A Complete Solution for Broad Microbial Identification Debbie Boldt-Houle, Ph.D
A Complete Solution for Broad Microbial Identification Debbie Boldt-Houle, Ph.D. Scientific Affairs Manager Abbott Molecular/Ibis Biosciences Objectives
• To gain an understanding of the PLEX-ID and the Ibis Technology • To gain an understanding of the wide range of PLEX-ID assays that are available, including: – Broad Bacteria – Vector-borne – Broad Viral 1 – Broad Fungal
Not For Use in Diagnostic Procedures Current Challenges to Microbial Detection:
• Over 1000* naturally occurring organisms known to infect humans – 217 virus species
– 538 bacterial species
– 307 fungi
– 66 parasitic protozoa
• Numerous strain variations of species (i.e., >300 recognized strains of Streptococcus pyogenes) • Potential bio-engineered organisms • Plant and animal microorganisms * Taylor et al, Phil. Trans. R. Soc. Lond. B (2001) 356, 983-989.
Not For Use in Diagnostic Procedures Mainstream Microorganism Detection Today • Culture techniques – Detect a subset of all pathogens – Can take multiple days (weeks) – Many organisms are slow growing or not culturable – False negative results due to conditions • Single agent nucleic acid tests – One test for each microorganism – Need many tests to cover disease state – Fail to detect newly emergent organisms There is currently no good routine method to detect organisms that have never been seen before
Not For Use in Diagnostic Procedures PLEX-ID offers a Unique Solution to Microbial Screening & Identification • Broad recognition of microorganisms – Bacteria, Viruses, Fungi, Parasites • Detects mixtures of microbes • High resolution genotyping and strain identification • Enables detection of novel microorganisms • Sensitivity of PCR with relative quantitation • Rapid identification in 6-8 hrs – culture often not required • Throughput up to 100 samples per 8 hours Changes the question “Is this in my sample?” to “What is in my sample?”
Not For Use in Diagnostic Procedures Ibis Technology: Unique Approach A Novel Combination of Powerful & Proven Technologies
Ribosomal PCR
universal primers sensitivity
ESI- Mass spectrometry PLEX-ID
Triangulation Automated & fast precision
specificity
Not For Use in Diagnostic Procedures What is the Ibis Technology?
• Coupling of broad PCR amplification with Electrospray Ionization Time-of-Flight Mass Spectrometry (ESI – TOF – MS) – Mass of PCR product (amplicon) is accurately determined by mass spec, converted to a unique base composition, then compared to a database of known targets for identification • When PCR ESI-MS is applied to microbial detection – Sensitive identification of known, unknown, and emerging microorganisms, usually from direct specimens
A28 G29 C25 T24
Not For Use in Diagnostic Procedures Foundation of the Technology: Design of PCR Primers Targeted to Universally Conserved Genes
Primers bind to conserved regions in ALL (or broad groups of) bacteria
Highly Variable Region
conserved conserved Informative region varies by type of bacteria (unique) Resulting PCR product acts like a “fingerprint” to identify the microbes
Not For Use in Diagnostic Procedures PCR ESI-MS Process Part 1: Nucleic Acid Prep and Broad Range PCR
9 Not For Use in Diagnostic Procedures PCR ESI-MS Process Part 2: MS-ESI Analysis and Signal Processing Spectral signal
Generation of ions Separation of ions Detection of ions
Mass Detection
Signal Processing Masses to Base Compositions
Not For Use in Diagnostic Procedures Converting Masses to Base Composition
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$7.63
A36 G33 C38 T32 Salmonella enterica Type II salamae
Not For Use in Diagnostic Procedures PCR ESI-MS Process Part 3: Triangulation Reconciling Base Counts to Target Identity
•Different primer pairs result in PCR products of different mass and base composition for any given microorganism
•Database contains over 750,000 base count entries
Unique ID from multiple primers
Not For Use in Diagnostic Procedures Primers Target Highly Conserved Regions
Broad Primers Covering Bacteria
Primers Covering Proteobacteria
Primers Covering Gamma Proteobacteria
Primers Covering Fusobacteria
Primers Covering Staphlococcus
Primers Covering Antibiotic Not For Use in Diagnostic Procedures Resistance Biodefense Organisms
Burkholderia mallei
Bacillus anthracis
Clostridium botulinum
Yersinia pestis
14 Not For Use in Diagnostic Procedures Public Health Organisms
Bordetella pertussis
Mycobacterium tuberculosis
Listeria monocytogenes
Salmonella enterica
15 Not For Use in Diagnostic Procedures Viral Identification • Viral coverage is achieved with broad-based virus family-specific primer pairs. *
*Housekeeping gene: RNA-dependent RNA polymerase
Not For Use in Diagnostic Procedures PLEX-ID Technology Features
• Suitable for many sample types: – Blood, throat swabs, nasal washes, tissue/cells, culture – Food, water, air, forensic, in-process quality controls
• Same experimental process PLEX-ID System for all: Desalting Mass Nucleic Spec Sample acid PCR Extraction Identify target by Convert amplicon comparing base mass to base composition to database composition of >700,000 entries
Not For Use in Diagnostic Procedures PCR/ESI-MS Workflow: Results in a Single 8-Hour Shift
Nucleic Acid Extraction Base Composition Broad Range PCR Desalting of Amplicons ESI-MS-TOF And Purification Triangulation
~2 hours 2-3 hours/plate 10 minutes/well 30 sec/well 1-2 minutes/sample
Open method DNA: 70 min/plate 2 hours per plate RNA viruses: Desalting is continually performed ahead of MS 3 hours per plate MS has two alternating ESI probes for increased throughput
6-8 hours
Not For Use in Diagnostic Procedures PLEX-ID using Ibis Technology Assay Capability
Broad bacterial or viral Unknown Sample identification
Genus/species confirmation
Species-level organism identification
Drug resistance virulence markers
Genotyping MLST/SNP/VNTR High-resolution molecular genotyping
Not For Use in Diagnostic Procedures PLEX-ID Crossing Broad Application Boundaries PLEX-ID Biosecurity Bioresearch Food Safety
• Biodefense Bacterial & • Flu Detection • Food-Borne Bacteria 1 Viral Surveillance • Broad Bacteria Detection (E. coli, Shigella, Salmonella, Bacterial agents: B. anthracis, • MRSA Genotyping Listeria) B. melitensis, B. mallei / B. • Broad Viral pseudomallei, C. botulinum, C. perfringens, C. burnetii, F. • Respiratory Virus tularensis, R. prowazekii, V. • Broad Fungal cholera, Y. pestis, E. coli O157:H7, S. flexneri, S. • Vector-borne Disease enterica • MDR TB • Pneumococcus Serotyping Viral agents: Variola virus, Ebola • C. Difficile virus, Influenza A virus H5N1, Venezuelan equine encephalitis virus 1 2011 release
20 Not For Use in Diagnostic Procedures PLEX-ID BROAD BACTERIA ASSAY PLEX-ID BROAD BACTERIA LO ASSAY
Not For Use in Diagnostic Procedures PLEX-ID Broad Bacteria / Broad Bacteria Lo Coverage and Targets
• Designed to detect & identify wide Coverage Target range of bacteria without a priori Broad Bacterial 16S rRNA (4) knowledge Broad Bacterial 23S rRNA (2) Broad Bacterial rpoC • Theoretically covers all major Gram + Firmicutes rplB (2) Bacteria, including intracellular Staphylococcus tufB organisms like Mycoplasma, speciation Chlamydia, and Rickettsia Gram+ Bacilli infB Bacillus speciation sspE
• Database currently contains more Gram- Enterobacteria valS than 5000 species base composition Gram- Gamma rpoB entries Proteobacteria Gram- rpoC Betaproteobacteria Gram- Beta and rpoB Gamma Proteobacteria
Not For Use in Diagnostic Procedures Broad Bacteria Assay layout
• 16 well assay with 16 16s rRNA Gram+ Fimicutes primer pairs per sample 16s rRNA Gram- Enterobacteria
• Unknown detections 16s rRNA Gram-Gammaproteobact. will be reported and linked to the closest 23s rRNA 23s rRNA match Gram+ Bacilli 16s rRNA
Broad Bacterial Gram-Betaproteobact.
Bacillus Gram-Beta/Gammaproteobact.
Gram+ Fimicutes Staphylococcus
Not For Use in Diagnostic Procedures Spectrum of one broad-range PCR well
Calibrant Peaks calculated as mean (A28C28G20T18) of the whole spectrum 16s rRNA primer 346 (from all stages of charge) Lactococcus 4000 lactis locus (A29C28G26T16) Lactococcus lactis locus 2000 (A30C29G26T14) Amplitude
0 28.5 30.25 31 molecular mass (kDa)
Not For Use in Diagnostic Procedures Multiple detections in one broad-range PCR well 16s rRNA primer Peaks calculated as mean 3000 from the whole spectrum S. aureus (from all stages of charge) (A30G29C30T29)
K. pneumoniae 1500 (A26G32C28T30) Amplitude
E. coli (A27G33C27T29)
0 35.6 36.1 36.6 molecular mass (kDa)
Not For Use in Diagnostic Procedures PCR/ESI-MS Broad Bacteria / Broad Bacteria Lo Data Usefulness of multilocus polymerase chain reaction followed by electrospray ionization mass spectrometry to identify a diverse panel of bacterial isolates* • 156 blinded bacterial isolates analyzed in the study – 142 (91%) were correctly identified to the genus level – 115 (74%) were correctly identified to the genus and species level – 14 (9%) isolates were misidentified • Misidentification generally due to missing database entries
“…Interestingly, based on the PCR/ESI-MS data, the system identified Campylobacter jejuni (ATCC 33560) as Streptococcus parasanguinis, which was confirmed by 16S rRNA PCR and sequencing. This is consistent with other data on several isolates obtained ATCC independently, suggesting at least certain lots of the bacteria could have been mislabeled.”
*Baldwin C, et al. Diagn Microbiol Infect Dis. 2009. 63(4): 403-408.
Not For Use in Diagnostic Procedures PCR/ESI-MS Broad Bacteria / Broad Bacteria Lo Data
Identification of Bacterial Plant Pathogens Using Multilocus Polymerase Chain Reaction/Electrospray Ionization-Mass Spectrometry* • 56 isolates were blinded – 52 (93%) of isolates were identified correctly to genus level – 41 (73%) of isolates were correctly identified to species and/or subspecies level – 4 (7%) of the isolates were incorrectly identified • 8 samples contained multiple bacterial species – 7 of these mixtures contained 3 species, 1 sample contained 5 species – In all, PCR/ESI-MS able to correctly identify most of mixture components to genus level (22 of 26 in total)
*Postinikova E, et al. Phytopathology. 2008. 98(11): 1156-64.
Not For Use in Diagnostic Procedures PCR/ESI-MS Broad Bacteria / Broad Bacteria Lo Data
*Postinikova E, et al. Phytopathology. 2008. 98(11): 1156-64.
Not For Use in Diagnostic Procedures PLEX-ID Broad Bacteria / Broad Bacteria Lo Summary • Detection and identification of bacterial species – BB for isolates, BB Lo for sensitive detection • Species unknown to the database will be detected and linked to the closest known species – Database contains more than 5000 species • Designed to characterize approximately 150 samples in 24 hours based on optimal workflow • You don’t need to know what you’re looking for!
Not For Use in Diagnostic Procedures PLEX-ID VECTOR-BORNE ORGANISMS
PLEX-ID Vector-Borne BPN ASSAY
Not For Use in Diagnostic Procedures PLEX-ID Vector-Borne BPN Assay
• Designed to detect and identify a wide range of microorganisms transmitted by vectors including ticks, mosquitoes and fleas • Coverage – Broad Bacterial with special focus on alphaproteobacteria and Spirochaetes: – Parasite: • Babesia genus – Nematode: • Dirofilaria immitis • Rapid results for a broad range of vector-borne microorganisms
Not For Use in Diagnostic Procedures Hard Ticks and Their Diseases Tick Species Diseases
Ixodes scapularis, •Lyme Disease Ixodes pacificus •Babesiosis Ixodes rincinus •Anaplasmosis
Dermacentor varabilus Dermacentor occidentalis •Tularemia Dermacentor andersonii
Amblyomma americanum •Ehrlichiosis
Not For Use in Diagnostic Procedures PLEX-ID Vector-Borne Organisms Plate Layout • 10 primer sets per sample plus control • Database covers 69 species including: – Bacteria • Francisella species such as F. tularensis and subspecies • Lyme disease Borrelia, such as B. burgdorferi, B. afzelii, B. valasiana, B. spielmanii, and B. lusitaniae and B. garinii • Rickettsia pathogens like R. rickettsii, R. typhi, and other non-pathogenic Rickettsia • Various alphaproteobacteria; Ehrlichia spp., Anaplasma phagocyophilum, Bartonella spp – Protozoa • Babesia protozoa such as B. microti, B. divergens, B. bovis,B. gibsoni, and B. canis
Not For Use in Diagnostic Procedures PLEX-ID Vector-Borne Organisms: Tested Organisms Anaplasma spp. Babesia spp. Coxiella spp. Rickettsia spp. Anaplasma phagocytophilum Babesia canis Coxiella endosymbiont Rickettsia rickettsii Anaplasma platys Babesia spp. coco Rickettsia typhi Babesia gibsoni Ehrlichia spp. Rickettsia conori Bartonella spp. Babesia odocoilei Ehrlichia canis Rickettsia prowazeki Bartonella alsatica Babesia divergens Ehrlichia chaffeensis Rickettsia ricksettii Bartonella arapensis Babesia duncani Ehrlichia ewingii Rickettsia australis Bartonella birtlesii Babesia microti Ehrlichia sp. panola mtn Rickettsia sibrica Bartonella bovis Rickettsia parkeri Bartonella chomelii Borrelia spp. Francisella spp. Rickettsia africae Bartonella doshiae Borrelia afzelii Francisella tularensis Rickettsia helvetica Bartonella elizabethae Borrelia burgdorferi Francisella endosymbiont Rickettsia felis Bartonella grahamii Borrelia garinii Rickettsia amblyommii Bartonella henselae Borrelia lonestari Leptospira spp. Rickettsia bellii Bartonella koehlerae Borrelia valaisiana Leptospira interregans Rickettsia endosymbiont Bartonella quintana Borrelia lusitaniae Leptospira biflexa Bartonella schoenbuchensis Borrelia spielmanii Leptospira borgpetersenii Dirofilaria immitis Bartonella tamiae Borrelia myamotoi Canine Heartworm Bartonella vinsonii Borrelia andersonii Borrelia parkeri Flaviviruses Borrelia hermsii Deer tick virus Borrelia coriaceae TBEV Borrelia turicatae JEV Borrelia bissettii Dengue virus Borrelia sp. LB-2001 Powassan virus
Not For Use in Diagnostic Procedures PLEX-ID Vector-Borne Assay: B. burgdorferi Screening for 299 Adult Ixodes Ticks from NY/CT
65% of ticks carried B. burgdorferi
No B. burgdorferi
106
B. burgdorferi 64.5%
193
Pilot version of Vector-Borne assay was used Crowder et al, 2010 PLOS One 5(5):e1065035
Not For Use in Diagnostic Procedures Additional Organisms: 299 Adult Ixodes Ticks From NY/CT
Many samples
Endosymbiont only showed two or three relevant microbes
B. burg +B. miya +B. odo B. burgdorferi B. burg +B. miya +B. mic B. burg +B. miya +A. phago B. burg +B. mic +B. odo B. burg +A. phago +Pow B. burg +B. mic +Pow B. burg +A. phago +B. mic B. miyamotoi +Powassan B. burg +B. miya B. burg +B. odo B. burg +Pow A. phagocytophilum B. burg +B. mic Powassan virus B. microti B. burg + A. phago B. odocoilei B. miyamotoi 36 Pilot version of Vector-BorneNot For Use assay in Diagnostic was used Procedures Organism Prevalence in 299 Adult Ixodes Ticks from NY/CT
• 193 Borrelia burgdorferi s.s. (64.5%) • 36 Anaplasma phagocytophilum (12.0%) • 22 Babesia microti (7.5%)* • 16 Powassan virus (5.6%)* • 11 Borrelia miyamotoi (3.7%) • 10 Babesia odocoilei (3.4%)*
200 ticks from NY (Connetquot/Shelter Island) and 99 ticks from CT (Bridgeport). *Due to insufficient sample volume, 15 ticks were not screened for Powassan virus and 6 ticks were not screened for Babesia species. Pilot version of Vector-Borne Assay was used
Not For Use in Diagnostic Procedures Borrelia Co-infections in US Ixodes Ticks
Borrelia/Babesia/Powassan 1% Borrelia/Anaplasma/Powassan Borrelia/Babesia/Anaplasma 1% 32% of Borrelia infected ticks 2% Borrelia/Powassan also contained another 4% relevant organism
Borrelia/Babesia 10%
Borrelia/Anaplasma 14%
Borrelia alone 68%
Pilot version of Vector-Borne assay was used 38
Not For Use in Diagnostic Procedures PLEX-ID Vector-Borne BPN Assay Summary
A single assay that is designed to: • Detect and identify Bacteria, Parasites (Babesia) and Nematodes associated with vectors such as ticks and mosquitoes • Detect endosymbionts associated with ticks and canine heartworm • Allow for analysis of direct specimens • Detect and characterize mixtures
Not For Use in Diagnostic Procedures PLEX-ID VECTOR-BORNE ORGANISMS
PLEX-ID Broad Viral 1 Assay
Not For Use in Diagnostic Procedures PLEX-ID Broad Viral I Assay
• Designed to detect and identify a range of viruses from several groups associated with disease in immunocompromised patients for research purposes
• Analysis can be performed directly from plasma or other biological specimens
Not For Use in Diagnostic Procedures PLEX-ID Broad Viral 1 Coverage
Not For Use in Diagnostic Procedures PLEX-ID Broad Viral I Assay
• Combining the power of nearly 20 single-target PCR tests – HSV-1 – HSV-2 – CMV – EBV – VZV PLEX-ID Broad – HHV-8 Viral I – BK virus – JC virus – Parvovirus B19 – Enterovirus A-D – Adenovirus A-F
Not For Use in Diagnostic Procedures PLX-ID Broad Viral I Assay
(8-well assay, partially multiplexed)
Assay detects: Herpesviruses Adenoviruses Parvovirus (PB19) Picornavirus (Enterovirus) Polyomaviruses
Sample: DNA & RNA from Plasma (and others)
Not For Use in Diagnostic Procedures PLEX-ID Broad Viral I Summary
• An assay that broadly detects members of several important and diverse viral groups from direct specimens – Combines many targeted tests into one • Allows for detection of mixtures and co-infected samples • Assay is high-throughput and rapid compared to viral culture which allows for a variety of research activities • Has the ability to detect newly emerging organisms within targeted groups – Pathogen discovery tool
Not For Use in Diagnostic Procedures PLEX-ID VECTOR-BORNE ORGANISMS
PLEX-ID Broad Fungal Assay
Not For Use in Diagnostic Procedures PLEX ID Broad Fungal Assay:
•Designed for the detection and identification of important yeasts and molds from culture isolates or samples
•Coverage of over 350 fungal families and identifies over 2000 fungal species
Wells Per Sample: 16
Primer Pairs Per Sample: 15
Samples Per Kit: 60
Input NA Volume: 10µl/well
Not For Use in Diagnostic Procedures Broad Fungal Assay coverage Achieved by broad-based rDNA and family specific primer pairs
Not For Use in Diagnostic Procedures Broad Fungal Assay Layout
Class Ascomycetes
Genus Fusarium Kingdom Fungi Kingdom Fungi
Order Mucorales
Genus Candida
Kingdom Fungi Genus Aspergillus
Genus Cryptococcus
Not For Use in Diagnostic Procedures PLEX-ID Broad Fungal Assay: Examples of Coverage
• Ajellomyces capsulatus • Penicillium marneffei (Histoplasma capsulatum) • Candida krusei • Aspergillus spp. • Candida parapsilosis • Aspergillus flavus • Candida tropicalis • Aspergillus fumigatus • Clavispora lusitaniae • Aspergillus • Coccidiodes immitis • Aspergillus terreus • Cryptococcus spp. • Bipolaris hawaiiensis • Cryptococcus neoformans • Blastomyces dermatitidis var. neoformans • Candida spp. • Cryptococcus neoformans • Candida albicans var. gattii • Candida dubliniensis • Exophiala dermatitidis • Candida glabrata • Pneumocystis carinii • Fusarium oxysporum • Pneumocystis jiroveci • Fusarium solani • Rhizopus spp.
Not For Use in Diagnostic Procedures PCR/ESI-MS Discrimination of Yeast Species
Gu et al 2011 Med Mycology early on-line: 1-7 Not For Use in Diagnostic Procedures PLEX-ID Broad Fungal Summary
• Broadly detects over 350 families of yeasts and molds – Identifies over 2000 species of fungi • Allows for detection of mixtures • Permits rapid species identification of fungi from sample
Not For Use in Diagnostic Procedures Current Applications for the Ibis Technology are Broad and Cross Multiple Industries
Biological Research Human ID Biosecurity Biopharma Other
Food Quality Microbial Detection Mito typing Biothreat Agent Quality Control detection & Food Safety & characterization STR profiling In Process testing identification Water safety testing
Not For Use in Diagnostic Procedures Ibis Technology Summary Molecular biology-based technology designed to: • Identify known and unknown microorganisms • Provide comprehensive microbial identification including bacteria, viruses, fungi and parasites. • Perform high resolution genotyping & subtyping • Determine drug resistance and virulence • Provide answers in less than one day
A Complete Solution for Broad Microbial Identification
Not For Use in Diagnostic Procedures Thank you for your time!
For more information, please contact: Greg Eppink [email protected] 419.345.8598
Not For Use in Diagnostic Procedures