Molecular Niches for Laboratory Diagnosis of Molecular Niches for Laboratory Diagnosis of Bloodstream InfectionsBloodstream Infections

Yi-Wei Tang, MD, PhD, FAAM, FIDSA

Chief of Clinical Microbiology ServiceMemorial Sloan-Kettering Cancer Center

Professor of Pathology and Laboratory MedicineWeill Medical College of Cornell University

““The three most important elements The three most important elements in practicing medicine are diagnosis, in practicing medicine are diagnosis,

diagnosis, and diagnosis.”diagnosis, and diagnosis.”

- William Osler, The Principles and Practice of Medicine, 1892

Clinical case presentation Current methodology Molecular “niches” Sepsis case-control study Take home messages

Advances in Laboratory Diagnosis of SepticemiaAdvances in Laboratory Diagnosis of Septicemia: : OutlinesOutlines

A 13-year-old previously healthy boy, presented with severe hypotension, tachycardia, and impending respiratory failure

Case PresentationCase Presentation

Case Presentation – cont.Case Presentation – cont.

The patient reported ten days ago a tick bite on his right medial ankle and an ATV accident resulting in mild right leg pain

Five days prior to admission, hip and knee pain progressively worsened and evolved into full body arthralgia without fever

24 hours prior to admission, the patient experienced nausea and vomiting, again without reported fever

On the morning of admission, the patient continued having episodes of nausea and vomiting with chest pain. He looked pale and blue, and collapsed in the parking lot

On admission, he was hypotensive and tachycardic and increased work of breathing

Blood cultures were drawn and the patient was given doxycycline and ceftriaxone

The patient was intubated secondary to severe shock and impending respiratory failure and transported via Life Flight to VCH

Case Presentation – cont.Case Presentation – cont.

The patient was sedated on arrival to the PCCU Vancomycin and gentamycin were added per

infectious disease consult Blood cultures revealed coagulase-positive cocci in

clusters at the second hospitalization day An ultrasound of the hip and knee were done to assess

for joint abscess, but revealed no source of infection At the third hospitalization day, blood, trach and

pleural fluid cultures were positive for MRSA The patient was continued vancomycin, clindamycin

and ceftriaxone

Case Presentation – cont.Case Presentation – cont.

Ceftriaxone was discontinued and rifampin was added at hospital day 4

Sedation was discontinued for complete neurological exam

The patient developed multiple organ failure, blood cultures remain positive for MRSA

In the afternoon of hospital day 6, he developed a fixed and dilated pupils

Brain death was documented and cardiopulmonary support was withdrawn

Case Presentation – cont.Case Presentation – cont.

Autopsy indicated S. aureus sepsis, with pre- and post-mortem cultures positive for MRSA

Severe diffuse necrotizing pneumonia with multifocal fresh infarcts

Shock-induced myocardial and hepatic injury Shock-related changes of spleen, nodal lymphoid

hyperplasia, hemorrhage and splenic subcapsular infarctions

Early infection of right hip, presumed secondary to pulmonary infection

The isolate was PVL positive and SCCmec type IV

Case Presentation – cont.Case Presentation – cont.

Case Presentation – EndCase Presentation – End

Can we do better, next time?

Bloodstream Infections Can be Lethal Bloodstream Infections Can be Lethal

Boucher & Corey. Clin. Infect. Dis. 46:S344-9, 2008

Antimicrobial Initiation Associated Survival Antimicrobial Initiation Associated Survival Following Onset of Septic Shock Following Onset of Septic Shock

Kumar et al. Crit. Care Med. 34:1589-96, 2006

Bloodstream Infection Detection: Bloodstream Infection Detection: Automated Blood Culture Instruments Automated Blood Culture Instruments

with Continuous Monitoringwith Continuous Monitoring

It works well, but it still takes an overnight growth, and …

BD BACTEC Trek ESPBacT/Alert 3D

Identification and Antimicrobial Identification and Antimicrobial Resistance: Current Procedures Resistance: Current Procedures

Organisms Seen by Gram Stain

Subculture “Pilot” AST

Identification Official AST

Identification report More tests

Pilot AST report

1

2

0

Days

?

Final AST report

“I am getting there…”

Niches for Rapid Detection and Identification Niches for Rapid Detection and Identification of Pathogens Causing Sepsis of Pathogens Causing Sepsis

Identification, antimicrobial resistance report

2

4

0

Hours

Positive blood culture broth (day 1)

Tang & Peterson, Lancet Infect. Dis. 14:94-6, 2014

Devices Approved by FDA for Rapid Identification Devices Approved by FDA for Rapid Identification of Staphylococci from of Staphylococci from Positive Blood Cultures Positive Blood Cultures

Niches for Rapid Detection and Identification Niches for Rapid Detection and Identification of Pathogens Causing Sepsis of Pathogens Causing Sepsis

Identification, antimicrobial resistance report

2

4

0

Hours

Original blood specimen (day 0)

Tang & Peterson, Lancet Infect. Dis. 14:94-6, 2014

Devices for Detection of Bacteremia Directly Devices for Detection of Bacteremia Directly from Bloodfrom Blood

Manufacturer Device name Pathogens covered References

Roche SeptiFast GN, GP, fungi, mecA Lehmann, et al. Med. Microbiol. Immunol. 197: 313-24, 2008

Mulzym SepsiTest GN, GP, fungi Wellinghausen et al. J. Clin. Microbiol. 47:2759-65, 2009

Seegene Magicplex GN, GP, fungi, mecA, vanA, vanB

Carrara L et al. J. Med. Microbiol. 62:1763-9, 2013

T2 Biosystems T2Candida Fungi (yeasts) Leely et al. Sci. Transl. Med. 5:182ra54, 2013

Abbott Mol Plex-ID GN, GP, fungi, mecA, vanA, vanB, KPC

Laffler et al. J. Clin. Microbiol. 51:3535, 2013

An Alternative System for Detection and An Alternative System for Detection and Identification of Bacterial PathogensIdentification of Bacterial Pathogens

David Ecker

Does not ask: “Is pathogen X, Y or Z in my sample?”Does not ask: “Is pathogen X, Y or Z in my sample?”

Asks instead: “Which pathogens are in this sample?”Asks instead: “Which pathogens are in this sample?”

An Alternative System for Detection and An Alternative System for Detection and Identification of Bacterial PathogensIdentification of Bacterial Pathogens

PCR-MS Combines Sensitivity PCR-MS Combines Sensitivity (PCR) and Specificity (MS)(PCR) and Specificity (MS)

Ecker et al. Nat. Rev. Microbiol. 6:553-8, 2008

Plex-ID

Performance of Performance of PCR/ESI-MS Assay for PCR/ESI-MS Assay for Detection of Ehrlichia Detection of Ehrlichia

Species in Blood Species in Blood (N=213)(N=213)

Eshoo et al. J. Clin. Microbiol. 48:472-8, 2010

Detection and Identification of Additional Detection and Identification of Additional Bacterial Pathogens by PCR/ESI-MS Bacterial Pathogens by PCR/ESI-MS

Eshoo et al. J. Clin. Microbiol. 48:472-8, 2010

Directly from whole blood specimens Rapid procedure done within six hours Multiple organisms covered in one

reaction Simultaneous detection and identification

Detection and Identification of Bacterial Detection and Identification of Bacterial Pathogens in Blood: Boom/PCR/ESI-MSPathogens in Blood: Boom/PCR/ESI-MS

Accountability of Specimens CollectedAccountability of Specimens Collected

Laffler et al. J. Clin. Microbiol. 51:3535, 2013

Organisms Detected Organisms Detected by PCR/ESI-MS and by PCR/ESI-MS and Blood Culture MethodsBlood Culture Methods

PCR/ESI-MS Blood Culture

86% 41%

Sensitivity was estimated when organism was detected by PCR/ESI-MS or blood culture

Total agreement: 78.6%

Laffler et al. J. Clin. Microbiol. 51:3535, 2013

Concordance of PCR/ESI-MS and CultureConcordance of PCR/ESI-MS and Culture

Category Culture +(N=464)

Culture(N=442)

Both culture and PCR/ESI-MS were in agreement in the number of detections and species identity

150 381

Culture and PCR/ESI-MS were in agreement on the primary pathogen; PCR/ESI-MS also detected one or more of the common contaminants

5 17

PCR/ESI-MS detected one or more additional organisms not identified by culture.

15 33

Culture reported one or more detections not made by PCR/ESI-MS. 6 0

Results do not fit in any of the other categories because of multiple detections 13 0

Both culture and PCR/ESI-MS were positive for one organism but disagreed in reported microbial species

13 0

Culture was positive but PCR/ESI-MS was negative 100 0

Coagulase-negative staphylococci 162 11

Laffler et al. J. Clin. Microbiol. 51:3535, 2013

Distribution of Q-scores and Genome Levels for Distribution of Q-scores and Genome Levels for Culture-Negative PCR/ESI-MS-Positive SamplesCulture-Negative PCR/ESI-MS-Positive Samples

Distribution of Q-scores and Genome Levels for Distribution of Q-scores and Genome Levels for Culture-Negative PCR/ESI-MS-Positive SamplesCulture-Negative PCR/ESI-MS-Positive Samples

Culture-negative, Ibis-positive (N=33)

Culture-positive, Ibis-multi-positive (N=15)

Clinical relevance Numbers (%) Numbers (%)

True bacteriemia and/or candademia with correct pathogen identification

10 30.3 3 20.0

True bacteriemia and/or candademia

21 63.6 7 46.7

Possible true bacteriemia and/or candademia

0 0.0 3 20.0

False positive most likely due to skin contamination

2 6.1 2 13.3

Clinical Relevance of Additional Positive Clinical Relevance of Additional Positive Results by PLEX-ID BCA AssayResults by PLEX-ID BCA Assay

Laffler et al. J. Clin. Microbiol. 51:3535, 2013

Detection of Bacteremia and Candademia in Detection of Bacteremia and Candademia in Blood Using PCR/ESI MS: Summaries Blood Using PCR/ESI MS: Summaries

Culture or PCR/ESI-MS alone provided poor diagnostic yield for diagnosis of septicemia

PCR/ESI-MS and blood culture reached an overall agreement of 72.4%

Among culture ()/PCR/ESI-MS (+) specimens, 93.9% were determined as true bacteremia

Among culture single (+) positive/PCR/ESI-MS multiple (+) specimens, 66.7% of the additional positive findings by PCR/ESI-MS were related to true and possible bacteremia

Adjunct with blood culture, PCR/ESI-MS enhances diagnostics of septicemia by shortening test turnaround time and improving yields

Take Home Messages: Beyond the BugsTake Home Messages: Beyond the Bugs

Beyond Culturable Bugs: Wider Pathogen Beyond Culturable Bugs: Wider Pathogen Spectrum and Coverage Causing SepsisSpectrum and Coverage Causing Sepsis

Beyond Bacteria: Sepsis Caused by ViremiaBeyond Bacteria: Sepsis Caused by Viremia

Hu et al. Proc Natl Acad Sci USA, 2013

Beyond the Pathogens: Gut Microbiome DiversitiesBeyond the Pathogens: Gut Microbiome Diversities

Ubeda et al. J. Clin. Invest. 2010

Beyond the Pathogens: Immune Beyond the Pathogens: Immune Repertoire DiversitiesRepertoire Diversities

Venet et al. Crit Care Med 2013

Beyond Bugs: SIRS, Septicemia, Sepsis, and Beyond Bugs: SIRS, Septicemia, Sepsis, and Septic ShockSeptic Shock

Ma et al. PLoS ONE 2013

Integrated, Rapid and Accurate Diagnosis of Integrated, Rapid and Accurate Diagnosis of Bloodstream Infections: Closer to RealityBloodstream Infections: Closer to Reality

Pathogens

Host Milieu

Empiric therapy Evidence-based therapy

AcknowledgmentsAcknowledgments

Thomas G. Laffler, Ph.D.Lendell L. Cummins, Ph.D.Colt McClain, M.D.Criziel D. Quinn, M.T.Michelle A. ToroHeather E. Carolan

Medical Technologists in Clinical Microbiology Laboratory and Molecular Infectious Diseases Laboratory, VUMC

Donna M. Toleno, Ph.D.Megan A. Rounds, Ph.D.Mark Eshoo, Ph.D.Charles W. Stratton, M.D.Ranga Sampath, Ph.D.Lawrence B. Blyn, Ph.D.

David J. Ecker, Ph.D.

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