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1/9/2014 Impact of New Diagnostic Technologies in the Clinical Microbiology Laboratory Randall J. Olsen, M.D., Ph.D. Director, Molecular Diagnostics Laboratory Co-Director, Microbiology Laboratory Department of Pathology and Genomic Medicine Houston Methodist Hospital Houston Methodist Research Institute Texas Society of Pathologists 93rd Annual Meeting Houston, TX January 17, 2014 Traditional microbiology • Culture conditions and growth characteristics • Staining pattern and microscopic morphology • Biochemical phenotype – Inexact – Time consuming – Limited to cultivable organisms A laboratory technologist reading culture plates. Strategies for innovation in the new microbiology lab Matrix Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) mass spectrometry Whole genome sequencing of microorganisms Rapid identification of pathogenic organisms Detection from patient specimens or primary cultures Rapid inferred antimicrobial susceptibility and virulence Implementation of new technologies may rapidly inform treatment decisions to improve patient care and decrease costs 1 1/9/2014 New technologies: MALDI-TOF Mass Spectrometry • Rapid identification of bacteria, yeast & fungi – Minimum of 24-96 h reduced turnaround time compared to conventional culture-based methods. – Even more dramatic results for certain organisms (i.e. Legionella and Mycobacterium species). • Test directly from positive blood cultures – Bypasses time-consuming subculture steps. Wimmer JL, J Clin Microbiol. 2012. Perez, K. Arch Pathol Lab Med. 2012. Gram-negative bacteremia causes significant morbidity and mortality • Account for 24% of nosocomial bloodstream infections in intensive care units. • Survival is directly associated with timely and appropriate initiation of antibiotic therapy. – For every hour that appropriate antibiotics are delayed, survival decreases by 7.6% • Treatment is increasingly becoming complicated by the emergence of antimicrobial resistance. Gaynes R. Clin Infect Dis. 2005. Kang CI. Antimicrob Agents Chemother. 2005. Kumar A. Crit Care Med. 2006. MALDI-TOF in our clinical laboratory Identify Gram negative bacteria directly from positive blood culture media • Transfer positive blood culture media to a serum separator tube. • Centrifuge to isolate the bacteria. • Transfer an aliquot of the bacteria from the pellet to the spotting plate. • Immediately perform organism identification using the MALDI-TOF. • Concurrently perform antimicrobial susceptibility testing using the pellet. 2 1/9/2014 Organism identification and antibiotic susceptibility testing directly from positive blood culture media. • 110 blood cultures that recovered a Gramnegative organism were tested. • Organism identification using the MALDI-TOF. – 98% concordance with the reference method – Reduced turnaround time by >24 hours Wimmer, J. J Clin Micro. 2012. Strategies to improve patient care Hypothesis: Combining new rapid MALDI-TOF technology with real-time clinical interpretation will significantly enhance patient care. Innovation plan: MALDI-TOF & antimicrobial stewardship • Rapid identification (MALDI-TOF) and antibiotic susceptibility testing directly from blood cultures that recover a Gramnegative organism. • Microbiology laboratory results are immediately reported to the infectious diseases pharmacist who is on-call 24/7. • Pharmacist interprets clinical applicability of the MALDI-TOF data and contacts the treating physician to recommend optimal antibiotic therapy. 3 1/9/2014 MALDI-TOF & antimicrobial stewardship Study Design Patient Eligibility and Inclusion: Bacteremia due to a Gram-negative organism Perez, K. Arch Pathol Lab Med. 2012. MALDI-TOF & antimicrobial stewardship significantly reduces turnaround times Perez, K. Arch Pathol Lab Med. 2012. MALDI-TOF & antimicrobial stewardship significantly reduces length of stay 14 12 p = 0.01 Days 10 8 p = 0.05 Pre- Intervention 6 4 Intervention 2 0 Hospital LOS ICU LOS Perez, K. Arch Pathol Lab Med. 2012. 4 1/9/2014 MALDI-TOF & antimicrobial stewardship significantly reduces costs • The MALDI-TOF antimicrobial stewardship program saved our hospital 1.97 million dollars during the study period. Pre- Intervention Intervention $50,000 $40,000 p = 0.009 $30,000 • The program is estimated to save our hospital approximately $18 million dollars annually. $20,000 $10,000 $0 Total hospital cost per patient Perez, K. Arch Pathol Lab Med. 2012. MALDI-TOF & antimicrobial stewardship • First investigation to evaluate the impact of MALDI-TOF paired with antimicrobial stewardship on clinical and economic outcomes. – Earlier initiation of targeted therapy informed by rapid identification and susceptibility testing significantly improved patient care and decreased LOS and expenditures. • Collaboration is key! – An innovative microbiology laboratory paired with an active pharmacy department was crucial to the success of our program. New technologies: Whole genome sequencing • First bacteria genome H. influenzae strain Rd Published in 1995 > 1 year > 1 million dollars • Today, 1 or a few genomes can be generated within 1 day 768 genomes can be generated in 3 weeks for ~$100 • The $10 microorganism genome will soon be a reality Flow cell for the Illumina MiSeq benchtop sequencer. 5 1/9/2014 Genome size and complexity • Virus: few kb -> ~200 kb • Bacterium: ~1 Mb -> 6 Mb; haploid • Saccharomyces: 12.1 Mb • C. elegans: 97 Mb • Fruit fly: 180 Mb • Human: 3,000 Mb; ~20,000 genes Whole genome sequencing in the clinical laboratory “A day in the life of the microbiology laboratory” 130 samples collected from 116 patient cultures on a single day Isolated colonies and mixed samples Aerobic bacteria, anaerobic bacteria, acid-fast bacilli and fungi Long, et al, J. Clin Microbiol, 2013 Workflow for whole genome sequencing of microorganisms Extract genomic DNA (Automated, Qiagen) 4 hours Prepare sequencing libraries (Automated, NexteraXT kit) 4 hours Perform sequencing reaction (Illumina MiSeq) Analyze data (Custom bioinformatics process) 6-39 hours (overnight) 1-6 hours 6 1/9/2014 Results: A day in the life of the microbiology laboratory Whole genome sequencing identified: – most “unknown” organisms (88.5% concordance with reference method) – Mycobacterium species in two samples 10 days before the conventional method – multiple pathogens present in several mixed samples Challenges: A day in the life of the microbiology laboratory Whole genome sequencing could not identify • 10 organisms due to their absence from the reference database • The lack of a comprehensive database of human pathogens was particularly problematic for medically important fungi Whole genome sequencing of microorganisms in the clinical lab First run: June 26, 2013 • ~400 genome sequences analyzed in the validation study • Bacteria, fungi and influenza A virus • ~100 genomes generated as an ordered test 7 1/9/2014 First organism to be identified by our clinical laboratory Mycobacterium abscessus subsp. massiliense • Single gene sequencing assays can not distinguish members of the M. abscessus group • This organism has higher treatment response rates to combination therapy since it usually lacks inducible resistance to clarithromycin Whole genome sequencing of microorganisms in the clinical lab Routine cases • Organisms that can not be identified using MALDI-TOF • • • • Mycobacterium species Fungus Uncommon pathogens not represented in the database Organisms requiring further classification for optimal patient care or public health efforts • Serotyping Salmonella enterica and Neisseria meningitidis Whole genome sequencing of microorganisms in the clinical lab Special cases • Organisms suspected of comprising an outbreak • Investigated a cluster of invasive group A Streptococcus infections in Wyoming (Brown, et al., Emerg Infect Dis, in press) • Investigated a perceived outbreak of multiple-drug resistant Klebsiella pneumoniae infections in our hospital 8 1/9/2014 different location/ different “outbreak patients” service same location/ different service Perceived outbreak of multipledrug resistant K. pneumoniae Patient Specimen Disposition kp1 Abd fluid expired kp2 LRT rehab facility kp3 LRT expired kp4 Urine inpatient not avail LRT expired kp5 LRT inpatient kp6 LRT inpatient We performed whole genome sequencing to investigate the possible clonal relatedness of strains recovered from these patients. Whole genome sequencing identified three distinct lineages Phylogenetic tree based on single nucleotide polymorphisms (SNPs) in the sequenced strains relative to the reference strain. Strain key: same location/ different service “outbreak patients” different location/ different service Epidemiologically linked strains kp3 & kp4 are distantly related! Whole genome sequencing of microorganisms in the clinical lab Special cases • Organisms with unusual antimicrobial resistance • Organisms causing unusually severe or unexpected clinical presentations • Investigated a fatal anthrax-like infection (Wright, et al., Arch Pathol Lab Med, 2011) 9 1/9/2014 Previously healthy male with severe pneumonia and death within 72 h BAL on presentation Blood culture with extremely high numbers of rod-shaped bacteria pure culture of Bacillus cereus group organisms Gram’s stain Pap stain India ink stain Wright, et al., Arch Pathol Lab Med, 2011 The Bacillus cereus group troika Bacillus anthracis Bacillus cereus Bacillus thuringiensis • • • Very closely related Difficult to unambiguously classify using conventional microbiology techniques Mobile genetic elements (plasmids) are crucial for virulence We performed whole genome sequencing to investigate the molecular basis of this severe, rapidly fatal infection. Whole genome sequencing identified the causative agent Identified etiologic agent of the fatal Anthrax-like infection B. cereus with anthrax toxin genes, not B. anthracis Ruled out the likelihood of bioterrorism No foreign DNA or evidence of other genetic manipulation Guided infection control response No cases of secondary transmission Gained new insight to molecular pathogenesis Launched a new line of investigation 10 1/9/2014 Economics of whole genome sequencing of microorganisms In a typical week, our Microbiology Laboratory recovers 8 Mycobacterium species, 1 fungus and 1 unusual bacterium Number of Type of organism strains Probe cost 4 Unknown Mycobacteria 600 4 Unknown Mycobacteria 600 1 1 Send out cost for rDNA sequencing Total cost for conventional ID 2400 325 3700 Unknown Fungus 150 150 Unknown Bacterium 325 Total 325 $6575 Cost of whole genome sequencing for 10 organisms: $1255 (includes all reagents, labor and controls) Take home message: Although some challenges remain, new technologies such as MALDI-TOF and whole genome sequencing can be readily integrated into clinical laboratories Enhance patient care Reduce turnaround times Reduce costs Acknowledgments • Department of Pathology and Genomic Medicine – – – – – – – – – Steven Beres, PhD Patricia Cernoch, MT(ASCP), SM, SV James Davis, PhD Heather Hendrickson, MT(ASCP) Manuel Hinojosa, MHA, MT(ASCP) Geof Land, PhD S. Wesley Long, MD, PhD Dustin Meuth, MBA James Musser, MD, PhD • Center for Biostatistics – Leif E. Peterson, PhD • Department of Pharmacy – – – – – William Musick, PharmD Judy Ikwuagwu, PharmD Michael Liebl, PharmD Dan Metzen, PharmD, MBA Katherine Perez, PharmD • Houston Methodist Operations – Clare Rose, MBA, FACHE 11