Bye-Bye Biochemicals:

Transcription

1 Bye-Bye Biochemicals: How New Technology is Revolutionizing Microbiology Kevin Alby Ph.D., D(ABMM), MLS(ASCP) Assistant Director Clinical Microbiology Laboratory, Hospital of the University of Pennsylvania Assistant Professor, Pathology and Laboratory Medicine, Perelman School of Medicine April 16 th, 2015

2 Disclosures No relevant disclosures 2

3 Traditional Identification 3

4 Modern Identification 4

5 Recent advances in modern identification MALDI-TOF MS Matrix assisted laser desorption/ionization time of flight mass spectrometry Primarily used for bacterial/yeast identification Some applications for fungi and mycobacteria Limited susceptibility information Multiplex nucleic acid amplification tests Allows for syndromic testing Increased number of assays available in sample to answer format 5

6 How can MALDI differentiate organisms? Differences in DNA encode for different protein products which are differentially fragmented under laser excitation. Globally, these produce different spectra which when compared against a reference database of spectra lead to organisms identification. 6

7 VITEK MS Nearly 200 FDA cleared targets (Gram positive, Gram negative, Yeast) Utilizes weighted bin matrix to determine identity Displays identifications with confidence value of

8 Making a weighted bin matrix one species Rychert, J Clin Microbiol, (7):

9 Applying a weighted bin matrix for ID Rychert, J Clin Microbiol, (7):

10 Bruker Biotyper 40 FDA cleared targets (all Gram negative) Comparative spectral algorithm Displays identification with score of

11 Comparative spectral algorithm Unk Ref Unk vs Ref Ref vs Unk Peak intensity 11

12 Comparative performance of MALDI systems Karpanoja et al. (Eur J Clin Microbiol Infect Dis, 2013) 97 Viridans streptococci by 16s, 36 to species level Biotyper 89% to group, 75% to species Vitek MS 93% to group, 97% to species Alby et al. (J Clin Microbiol, 2013) 203 glucose non fermenting GNRs by 16s from CF patients Biotyper 96.6% agreed with previous identification Vitek MS 93.6% agreed with previous identification Martiny et al. (J Clin Microbiol, 2012) 986 isolates ID d by standard laboratory procedures (including Biotyper RUO) to species level Biotyper 92.7% agreement Vitek MS 93.2% agreement Mather et al. (J Clin Microbiol, 2014) 198 mycobacterial strains by 16s/rpoB/hsp65 Biotyper 79.3% to species, 93.9% with lower threshold Vitek MS 94.4% to species McElvania TeKippe and Burnham (Eur J Clin Microbiol Infect Dis, 2014) 174 unusual/difficult organisms Biotyper 92.3% to genus, 85.1% to species Vitek MS 86.3% to genus, 85.1% to species 12

13 Improved identification information Period Definitive IDs Probable IDs 7/12-9/12 (Pre) N=5095 1/13-3/13 (Post) N= (81.8%) 926 (18.2%) 4500 (92.2%) 383 (7.8%) Examples of probable IDs Coagulase-negative Staphylococcus Enterococcus spp. Candida spp. Clostridium not perfringens or septicum Pseudomonas not aeruginosa 13

14 Increased identification of rare species Seng, J Clin Microbiol, (7):

15 We still need to practice microbiology! From a male urine culture After 24 h of incubation, growth of small colonies was noted on sheep blood agar, with no growth evident on MacConkey agar At 48 h, a pure culture of gray, medium-sized colonies was growing equally well on blood and MacConkey agars. The oxidase positive GNR was identified by MALDI-TOF MS [Bruker] as [Burkholderia spp] The Washington State Public Health Laboratory identified the organism as B. pseudomallei with a speciesspecific PCR assay. 21 employees were determined to have risk of exposure 15

16 We still need to practice microbiology! Phenotype MALDI ID Reported Identification Aerobic oxidase negative lactose fermenting GNR Ralstonia insidiosa Klebsiella pneumoniae Aerobic alpha-hemolytic GPC Facklamia hominis Enterococcus faecium Aerobic beta-hemolytic large GPR Bacillus anthracis Bacillus cereus group Aerobic slow growing small GPR Mycobacterium kansasii Actinomyces gerencseriae Anaerobic small GPR Streptococcus pseudopneumoniae Actinobaculum schaalii Anaerobic GPC Bacillus sporothermodurans Ruminococcus gnavus Anaerobic GPR Serratia rubidaea Anaerobic GPR Aerobic yellowish GPR Corynebacterium jeikeium Kocuria rhizophila Anaerobic GPR Staphylococcus cohnii ssp cohnii Anaerobic GPR Anaerobic GPC in chains Staphylococcus epidermidis Anaerobic GPC Anaerobic GPR Ochrobactrum anthropi Anaerobic GPR Alby, J Clin Microbiol, (1):

17 Other applications for MALDI-TOF in clinical microbiology Direct from specimen testing Blood cultures Urine cultures Outbreak tracing Antimicrobial resistance testing Hydrolysis Isotope incorporation Global protein production 17

18 Direct detection from urine Ferreira, J Clin Microbiol, (6):

19 Direct detection from urine Ferreira, J Clin Microbiol, (6):

20 Detection of specific KPC carrying plasmid Lau, J Clin Microbiol, (8):

21 Detection of resistance via global protein expression Mero sensitive strain Mero resistant strain Lange, J Clin Microbiol, (12):

22 Nucleic Acid Amplification What I am going to cover Massively multiplexed technologies Multistep Luminex, Genmark Sample-to-answer Nanosphere, Biofire What I am not going to cover Non-PCR based methods of amplification Hologic/Gen-probe Illumigene Basic multiplex sample to answer or direct methods Cepheid, Focus, Quidel, BD MAX 22

23 Hydrolysis Probe (Taqman) Chemistry Easy to design Easy to adopt Amenable to multiplexing Limited to 4-6 reactions Koch, Nat Rev Drug Discov, (9):

24 Luminex Technology Beads are coated with complimentary oligonucleotides Beads hybridize with labelled reaction product Beads are queried to determine bead identity as well as presence/absence of label Luminexcorp.com 24

25 Genmark Technology Genmarkdx.com 25

26 Nanoparticle Detection Target specific oligonucleotides are hybridized to specific locations on a glass slid Gold nanoparticles with complementary oligonucleotides anneal to target sequences Silver is utilized to amplify the signal Georganpoulou PNAS

27 FilmArray Technology First round PCR is performed in a general well Products are diluted 100-fold Second round nested PCR takes place in individual wells Rand, J Clin Microbiol, (7):

28 FilmArray v Genmark v Luminex for the detection of respiratory viruses Popowitch, J Clin Microbiol, (5):

29 Comparative workflow of the three systems Popowitch, J Clin Microbiol, (5):

30 FilmArray v Verigene Virus n FilmArray Verigene TP TN FP FN Invalid TP TN FP FN Invalid 09H1N a 1 b 15 1 a sh3n sh1n Flu B a 3 RSV hmpv PIVR1 5 5 PIVR2 4 4 PIVR3 5 5 AdV RSV/AdV 1 1 c 1 c 1 No virus Total Butt, J Clin Virol, (3):

31 Impact of respiratory viral panel testing Rogers, Arch Pathol Lab Med, Epub PMID

32 Impact of molecular testing on infectious gastroenteritis Wessels et al (Clin Microbiol Infect; 2014) Looked at 393 fecal samples collected over a 2 month period All samples received bacterial culture, real time PCR for virus or parasites was done upon request. Molecular panel (GPP) was done on all samples. 40 positives were detected using standard algorithm All were detected using the molecular panel 43 additional positives were detected using a molecular panel approach 32 (74.4%) were confirmed by a 2 nd method (pathogen specific real-time PCR) Salmonella (4) and E. histolytica (5) were the most discordant targets 32

33 Increased positivity due to molecular methods Routine methods = positives in 8.3% of samples FilmArray (A) = positives in 33% of samples Luminex (B) = positives in 30% of samples Khare, J Clin Microbiol, (10):

34 Comparison of Luminex and FilmArray GI Khare, J Clin Microbiol, (10):

35 Effect of non culture methods on public health Majority are EIA detection of STECs Data is before widespread FDA clearance of multiple molecular platforms Iwamoto, MMWR Morb Mortal Wkly Rep, (9):

36 Conclusions New technology is rapidly changing the way microbiology is performed MALDI-TOF allows for rapid identification of a wide variety of organisms in culture Multiplex panels allow for clinicians to test for a wide variety of agents causing similar illnesses Classically trained microbiologists are invaluable resources Detection of inconsistent/incorrect MALDI-TOF identifications Syndromic testing comes at a cost Large increase in the number of positive specimens How many are true positives? Detection of low-level colonization/persistence Impact on public health 36

37 Clinical Microbiology Portal Engaging, enlightening, empowering our clinical community The only comprehensive online resource for clinical microbiologists. 37