, NY ª. Research support: •. BRAHMs
Diagnostics ..... Xpert MRSA/SA : Cepheid Diagnostics. Broad-based Assays*.
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 • • •
Research support: • • • • • • • •
BD Diagnostics, Franklin Lakes, NJ, ª Iris Diagnostics, Chatsworth, CA, ª Siemens Healthcare Diagnostics, Tarrytown, NY ª
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
Christian Gram (1853 – 1938)
Robert Koch (1843 – 1910)
Friedrich Loeffler (1852 – 1915)
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
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.
Pace N R. Science 1997;276:734-740
Clarridge J E. Clin. Microbiol. Rev. 2004;17:840-862
The Clinical Continuum Exposure
Clinician processing information
Incubation Period
AST results reporting
Disease manifestations
Intervention !
Performance of AST
Seek provider care
Organism identification Clinical evaluation Specimen processing Care plan
Specimen transport Specimen collection Specimen ordering
Implementation
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 ! Laboratory interventions that decrease TAT can be effective • 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
The Evolution of Molecular Diagnostic Methods
Technological developments and improvements
Commercial profit
“If you build it they will come…”
The Evolution of Technology in Clinical Microbiology DNA microarray developed DNA discovered
1953
PCR developed
High-throughput sequencing developed MassTagPCR developed
1983
1995
2005
2012
2001 Cost of sequencing $ 5,000 per Megabase
Cost of sequencing $ 0.50 per Megabase 2008 Cost of sequencing $ 15 per Megabase
Adapted from: Lipkin W . Nature Reviews, Microbiology 2013; 11: 133 - 141
Can and will molecular tests replace traditional microbiology methods?
Can and will molecular tests replace traditional microbiology methods?
The questions & concepts to consider
Baron EJ 2011; J Clin Microbiol 49: S43
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 ? What pre-analytical and analytical changes need to be implemented ? Are the decreased TAT and improved sensitivity worth the additional cost ?
And there are more questions…. • 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 Zhang W, Versalovic J 2007. J Molecular Diagn 9: 572
Is ribosomal RNA gene sequencing the new or current “gold standard” in microbial identification? How does the emerging molecular technology affect smaller clinical microbiology laboratories ?
Applications for Molecular Diagnostics 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 disease outbreak or emerging pathogens: e.g. H1N1 influenza , E. coli O104:H4
Specific Pathogen Identification, for organisms associated with nosocomial transmission (infection control): e.g. MRSA , C. difficile Specific Pathogen Identification for disease surveillance and other epidemiologic purposes (e.g. microbiome project)
*List of organisms is not all inclusive
VRE Surveillance (vanA, vanB)
Respiratory Virus Testing (Influenza, RSV, hMPnV, etc.)
MRSA Surveillance Enterovirus and HSV
Clostridium difficile (diagnosis; tcdA, tcdB)
Molecular Microbiology*
Norovirus Bordetella pertussis
Enteric Pathogen Testing
Mycobacterium tuberculosis
Group B Streptococcus
Neisseria gonorrhea ; Chlamydia trachomatis
*List of technologies is not all inclusive
Film Array Technology
AdvanDx: PNA-FISH
(non-amplified DNA probes)
( Idaho Technology, Inc. / BioFire)
MALDI-TOF MS
SeptiFast multiplex PCR
(Bruker)
(Roche)
Molecular Microbiology* Cepheid: GeneXpert (modular real-time PCR)
BD: BD Max™
(automated specimen processing, real-time PCR)
Nanosphere
(Verigene Clinical Microbiology)
Clinical Application for Molecular Diagnostics in Microbiology Direct Pathogen Detection from a Clinical Specimen Broad Based Assays Novel Organism/Pathogen Discovery
Bacteremia / Sepsis A common problem with significant clinical and cost ramifications ! • approx. 750,000 episodes annually in U.S. – ~250,000 nosocomial episodes of BSI • Mortality rate: 14% (community-onset BSI) - 34% (nosocomial BSI) – risk of death from septic shock increases by 7% with every hour until start of appropriate/targeted therapy – 10th leading cause of death – Substantial reduction of quality of life in survivors
• Attributable cost: ~ $ 9.6 billion
Danai et al. Chest 2006; 129: 1432-1440 Martin et al. NEJM 2003; 348: 1546-1554 Wisplinghoff et al. CID 2004; 39: 309-317
The Traditional Approach A two bottle system with blood specimen split evenly between an
AEROBIC and an ANAEROBIC bottle. Traditional Laboratory Methods rely on Cultivation of Pathogens! • preliminary results within 1-3 days • definitive results often require more than 3-5 days • ineffective for modification / de-escalation of antimicrobial therapy • contributes to increased mortality and emergence of MDR organisms
Rapid Identification of BSIs and other Infections – the current situation Wolk et al. 2011. J Clin Microbiol 49 (9S): S62-S57 ; Tenover 2010. Ann NY Acad Sci 2013: 70-80
Clear benefits of rapid reporting of Gram Stain results and timely reporting of AST results Doern et al. 1994. J Clin Microbiol 32: 1757-1762 Barenfanger et al. 1999. J Clin Microbiol 37: 1415-1418 Barenfanger et al. 2008. Am J Clin Pathol 130: 870-876
Peptide Nucleic Acid Fluorescent in situ Hybridization (PNA-FISH) • Fluorescence tagged peptide nucleic acid probe (AdvanDx) - detects S. aureus-specific 16SrRNA
• Differentiates S. aureus from staphylococci other than S. aureus, directly from blood cultures • Sensitivity 99-100%; Specificity 96-100% Chapin K, et. al. 2003. J Clin Microbiol 41:4324
Oliveira K, et. al. 2002. J Clin Microbiol. 40:247
Gonzalez V, et. al. 2004. Eur J Clin Microbiol Infect Dis 23:396
Forrest et. al. 2006. J Antimicrob. Chemother. 58:154-58
Other PNA FISH Assays • C. albicans and C. glabrata • Yeast Traffic light PNA FISH: Candida albicans and/or Candida parapsilosis, Candida tropicalis and Candida glabrata and/or Candida krusei
• Enterococcus faecalis vs. Enterococcus not faecalis (faecium) • GNR: P. aeruginosa vs. E. coli • GNR: E. coli +/- K. pneumoniae versus P. aeruginosa (traffic-light probe)
PNA-FISH & Coagulase-negative Staphylococci Forrest et al. 2006. J Antimicrob Chemother 58: 154-158
Implementation of PNA-FISH for CoNS in BCs in conjunction with AMT • lower hospital costs ( approx. $ 4,000 less per patient) • decreased length of stay (approx. 2 days per episode) • decreased use of vancomycin Ly et al. 2008. Clin Risk Manag 4: 637-640 similar results for LOS and cost
Forrest et al. 2008. Antimicrob Agents Chemother 52: 3558-3563
earlier initiation of appropriate antimicrobial therapy for HA E. faecium bacteremia
Holtzman et al. 2011. J Clin Microbiol 49 (4): 1581-1582
Impact of PNA-FISH for CoNS in BCs in the absence of AMT • accurate performance of PNA FISH test, with high sensitivity & specificity • no active reporting by laboratory & no AMT support/guidance
• NO reduction in LOS (p = 0.35) and vancomycin use (p = 0.49) between control and study group patients !
Newer, improved PNA FISH Assays: QuickFISH* (*not all assays are currently FDA approved in the U.S.)
• probe quenching complexes eliminate need to wash away excess probe • upon heating, quencher & probe will separate, allowing fluorescent probe to hybridize with target rRNA • upon cooling, unused probe will again combine with quencher • 5 min “hands-on” time ; 20 min TAT for results Will physicians act upon 2nd CAV call ? 1.5-2 h 12-24 h
12-24 h
Gram Stain CAV call
Gram Stain + QuickFISH CAV call
PNA FISH + 2nd CAV call
10-72 h
8-72 h
Definitive ID + AST (LIS report)
Definitive ID + AST (LIS report)
Additional studies will hopefully show difference in clinical utility.
Molecular Amplified Technologies MRSA vs. MSSA • GeneOhm™ StaphSR : BD GeneOhm™ • Xpert MRSA/SA : Cepheid Diagnostics
Broad-based Assays* Assays performed directly on blood • SepsiTest : Molzym, Bremen, Germany • LightCycler SeptiFast : Roche, Mannheim, Germany *not available in the U.S.
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
• Sensitivity 100%, Specificity 98.4%, PPV 92.6%, NPV 100%
• MSSA detection
• Sensitivity 98.9%, Specificity 96.7%, PPV 93.6%, NPV 99.5%
Other studies noted some limitations….. • failure to detect certain SCCmec types • misidentification of revertant strains (deleted or nonfunctional mecA genes) • sensitivity 95.5%, specificity 95.6% in seeded study Snyder JW, et al. J Clin Microbiol 2009; 47: 3747-3748
Groebner SM, et al. J Clin Microbiol 2009; 47: 1689--1694
Additional Considerations • TAT 2.5 h and expense to laboratory • likely to perform batch testing Freezing of reagent master mix (up to 6 months) will decrease reagent waste and cost without compromising accuracy of test results Munson E, et al. J Clin Microbiol 2010; 47: 3747-3748 Riedel S, Carroll KC. J Infect Chemother 2010; 16: 301-316
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
% (no. of positive samples/total no.) Source and organism 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)
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)
BC
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” No. of specimens
Assay
Result
LCSF ST
Comparison to BC
Positive
Negative
Positive
3
8
Negative
4
60
Positive
2
10
Negative
5
58
Sensitivity (%) [95% CI]
Specificity (%) [95% CI]
42.9 [15.8, 75.0] 88.2 [78.5, 93.9] 28.6 [8.2, 64.1]
85.3 [75.0, 91.8]
Comparison to designed gold standard LCSF ST
Positive
7
4
Negative
4
60
Positive
3
9
Negative
5
58
63.7 [35.4, 84.8] 93.8 [85.0, 97.5] 37.5 [13.7, 69.4] 86.6 [76.4, 92.8]
LCSF, LightCycler® SeptiFast; ST, SepsiTest™; BC, blood culture; CI, confidence interval
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. 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
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.
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 Marlowe EM, Bankowski MJ. J Clin Microbiol 2011; 49: S53-S56
Method
Sensitivity Specificity
TAT
Cost
Technologist skill level
Culture
Low*
100 %
18-48 h
low
moderate
Molecular
high
40% in pediatric patients)
C. difficile – Laboratory Diagnosis Bartlett J. ICHE 2010; 31: S35 Stamper P, et al. J. Clin. Microbiol. 2009; 47: 373
Test
Target detected
TAT
Cytotoxin
Toxin B
1-3 days
95
90-95
Toxin Culture
Toxigenic C. difficile Toxin A or Toxin A&B C. difficile
3-5 days
>95
80-90
hours
75-80
97-98
hours
95-100
70-80
C. difficile and Toxin A/B Toxigenic C. difficile
hours
95-100
97-98
hours
>98
80-99
EIA Toxin A or A/B EIA GDH EIA GDH and Toxin A/B RT-PCR
GDH: glutamine dehydrogenase
Sensitivity Specificity (%) (%)
C. difficile: molecular tests BD GeneOhm
(Becton, Dickinson & Co.)
GeneXpert C. difficile (Cepheid)
tcdB
tcdA, tcdB, tcdC deletion 117
PCR molecular beacon
PCR, Taqman (one cartridge)
TAT 2-3 h
TAT 1 h
Illumigene
AmpliVue C. difficile
(Meridian Bioscience)
(Quidel Diagnostics)
tcdA
LAMP methodology TAT 1 h
Conserved DNA region A+B+ and A-B+ strains Hand-held device Isothermal helicase-dependent amplification TAT 20 min
Performance Characteristics Molecular C. difficile Tests
Viala C, et al. J Microbiol Methods 2012; 90: 83-85
Result
Gold standard (no. samples) Positive
Negative
Sensitivity (%) [CI 95%]
Specificity (%) [CI 95%]
Accuracy (%)
XPert C. difficile
Positive Negative
44 1
1 48
97.8 97.9 97.9 [93.5–102.1] [93.9–101.9]
BD GeneOhm Cdiff
Positive Negative
43 2
1 48
95.5 97.9 96.8 [89.4–101.5] [93.9–101.9]
illumigene C. difficile
Positive Negative
39 6
49
86.7 [76.8–96.6]
CI, Confidence Interval
100
93.6
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 MALDI-TOF MS
Matrix assisted laser desoprtion ionization-time of flight mass spectrometry
PCR/ESI-MS
PCR combined with electrospray ionization mass spectrosmetry
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
Applications for Molecular Diagnostics 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 disease outbreak or emerging pathogens: e.g. H1N1 influenza , E. coli O104:H4
Specific Pathogen Identification, for organisms associated with nosocomial transmission (infection control): e.g. MRSA , C. difficile Specific Pathogen Identification for disease surveillance and other epidemiologic purposes (e.g. microbiome project)
Dunne W.M., Pinckard J.K., Hooper L.V. Clinical Microbiology in the year 2025. J. Clin. Microbiol. 2002; 40 (11): 3889-3893
“Advanced diagnostic technology will continually rely on the basic principles and practice of culture and identification.” 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
