Molecular

Tes,ng and Infec,on Preven,on

Daniel J. Diekema, MD, D(ABMM) University of Iowa Carver College of Medicine Disclosures: Research funding from bioMerieux, Cerexa, Merck, Pzer, Purthread

Objec,ves
Know which available molecular diagnos,c tests hold promise for improving the diagnosis and preven,on of HAIs Understand the limita,ons of molecular diagnos,cs, and how those limita,ons may impact diagnosis and surveillance ac,vi,es Be able to discuss with your lab director the pros and cons of introducing certain molecular assays.

Caveats: In the interest of ,me

Will emphasize applica,on to healthcare epi
most commonly encountered issues MDROs, C. dicile, molecular typing

Will not discuss specic products in detail

Subject to rapid change, oSen more than one using a specic approach

Emphasize technology available to most labs

Major uses of molecular tes,ng for infec,on preven,on

Detec,on of pathogens and resistance (MDRO)
Allow earlier interven,on Focused therapy, isola,on, decoloniza,on

Typing to assess gene,c relatedness

Inves,gate pathogenesis, transmission, outbreaks PFGE, ribotyping, rep-PCR, mul,locus sequence typing, spa typing (S. aureus)

Molecular detec,on of pathogens

Direct from sample or from culture growth Advantages in comparison with culture:
Faster analy,c turnaround ,me (TAT) Increased sensi,vity (some,mes) Best alterna,ve for dicult-to-grow pathogens

PCR, or nucleic acid amplica,on tes,ng (NAAT) methods

Molecular detec,on of MRSA from nares This should be easy, right?

Wrong! Why?
Mixed samples include coagulase-nega,ve staphylococci that carry mecA gene
False posi,ve results (MSSA + MR-CoNS) Must nd a target linking MR (mecA) with SA SCCmec/orfX junc,on

Huletsky et al. J Clin Microbiol 2004;42:1875.

But problems remain

Single target may remain aSer loss of mecA (mecA dropouts)
Found in 5-10% of all + samples in some centers
Arbefeville SS, et al. J Clin Microbiol 2011;49:2996-9.

Some MR-CoNS may test posi,ve

Malhotra-Kumar et al. J Clin Microbiol 2010;48:4598.

Posi,ve predic,ve value for these assays can therefore be lower than culture
Herdman, et al. J Clin Microbiol 2009;47:4102. New tes;ng approaches include mecA primers, to address bullet 1, but remain vulnerable to mixed popula;ons or false + MR-CoNS

False posi,ves arent the only issue

mecALGA251
Undetected by current PCR Widespread in UK among LA-MRSA Mul,ple spa and MLST types

Im sure this wont ever happen again

Garcia-Alvarez, et al. Lancet Infect Dis 2011;11:595-603. Shore AC, et al. An,microb Agents Chemother June 2011

The moral of this story: Dont toss those agar plates!

Less vulnerable than PCR to gene,c variability
Emerging clones, empty casseoe variants

Improved PPV, helps detect assay problems Organism is available for AST, typing, etc. Greater exibility of use (site, sample) Adding a molecular test to culture, rather than replacing culture

The importance of MSSA detec,on

Focus periop decoloniza,on of SA carriers Tailor an,microbial prophylaxis for MRSA carriers

Bode et al. N Engl J Med 2010;362:9-17

A new test method of interest, nares test not yet available:

Uses bacteriophages to amplify signal (must be species-specic) Simple immunoassay detects phage Ag Tes,ng +/- an an,bio,c can serve as a method of suscep,bility tes,ng Only current FDA-approved assay is for blood cultures + for Gram posi,ve cocci
www.microphage.com

From blood cultures or nares samples

Method PCR (various)* Bacteriophage** TAT 1-4 5-6 Sens 90-98 92 Spec 91-99 98 Equipment Per test 30-150K n/a n/a 25-50 ? 5

Rapid detec,on of MRSA/MSSA

Chromogenic agar 18-48 60-90 90-100

*Commonly used commercial assays: home brew PCR is far less expensive **blood culture only, nares test in development ***TAT is analy=c TAT only! Bhowmick, et al. 50th ICAAC, Boston, MA, 2010, Abstract D-155. Carroll KC. Mol Diagn Ther 2008;12:15 Malhotra-Kumar et al. J Clin Microbiol 2010;48:4598. Reyes RC, et al. Diagn Microbiol Infect Dis 2008;60:225 Pape J, et al. J Clin Microbiol 2006;44:2575.

Scenario
Your lab director comes to your monthly infec,on control mee,ng to ask for your support to bring in a new real-,me PCR test for MRSA direct from nasal swabs. Your hospital currently screens all admissions to selected units, has a 4% admission prevalence, and has seen a 60% decline in MRSA HAI rate over the past 5 years. The current screening method is chromogenic agar, with ~24 hr TAT.

The lab director asks: will adop,ng the new test be cost-eec,ve? Will it result in improved preven,on of MRSA transmission and infec,on?

Best way to answer the ques,on:

Prospec,ve trial with concurrent controls! Culture vs. NAAT (PCR with reduced TAT) Outcomes to include MRSA transmission (acquisi,on) and infec,on events At least 3 such trials have been published, all use unit-based crossover designs:
1. Jeyaratnam et al. BMJ 2008;336:927-930. 2. Aldeyab et al. J Hosp Infect 2009;71:22-8. 3. Hardy et al. Clin Microbiol Infect 2010;16:333.

Summary of controlled trials

Two show no dierence in MRSA outcomes1,2
But overall TAT only reduced to 19-22 hours

Hardy, et al, showed benet for PCR3:

Pts on cx units 1.5X more likely to acquire MRSA Almost 2-fold more unscreened in culture arm Only 17% of colonized were properly isolated 71% of PCR + decolonized (vs. 41% in cx + arm)
Really a trial of decoloniza,on (not of ADI)
1. Jeyaratnam et al. BMJ 2008;336:927-930. 2. Aldeyab et al. J Hosp Infect 2009;71:22-8. 3. Hardy, et al. Clin Microbiol Rev 2010;16:333-39.

What is your true turnaround ,me?

Pre-analy,c Collec,on Transport Analy,c Processing Tes,ng Post-analy,c Repor,ng Interven,on

How long is each step? What por,on of TAT is aoributable to the test? Are PCR tests batched? Is the eerent arm of your program func,oning? It isnt as simple as 2 hours versus 24-72 hours.

MRSA/MDRO preven,on: So much more than a lab test

Environmental disinfec,on prac,ces Hand hygiene adherence (40% or 90%?) Contact precau,ons adherence Hospital design: % single rooms Adherence to CLABSI/VAP/SSI bundles Decoloniza,on prac,ces (CHG, mupirocin) Pa,ent popula,on (risk factors) Admission MRSA prevalence

Consider two units

A. 40% adherence to HH and CP, poor implementa,on of DAI preven,on bundles B. 90% adherence to HH and CP, nearly 100% adherence to DAI bundle measures Reduce TAT to zero in unit A, you s,ll have no impact on MRSA disease. In unit B, MRSA disease falls 85-90% before screening can be implemented.

Has your laboratory adopted a rapid molecular test for detec,on of MRSA and/or MSSA direct from clinical samples?

ClinMicroNet Survey, 2011

24 (75%): cost 3 (9%): performance 4 (13%): both

32 (46%)

38 (54%)

35 (92%) for nares 13 (34%) for BSI

Diekema D, Peterson L. ClinMicroNet survey.

Has your laboratory adopted a rapid test and then gone back to a culture-based test? Thirteen (19%) of the labs reported having gone back to an agar-based method from a rapid detec,on test
Eight went completely back (all tes,ng) Five s,ll did some rapid molecular tes,ng
Physician preference Agar-based for one unit with endemic empty casseoe Went back for nares but not BSI

Reasons: Cost (7), performance (3), cost + perf (3)

Empty casseoes, failure to demonstrate savings/impact
Diekema D, Peterson L. ClinMicroNet survey.

Quote excerpts from survey responses

May go back to culture from PCR b/c promised cost savings haven't been demonstrated Did a comparison and PCR not beoer, at higher cost Adopted PCR but had no impact on MRSA rates in ICU Concerns about sensi,vity and applica,on to non-nares sites Cost, plus not validated for non-nares samples No di in performance in our hands, TAT not dierent enough Actual TAT in our lab not much dierent for chrom vs PCR Would cost over 500K per year, no proof that faster TAT causes fewer MRSA infxs We save about 450K per year by using chromogenic agar No study showing reduc,on in MRSA transmission for molecular vs 24 hr chromogenic culture detec,on

Lessons learned from molecular tes,ng for S. aureus

Gene,c variability and evolu,on will be an ongoing challenge for NAAT tests Culture back-up remains important Analy,c TAT may dier from actual TAT
Know how your lab will incorporate the test

Cost saving es,mates must be individualized: NAAT may not be the right choice for all

Vancomycin-resistant enterococci
Another MDRO with well-characterized resistance genes (vanA/B/C/D/E/G/L/M) vanA and vanB detec,on most important
Mobile gene,c elements, E. faecium and E. faecalis

Problem for rapid detec,on: reservoir

Rapid VRE detec,on

Molecular aorac,ve given TAT of culture Some obstacles to keep in mind:
lower limit of detec,on (varies, ~10-100 cfu/ml)
eect of an,microbials on detec,on!

vanB is present in selected gut anaerobes

What does a nega,ve VRE screen mean?

Before versus aSer an,microbial exposure

What does a posi,ve VRE screen mean?

Ballard et al. An,microb Agents Chemother 2005;49:77-81. Gazin et al. Eur J Clin Microbiol Infect Dis 2012;31:273-76. Donskey et al. N Engl J Med 2000;343:1925-32.

Performance of vanA/vanB screen vs. broth enriched culture

Posi,ve predic,ve value in mul,center evalua,on ~50% For vanB, PPV in one study was <10% for 2 di assays Dont toss those agar plates!
Xpert vanA/vanB package insert (www.cepheid.com) Gazin et al. Eur J Clin Microbiol Infect Dis 2012;31:273-76.

Lessons learned from molecular tes,ng for VRE

Tes,ng samples from complex organism popula,ons (direct from samples of non- sterile environment) is challenging Other species share resistance determinants
mecA: found in S. aureus + coag nega,ve staph vanB: found in VRE + selected anaerobes

Molecular detec,on of MDR-GNR Even more complicated!

Peleg and Hooper. N Engl J Med 2010;362:1804-13.

Rapid emergence and spread of carbapenemases (NDM, KPC, VIM, IMP)

Obstacles to widely available molecular detec,on of MDR-GNR

Mul,plicity of targets, emerging new targets
Dicult to keep up with FDA-approved methods

Gene presence gene expression

e.g. chromosomal cephalosporinase in E. coli

Some important phenotypes are complex

Carbapenem R from stably de-repressed ampC cephalosporinase + porin muta,on

The realm of referral, reference, research labs

Current approaches
PCR targe,ng most common problems
e.g. home brew KPC detec,on assays common on isolated colonies, limited targets

For screening, microarray most promising

Specic nucleic acid probes Only hybridized probes amplied

Naas, et al. J Clin Microbiol 2011;Feb 16 (online ahead of print)

MDR-GNR NAAT tes,ng: Summary

Complexity/variety of resistance determinants is a challenge for test development For now, screening for carriage best done with selec,ve culture (e.g. chromogenic agar)
Labor intensive, and ideal screening sites, assay performance s,ll uncertain

Performance of any new assay in mixed and complex samples (e.g. stool, blood culture broth, respiratory secre,ons) will be key

Molecular detec,on of C. dicile

The right target for molecular tes,ng:
Dicult/laborious to culture Standard tests (EIA) perform poorly (60-70% sens) Toxin gene presence correlates with pathogenicity

Toxin EIAs: Poor sensi,vity and PPV

Goldenberg SD, et al. J Hosp Infect 2011;79:4-7.

Several NAAT tests now available

Most target tcd B gene (gene, not toxin!) Important to test only those with disease Sensi,vity and specicity in mid-90s Repeat tes;ng is not necessary

Table adapted from: Carroll KC. Anaerobe 2011;17:170-174.

Two step: Glutamine dehydrogenase PCR/NAAT

A less expensive approach?

Problem: GHD EIA isnt sensi,ve enough (? Mid-80%)

Chapin K, et al. J Mol Diagn 2011;13:395-400. Selvaraju, et al. Diagn Microbiol Infect Dis 2011;71:224-29. Carroll KC. Anaerobe 2011;17:170-174.

Best tes,ng prac,ces are essen,al!

Diagnos,cs 101
PPV depends upon disease prevalence Prevalence drops if test is performed on those with low pre-test probability of disease
e.g. at a 4-5% posi,vity rate, PPV ~50% Usually reects poor tes,ng prac,ces

NO tes,ng of non-diarrheal stool (C-T-C) NO repeat tes,ng within 7 days NO test of cure

What is your prevalence?

Deshpande, et al. Clin Infect Dis 2011;53:e81-e90.

Pooled sensi,vity = 90% Pooled specicity = 96%

How about those C. dicile rates!

The impact of sensi,vity

N samples N (%) posi,ve CDI rates*

EIA 2579 167 (6.5) 4.9

PCR 2534 382 (15.1) 10.3

*cases/10,000 pa,ent days

Fong KS, et al. Infect Cont Hosp Epidemiol 2011;32:932.

C. dicile tes,ng: summary

PCR/NAAT seems the best single op,on Prepare for CDAD rate increase of up > 50% Strict enforcement of test rejec,on policies
NPV >99% allows this, preserves good PPV

Consider review of clinical tes,ng approach

> 3 diarrhea stools per day, an,bio,c use history

Periodically assess posi,vity rate

If <15%, assess rejec,on polices/tes,ng prac,ce

Use of Molecular Typing

Study pathogenesis of infec,on
Colonizing and infec,ng strains Contamina,on versus pathogen

Assess extent and mode of pathogen transmission

Eec,veness of infec,on control eorts Outbreak inves,ga,on

Detect epidemiologically important pathogen (e.g. NAP1/BI C. dicile) Requires maintaining an organism bank

Methods are all based upon detec,ng dierences in nucleo,de sequence

Banding paoerns: size of fragments produced by amplica,on and/or enzyma,c diges,on of genomic DNA
PFGE, RFLP, ribotyping, AFLP, rep-PCR, RAPD, MLVA, etc.

DNA sequencing: polymorphism of DNA sequences

Mul,locus sequence typing (MLST), spa (S. aureus) Whole genome sequencing

DNA hybridiza,on: using nucleo,de probes

Spoligotyping (macroarrayM.tb), microarrays
Li, Raoult and Fornier. FEMS Microbiol Rev 2009;33:892-916.

Criteria for Evalua,ng Typing Systems

Typeability: Ability to obtain an unambiguous result for each isolate analyzed Reproducibility: Ability to give the same result each ,me an isolate is tested Discriminatory power: Ability to dieren,ate among unrelated strains Cost ($$, labor, ease of use, etc.) Turnaround ,me

Choosing a Typing Method

PFGE: excellent discrimina,on, exibility
Labor intensive, longer TAT (2-3 days)

Several PCR methods provide good discrimina,on w/ faster TAT (rep-PCR, MLVA) Method used determined by:
Purpose (local outbreak/cluster, vs. library) Organism (some specic for bug (e.g. spa typing))

Combina,on of methods may provide greatest discrimina,on (e.g. MRSA)

Comparing common typing methods

Method Reproducibility Discrimina,on PFGE MLVA Ribotype MLST WGS
Intra (Y)/Inter (V)

Cost $$ $$ $ $$$ $$ $$$

TAT 2 d 4 h 4 h 1 d 2-3 d ??

Excellent Good Very Good Fair-Good Fair Excellent

rep-PCR Yes (auto only) Yes Yes Yes Yes

When will rapid throughput sequencing come to the clinical lab? How will we handle all the data? Resistance detec,on, typing, virulence, etc., etc., etc.

Typing data informs good epidemiology (it doesnt replace it)

Indis,nguishable immediate common source Same: PFGE spa MLST SCCmec rep-PCR

Zazakova, et al. N Engl J Med 2005;352:468.

Typing data informs good epidemiology (it doesnt replace it)

Dierent (type or species) does not rule out transmission
e.g. spread of plasmid-mediated ESBLs

Regional KPC spread Most were KPN Interspecies to E. coli on at least 2 occasions
Won, et al. Clin Infect Dis 2011;53:532.

Molecular typing: Summary

Many acceptable methods available Discuss per,nent issues with your lab director
TAT: need for in house versus send out tes,ng Local use for cluster/outbreaks only? Need for library method (e.g. MLST, spa)

Most well served by PFGE, rep-PCR, MLVA Most important point is to interpret results in context (bug, gene,cs of R, epidemiology)

Proteomics
Matrix assisted laser desorp,on/ioniza,on ,me of ight (MALDI-TOF) Produces detailed protein ngerprint Accurate species ID
Rapid, inexpensive Requires isolated colony

Other poten,al applica,ons?

Strain typing Resistance detec,on Analysis of mixed samples

Summary
Molecular tes,ng increasingly oers faster, more sensi,ve detec,on of HAI pathogens No test is perfect: understand limita,ons and implica,ons (diagnosis, surveillance, cost) Close collabora,on with lab director
Regular rounds in the micro lab Lab par,cipa,on on infec,on control commioee Know implica,ons of IC ini,a,ves on the lab