Molecular Niches for Laboratory Diagnosis of Bloodstream Infections

Molecular Niches for Laboratory Diagnosis of Bloodstream Infections Yi-Wei Tang, MD, Ph. D, FAAM, FIDSA Chief of Clinical Microbiology Service Memorial Sloan-Kettering Cancer Center Professor of Pathology and Laboratory Medicine Weill Medical College of Cornell University

“The three most important elements in practicing medicine are diagnosis, and diagnosis. ” - William Osler, The Principles and Practice of Medicine, 1892

Advances in Laboratory Diagnosis of Septicemia: Outlines ¨ Clinical case presentation ¨ Current methodology ¨ Molecular “niches” ¨ Sepsis case-control study ¨ Take home messages

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

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

Case Presentation – cont. ¨ 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. ¨ 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. ¨ 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. ¨ 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 – End ¨ Can we do better, next time?

Bloodstream Infections Can be Lethal

Boucher & Corey. Clin. Infect. Dis. 46: S 344 -9, 2008

Antimicrobial Initiation Associated Survival Following Onset of Septic Shock Kumar et al. Crit. Care Med. 34: 1589 -96, 2006

Bloodstream Infection Detection: Automated Blood Culture Instruments with Continuous Monitoring It works well, but it still takes an overnight growth, and … BD BACTEC Bac. T/Alert 3 D Trek ESP

Identification and Antimicrobial Resistance: Current Procedures “I am getting there…” Organisms Seen by Gram Stain 0 Days 1 2 Subculture “Pilot” AST Identification Official AST Identification report Final AST report More tests ? Pilot AST report

Niches for Rapid Detection and Identification of Pathogens Causing Sepsis Positive blood culture broth (day 1) 0 Hours 2 4 Identification, antimicrobial resistance report Tang & Peterson, Lancet Infect. Dis. 14: 94 -6, 2014

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

Niches for Rapid Detection and Identification of Pathogens Causing Sepsis Original blood specimen (day 0) 0 Hours 2 4 Identification, antimicrobial resistance report Tang & Peterson, Lancet Infect. Dis. 14: 94 -6, 2014

Devices for Detection of Bacteremia Directly from Blood Manufacturer Device name Pathogens covered References Roche Septi. Fast GN, GP, fungi, mec. A Lehmann, et al. Med. Microbiol. Immunol. 197: 313 -24, 2008 Mulzym Sepsi. Test GN, GP, fungi Wellinghausen et al. J. Clin. Microbiol. 47: 2759 -65, 2009 Seegene Magicplex GN, GP, fungi, mec. A, van. B Carrara L et al. J. Med. Microbiol. 62: 1763 -9, 2013 T 2 Biosystems T 2 Candida Fungi (yeasts) Leely et al. Sci. Transl. Med. 5: 182 ra 54, 2013 Plex-ID GN, GP, fungi, mec. A, van. B, KPC Laffler et al. J. Clin. Microbiol. 51: 3535, 2013 Abbott Mol

An Alternative System for Detection and Identification of Bacterial Pathogens David Ecker

An Alternative System for Detection and Identification of Bacterial Pathogens Does not ask: “Is pathogen X, Y or Z in my sample? ” Asks instead: “Which pathogens are in this sample? ”

PCR-MS Combines Sensitivity (PCR) and Specificity (MS) Plex-ID Ecker et al. Nat. Rev. Microbiol. 6: 553 -8, 2008

Performance of PCR/ESI-MS Assay for Detection of Ehrlichia Species in Blood (N=213) Eshoo et al. J. Clin. Microbiol. 48: 472 -8, 2010

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

Accountability of Specimens Collected Laffler et al. J. Clin. Microbiol. 51: 3535, 2013

Organisms Detected by PCR/ESI-MS and Blood 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 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 Culture and PCR/ESI-MS were in agreement on the primary pathogen; PCR/ESI -MS also detected one or more of the common contaminants 150 381 5 17 PCR/ESI-MS detected one or more additional organisms not identified by culture. Culture reported one or more detections not made by PCR/ESI-MS. 15 33 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 Culture was positive but PCR/ESI-MS was negative 13 0 100 0 Coagulase-negative staphylococci 162 11 Laffler et al. J. Clin. Microbiol. 51: 3535, 2013

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

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

Clinical Relevance of Additional Positive Results by PLEX-ID BCA Assay Culture-negative, Ibis- Culture-positive, Ibispositive (N=33) multi-positive (N=15) Clinical relevance True bacteriemia and/or candademia with correct pathogen identification True bacteriemia and/or candademia Possible true bacteriemia and/or candademia False positive most likely due to skin contamination Laffler et al. J. Clin. Microbiol. 51: 3535, 2013 Numbers (%) 10 30. 3 3 20. 0 21 63. 6 7 46. 7 0 0. 0 3 20. 0 2 6. 1 2 13. 3

Detection of Bacteremia and Candademia in 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 Bugs

Beyond Culturable Bugs: Wider Pathogen Spectrum and Coverage Causing Sepsis

Beyond Bacteria: Sepsis Caused by Viremia Hu et al. Proc Natl Acad Sci USA, 2013

Beyond the Pathogens: Gut Microbiome Diversities Ubeda et al. J. Clin. Invest. 2010

Beyond the Pathogens: Immune Repertoire Diversities Venet et al. Crit Care Med 2013

Beyond Bugs: SIRS, Septicemia, Sepsis, and Septic Shock Ma et al. PLo. S ONE 2013

Integrated, Rapid and Accurate Diagnosis of Bloodstream Infections: Closer to Reality Pathogens Sepsis Host Empiric therapy Milieu Evidence-based therapy

Acknowledgments Thomas G. Laffler, Ph. D. Donna M. Toleno, Ph. D. Lendell L. Cummins, Ph. D. Megan A. Rounds, Ph. D. Colt Mc. Clain, M. D. Mark Eshoo, Ph. D. Criziel D. Quinn, M. T. Charles W. Stratton, M. D. Michelle A. Toro Ranga Sampath, Ph. D. Heather E. Carolan Lawrence B. Blyn, Ph. D. David J. Ecker, Ph. D. Medical Technologists in Clinical Microbiology Laboratory and Molecular Infectious Diseases Laboratory, VUMC

Hospital Ranking: 1 (2014) University Ranking: 16 (2014) School Ranking: 15 (2014) “The Best Cancer Care, Anywhere”