Perspectives

Nature Reviews Microbiology | AOP, published online 3 June 2008; doi:10.1038/nrmicro1918 Perspectives

related species; facilitate outbreak investiga- I n n ovati o n tions and forensic analysis; facilitate rapid information sharing through a centralized Ibis T5000: a universal biosensor database; allow immediate testing of samples, as well as high-throughput testing; and approach for microbiology have low per-sample analysis costs. The technology that we describe in this article was designed to meet these David J. Ecker, Rangarajan Sampath, Christian Massire, Lawrence B. Blyn, specifications. We have coupled broad Thomas A. Hall, Mark W. Eshoo and Steven A. Hofstadler amplification by PCR with electrospray ionization mass spectrometry (ESI–MS) Abstract | We describe a new technology, the Ibis T5000, for the identification of in a system that we call the Ibis T5000. pathogens in clinical and environmental samples. The Ibis T5000 couples nucleic The basic principle of operation is shown acid amplification to high-performance electrospray ionization mass spectrometry in FIG. 1. In brief, multiple pairs of prim- and base-composition analysis. The system enables the identification and ers are used to amplify carefully selected quantification of a broad set of pathogens, including all known bacteria, all major regions of the genome of the microorganism of interest. Following amplification, groups of pathogenic fungi and the major families of viruses that cause disease in a fully automated ESI–MS analysis is humans and animals, along with the detection of virulence factors and antibiotic performed. The mass spectrometer effec- resistance markers. tively weighs the amplicons, or mixture of amplicons, with sufficient mass accuracy A walk into a clinical microbiology labora- health-care system. The CDC’s PulseNet that the composition of nucleotides (A, tory today can be like taking a step back programme (see Further information) for G, C and T) can be deduced for each in time: culture plates, incubators and the comparison of PFGE patterns perhaps amplicon present. The base composition microscopes dominate the ‘labscape’. It can presages the future of tracing and follow- is compared with a database of calculated take days to fully characterize the infectious ing accidental or intentional outbreaks of base compositions that is derived from the agents in a patient’s sample, beginning with infectious disease. However, PFGE is time sequences of known organisms to deter- culturing, followed by biochemical tests. consuming, requires a high level of skill mine the identities of any microorganisms Public-health laboratories also deal with the and the pattern results can vary between that are present. Thus, analysis using the detection and management of infectious laboratories. This demonstrates the primary Ibis T5000 provides detailed information diseases, but from the perspective of ben- hurdle that prevents rapid and reliable data which is analogous to that obtained using efiting society rather than the individual. sharing between clinical microbiology labo- a microarray or parallel DNA-sequencing Epidemiological investigations begin with ratories, public-health laboratories and bio- instrument. The Ibis T5000 mass spec- laborious methods, such as culturing, fol- defence agencies: the lack of a reproducible, trometer analyses each PCR reaction in lowed by pulse-field gel electrophoresis high-throughput, cost-effective technology less than 1 minute, using no consumable (PFGE) or sequencing. Government that provides digital signatures to detect and products, and is completely automated. We biodefence and homeland-security agencies identify microorganisms. have developed assays for use with the Ibis have a mandate to detect infectious disease The development of this type of technol- T5000 that enable the simultaneous iden- agents to protect against, or respond to, ogy is a daunting proposition. According to tification and quantification of all known acts of bioterrorism. The US government, literature reports, more than 1,400 species bacteria, all major groups of pathogenic for example, has built a large surveillance of microorganism are known to cause fungi and the major families of viruses infrastructure to monitor the environment disease in humans1. Moreover, each of that cause disease in humans. Here, we in major cities for the presence of biologi- these species can have hundreds of strain or describe how this technology can be used cal weapons that might be released in an genotypic variations with different proper- for pathogen detection for the benefit of aerosol attack. In these cities, individuals ties of virulence, transmissibility or drug individuals and society. with acute respiratory distress or fevers are resistance. The ideal diagnostic technol- treated in hospital emergency departments ogy to serve individuals, broad public- The Ibis T5000 universal biosensor and doctors’ offices. If an individual was health interests and biodefence should The mass spectrometer component of the infected with a microorganism of bioterror- provide universal pathogen-identification Ibis T5000 enables more information to ism significance, it would not be discovered capability; identify all organisms that are be extracted from PCR reactions than can be through the government’s efforts, as the present in a quantitative manner; identify obtained with standard individual probes. government’s environmental surveillance emerging, previously uncharacterized This enriched information extraction programme is not integrated into the organisms and determine the most closely occurs in two dimensions simultaneously.

9 Conserved Variable Conserved species, and primers for bacteria can be as RNase P, and housekeeping proteins . designed that encompass the entire bacte- For this strategy, it is essential that the rial domain of life. Second, a large amount broad-range primers flank regions of vari- Primer design of information is obtained from each indi- ability such that the base composition of vidual amplicon by mass spectrometry. The the resulting amplicon is information rich. mass spectrometer weighs each amplicon It would be ideal if an amplicon from a with sufficient accuracy that the composi- single pair of broad-range primers would tion of nucleotides (As, Gs, Cs and Ts) can provide sufficient information to identify PCR be unambiguously determined. Although all bacteria and determine the species but, not as information rich as the sequence because some bacterial species have the (the linking order is not determined using same base composition in conserved primer ESI–MS), for many diagnostic purposes, regions, a number of amplicons from dif- the nucleotide composition of a nucleic ferent regions of the microbial genome acid can have the same practical value. must be analysed. Primer choices are made For example, when a small set of primers based on their ability to provide maximal ESI–MS is strategically chosen, approximately six parsing using base composition as a metric. PCR reactions can yield sufficient infor- Microorganisms are distinguished using the mation to identify the bacteria that are aggregate information from the base com- present to the species level2. For viruses, position of a number of amplicons; we refer primers can be designed to encompass to this as triangulation. The resolving power broad genera, such as Alphaviruses3 or of base-composition analysis is compared Mastadenoviruses4, or even whole virus with that of sequencing in BOX 1. families, such as the Orthomyxoviridae5 Analysis of a sample using multiple broad 6 Base-composition or Coronaviridae . When primers are PCR reactions has several other important assignment designed to amplify all known members benefits. The first relates to coverage of within a target group, previously uncharac- diverse organisms. Broad-range primers terized members are also detected. This is designed to amplify DNA from all bacteria, a crucial advantage of the Ibis T5000 tech- even those targeted to ribosomal DNA, do nology relative to probe-based molecular not match all organisms perfectly and a methods, for which anticipation of the certain number of mismatches between the target nucleic acid sequence is required to PCR primers and some genomes is inevita- design the probe. ble. This was considered in the development Figure 1 | Flow scheme for Ibis T5000 analysis. of PCR conditions for the Ibis T5000, and Like any other methodology that uses PCR, Bacterial surveillance the conditions are permissive in the first few Nature Reviews | Microbiology the nucleic acids must first be purified from the Detection and identification of multiple cycles to ensure hybridization of a partially clinical or environmental sample. Target sites bacterial species in a clinical sample, such mismatched oligonucleotide primer to on microbial genomes that are common across as blood, cerebrospinal fluid or sputum, is the target. Great care is taken to match the broad groups of microorganisms and that flank challenging. The conventional molecular 3′ ends of the primers to the targets; this regions of high information content are approach is to design specific PCR primers region is the most sensitive to mispairings selected for primer design. Broad-range PCR is conducted and, following an automated desalting that target specific pathogens. However, during PCR initiation. However, priming step, amplified nucleic acids are electrosprayed this quickly becomes unwieldy because failures owing to mismatches between prim- into a time-of-flight (TOF) mass spectrometer of the large number of possible infecting ers and targets will occur for some organisms and the spectral signal is processed to identify pathogens. It is not possible to identify for any given primer pair. The use of multiple the masses of the amplicons that are present rare, emerging or unanticipated pathogens PCR reactions that are targeted to different with sufficient mass accuracy to unambiguously using this conventional method, because calculate the nucleotide base compositions the organism’s identity must be known Figure 2 | The bacterial domain of life present in each amplicon. The information from ▶ in advance to design the test. In the Ibis depicted using 373 sequenced species and all wells is used in a mathematical process that T5000 system, broad-range primers that can the coverage of 16 primer pairs. The colour is known as triangulation to convert the base- hybridize to virtually all bacterial genomes, coding depicts the specificity of the broad-range composition data to a list of the organisms that primers that are used in bacterial identification. are present and their relative and absolute rather than specific primers, are used. All the species shown are amplified by the six quantities. ESI–MS, electrospray ionization Broad-range priming is possible because ribosomal-DNA-targeted primers (346, 347, 348, mass spectrometry. all bacteria share a number of common nucleic acid sequences that are of sufficient 349, 360 and 361); exceptions are shown boxed length to support the binding of PCR prim- by a red dashed line, along with an indication of the actual subset of primer pairs that is predicted First, a large number of PCR amplicons can ers. These broad-range primers are used to yield amplicons. Other colours depict cover- be analysed. This enables the use of PCR to amplify a product from all bacteria or age of the division- or group-specific primers primers that amplify groups of organisms in groups of phylogenetically related bacteria that target genes which encode conserved mixed populations, rather than single spe- (FIG. 2). The most notable region for broad housekeeping genes. For clarity, diverse cies. For example, primers for viruses can be priming is ribosomal DNA7,8, but there are branches that are covered by ribosomal primer designed to encompass entire viral families also broad-priming opportunities in genes pairs only (the Aquificae, Thermotogae and that comprise hundreds of characterized that encode other structural RNAs, such Chloroflexi phyla) are not represented.

Rhizobium spp. (2) Caulobacter crescentus Agrobacterium tumefaciens Escherichia coli Hyphomonas neptunium Sinorhizobium spp. (2) Klebsiella pneumoniae Mesorhizobium spp. (2) Maricaulis maris Pasteurella multocida Salmonella spp. (4) Ochrobactrum anthropi Actinobacillus spp. (3) Shigella spp. (4) Nitrobacter spp. (2) Rhodobacter spp. (2) Haemophilus spp. (3) Yersinia pestis Parvibaculum lavamentivorans Paracoccus denitrificans Mannheimia spp. (2) Other Yersinia spp. (2) Silicibacter spp. (2) Brucella spp. (4) Enterobacter spp. (2) Jannaschia sp. Citrobacter koseri Bartonella spp. (3) Vibrio spp. (5) Roseobacter denitrificans Erwinia spp. (2) Bradyrhizobium spp. (3) Photobacterium Serratia spp. (2) Sphingomonas wittichii Xanthobacter autotrophicus profundum Miscellaneous symbionts (6) Novosphingobium sp. Rhodopseudomonas palustris Sphingopyxis alaskensis Aeromonas spp. (2) Colwellia psychrerythraea Erythrobacter litoralis Acidiphilium cryptum Idiomarina loihiensis Zymomonas mobilis Rhodospirillum rubrum Marinobacter aquaeolei Magnetospirillum spp. (2) Pseudomonas spp. (7) Pseudoalteromonas spp. (2) Bacteroides Psychromonas ingrahamii Bacteroides spp. (3) Gluconobacter oxydans Psychrobacter spp. (3) Saccharophagus degradans Parabacteroides distasonis Granulibacter bethesdensis Acinetobacter spp. (2) Shewanella spp. (12) Prevotella intermedia Porphyromonas gingivalis Legionella pneumophila Nitrosococcus oceani Rickettsia spp. (8) Coxiella burnetti Halorhodospira halophila Sphingobacteria Anaplasma spp. (2) Alkalilimnicola ehrlicheii Ehrlichia spp. (3) Flavobacteria Cytophaga hutchinsonii Not 349 Salinibacter ruber Neorickettsia sennetsu Flavobacterium spp. (2) Orientia tsutsugamushi Francisella tularensis Gramella forsetii Miscellaneous symbionts (3) Thiomicrospira crunogena Chlamydiae Gamma Xanthomonas spp. (3) Alpha Chlorobia Chlamydia spp. (2) Xylella fastidiosa Chlorobium spp. (3) Chlamydophila spp. (4) Pelodictyon luteolum Parachlamydia sp. Prosthecochloris vibrioformis Not 347, 360 Nitrosomonas spp. (2) Not 347 Beta Proteobacteria Thiobacillus denitrificans Methylobacillus flagellatus Spirochaetes Azoarcus spp. (2) Borrelia spp. (3) Dechloromonas aromatica Treponema spp. (2) Delta Leptospira spp. (2) Epsilon Chromobacterium violaceum Neisseria spp. (2) Desulfovibrio spp. (2) Burkholderia spp. (8) Cyanobacteria Campylobacter spp. (5) Lawsonia intracellularis Anabaena variabilis Desulfotalea psychrophila Bordetella spp. (4) Nostoc spp. (2) Helicobacter spp. (3) Bdellovibrio bacteriovorus Polynucleobacter sp. Trichodesmium erythraeum Wolinella succinogenes Bacteriovorax marinus Ralstonia spp. (3) Acidovorax spp. (2) Synechococcus spp. (9) Nitratiruptor sp. Anaeromyxobacter spp. (2) Herminiimonas arsenicoxydans Synechocystis sp. Sulfurovum sp. Myxococcus xanthus Janthinobacterium sp. Thermosynechococcus elongatus Not 346 Prochlorococcus marinus Pelobacter spp. (2) Methylobium petroleiphilum Gloeobacter violaceus Fusobacteria Geobacter spp. (3) Polaromonas spp. (2) Syntrophobacter fumaroxidans Rhodoferax ferrireducens Fusobacterium nucleatum Syntrophus aciditrophicus Verminephrobacter eiseniae Actinobacteria Not 346 Mycobacterium spp. (11) Acidothermus cellulolyticus Clostridium difficile Arthrobacter spp. (2) Clostridium botulinum Bacillus anthracis Bacillus thuringiensis Corynebacterium spp. (4) Other Clostridium spp. (7) Bacillus cereus Nocardia farcinica Misc. spp. (9) Bacillus spp. (5) Rhodococcus sp. Clostridia Broad coverage with ribosomal primer Streptomyces spp. (2) Staphylococcus spp. (4) Firmicutes pairs 346, 347, 348, 349, 360 and 361 Propionibacterium acnes Listeria spp. (3) unless otherwise indicated Bacilli Oceanobacillus iheyensis Nocardioides sp. Colour key for the primer pairs that target Thermobifida fusca Geobacillus spp. (3) housekeeping genes Saccharopolyspora erythraea Salinispora tropica Mollicutes Primer 352 Primer 359 Kineococcus radiotolerans Frankia spp. (2) Mycoplasma spp. (12) Primer 354 Primer 362 Ureaplasma parvum Streptococcus spp. (9) Clavibacter michiganensis Primer 355 Primer 363 Leifsonia xyli Mesoplasma florum Lactococcus lactis Tropheryma whipplei Spiroplasma kunkelii Pediococcus pentosaceus Primer 356 Primer 367 Candidatus Phytoplasma spp. (2) Symbiobacterium thermophilum Enterococcus faecalis Primer 358 Primer 449 Rubrobacter xylanophilus Not 347 and 348 Lactobacillus spp. (10) Bifidobacterium spp. (2) Leuconostoc mesenteroides Not 349 Oenococcus oeni

Box 1 | Comparison of the resolving power of base-composition and sequence analysis more than 100 to 1, the lower abundance in bacteria microorganisms might not be detected because they exceed the dynamic range of the competitive PCR reactions. However, Unresolved species when two different microorganisms are Burkholderia mallei and Burkholderia pseudomallei individually amplified by non-overlapping Bordetella bronchiseptica and Bordetella parapertussis Brucella melitensis, Brucella abortus, Brucella suis and Brucella ovis division-wide primers, the microorganisms Rickettsia conorii and Rickettsia sibirica do not compete with each other, because Mycobacterium sp. ALS, Mycobacterium sp. KMS and Mycobacterium sp. MCS they are amplified in different PCR reac- Shewanella putrefaciens and Shewanella sp. W3-75-3 tions. The mass spectral information from Shewanella sp. ANA-3 and Shewanella sp. MR-7 several reactions is used to triangulate Dehalococcoides sp. BAV1 and Dehalococcoides sp. CBDB1 the identities of the organisms that are present. Notably, using the triangulation 405 species 400 384 386 species strategy, none of the primers is designed to Sequence 360 be specific for any one microorganism, but 354 Listeria inocua and instead the primers are designed to cover the bacterial tree of life using a redundant, 300 Listeria monocytogenes nested-coverage approach. This enables Base composition Mycobacterium tuberculosis identification of any bacterial species, even and Mycobacterium bovis previously unknown organisms, with a

terized species terized 200 single test. ac Yersinia pestis and Yersinia pseudotuberculosis An example of a broad-range primer Shigella boydii, Shigella sonnei and Escherichia coli analysis of a complex mixture of microorganisms is shown in FIG. 3. The spectrum 100 Bacillus anthracis and Bacillus thuringiensis was obtained by using a ribosomal-DNA- Number of char Burkholderia cepacia and Burkholderia cenocepacia targeted primer pair for PCR of a sputum sample from a patient with cystic fibrosis. Spectral signals from six distinct microor- 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 ganisms were present and microorganism Number of primer pairs identification was based on triangulation 361 348 360 347 349 346 354 358 362 449 363 352 359 367 356 355 of the signals from eight broad-range 16S 16S 23S 16S 23S 16S rpoC valS rpoB rplB rpoC infB rpoB tufB rplB sspE primers. Use of integrated information Ribosomal primers Housekeeping-gene primers from multiple broad-range primers allows Additional primer-pair identification and gene location the identification of virtually all organisms that are present in a sample. Crucially, the time to identification is 4–6 hours, and We analysed the resolution of 405 unique bacterial species that was provided by the regions amplified by the 16 primer pairs that are used for bacterial surveillance2 (see the figure). Six of these therefore appropriate action can begin loci are found within the ribosomal subunits and are expected to yield ampliconsNature Re fromviews virtually | Microbiolog all y promptly. bacteria. By contrast, the ten primer pairs that target essential housekeeping genes typically cover distinct phyla (FIG. 2) and provide additional resolution within the densest parts of the bacterial Bacterial genotyping tree. We first used a computational simulation of PCR to extract the representative sequence and The Ibis T5000 method identifies micro- base composition (if any) from each loci for each of the 405 unique, completely sequenced (by organisms by measuring the base com- September 2007) bacterial species (see Further information for a link to complete microbial position of selected genomic regions and genomes from the NCBI database). Thus, we associated each species with a unique 16-loci comparing these digital fingerprints with a signature. As the genomes of some species are not amplified by certain primer pairs, on average, database of microorganism signatures that there were 7.2 loci with base-composition and sequence signatures. We then determined the are derived from known sequences. These number of species that were unambiguously identified by either sequence or base-composition analysis using a variable number of these loci, starting with the six ribosomal loci and adding one digital signatures provide a powerful means locus at a time. Primer pairs were initially sorted by decreasing breadth of coverage, starting with to identify microorganisms; digitization the ribosomal primer pairs (in blue). The resolution of base-composition analysis approximates that also enables the technology to be applied to of sequence analysis after the addition of the third primer pair, leaving only six extra pairs of closely outbreak investigations, forensic analyses, related organisms that are not separated; this difference disappears after the addition of the ninth epidemiological tracking and real-time primer pair, and neither base-composition nor sequence analysis of the selected genomic regions integration of data from various locations. distinguishes the remaining 19 closely related species. Because regions of high variability in the microbial genomes are amplified, a certain amount of intra-species variability is often regions of the microbial genome provides an extremely broad coverage, such as that observed. For example, primer pairs tar- inherent redundancy that protects against provided by the ribosomal-DNA-targeted geted to variable regions of housekeeping some organisms being missed owing to mis- primers, the microorganisms that are genes that are common to all bacteria will matches from single primer pairs. present in a sample compete during the yield a set of base compositions that vary Triangulation also increases the amplification process. When organisms slightly — usually one or two mutations dynamic range of detection limits. With differ in concentration by a ratio of in a few of the target sites — in different

strains of the same species. These varia- a Calibrant tions provide distinguishing fingerprints that can be used to track the spread of microorganisms in a hospital setting or follow an epidemiological trail. However, ce to use the Ibis T5000 technology specifi- cally for epidemiology, a set of genus- or e abundan iv

species-specific primer pairs should be at designed that target genomic regions that Rel are optimized to distinguish strains within the same species. Genes are selected that provide two levels of evolutionary ‘clock speed’ for strain genotyping. The first are the housekeeping genes that are used in 31094.78 31414.19 31733.60 32053.00 32372.41 32691.82 33011.22 33330.63 33650.03 33969.44 34288.85 Mass (Da) multilocus sequence typing (MLST)10 and the second are species-specific variable b 1a. A23, G22, C31 and T32 1b. A32, G31, C22 and T23 Gemella haemolysans number of tandem repeats (VNTRs)11. 2a. A24, G25, C30 and T29 2b. A29, G30, C25 and T24 Staphylococcus aureus 3a. A24, G26, C30 and T28 3b. A28, G30, C26 and T24 Streptococcus sp. MLST and VNTR analyses are convention- 4a. A20, G29, C33 and T27 4b. A27, G33, C29 and T20 Pseudomonas aeruginosa ally conducted using PCR, followed by 5a. A22, G26, C37 and T25 5b. A25, G37, C26 and T22 Arthrobacter chlorophenolicus 6a A23, G26, C36 and T25 6b. A25, G36, C26 and T23 Stomatococcus mucilaginosus sequencing or gel electrophoresis, but both types of targets can be analysed using 3a e 3b the high-throughput Ibis T5000. This approach has been used successfully to track a group A Streptococcus outbreak in a 2 e abundanc military training camp and to investigate iv

at 1b 6b

the source of an outbreak of Acinetobacter Rel 12,13 1a 2b 6a spp. in hospital settings . 2a 5b 4a 4b 5a Virus identification and genotyping The Ibis T5000 is also useful for identify- ing and tracking viruses. Despite higher mutation rates and greater sequence 32890.08 33035.09 33180.09 33325.10 33470.10 33615.10 33760.11 33905.11 34050.12 34195.12 34340.12 variability than bacteria, conserved Mass (Da) primer target sites can be identified that Figure 3 | The spectrum of amplicons obtained from a sputum sampleNa fromture Re avie patientws | Microbiology with enable priming of entire genera or even cystic fibrosis using a primer pair that targets ribosomal DNA. a | Shows a broad mass range that includes each strand of the internal calibration standard. b | Shows an expanded view of the spectral complete virus families. RNA-dependent region that excludes the calibration standard. Each amplified product has two peaks that correspond RNA polymerase is a housekeeping gene to the two strands of the amplicon. The organisms that are assigned to each peak are based on the that is common to all RNA viruses and collective interpretation from eight broad-range primers. provides several target-site opportunities for developing primers that amplify multiple species within a virus family. This Microorganism quantitation calibrated and the average over all PCR strategy is powerful because a single PCR Quantitation in the Ibis T5000 process reactions is reported for each sample9. The reaction analysed by mass spectrometry is achieved by using an internal calibra- calibration standard also serves as an internal can be used to detect and identify tens tion standard. A nucleic acid with a target positive control. If the calibration standard to hundreds of related viral species. As sequence that is similar to the primer target is not amplified, something in the particular shown in FIG. 4, despite the inherent vari- sites is added to each PCR reaction at a pre- sample might be inhibitory or the PCR might ation of base composition that is found in cisely known concentration. The calibration have failed for some other reason. Thus, the different flavivirus entries in GenBank, standard is amplified along with the microbial calibration standard is valuable as a qual- which generates a ‘cloud’ of base composi- nucleic acids that are present. The amplicon ity-control check for PCR inhibition and the tions for each species, each species can be that is generated from the calibration stand- subsequent steps in mass spectrometry and distinguished. Because at least two sets of ard contains a small deletion that unambigu- signal analysis. primer pairs targeted to different regions ously distinguishes it from the amplified of the viral genome are generally used microorganism nucleic acid in the mass spec- High-information-content diagnostics for each virus group, potential misclas- trum (FIG. 3a). Comparison of spectral peak Key to the use of high-information-content sification is avoided, as two regions taken heights from the calibration standard and tests in a diagnostic setting are practical together provide unambiguous species the amplified microorganism nucleic acids considerations, such as the complexity and identification. This strategy has been enables determination of the concentration. the skill set that are required to run the assay, used effectively for broad detection and This internal calibration method is accurate, costs, throughput and interpretation of strain typing of adenoviruses4, influenza within fourfold of the actual concentration, in complex data. Although several current tech- viruses5, alphaviruses3, coronaviruses6 and part because each sample is analysed in multi- nologies, including microarrays and parallel orthopoxviruses9. ple PCR reactions that are all independently sequencing methods, can theoretically

C including bioterrorist agents, rather than only 34 36 38 40 42 44 Size by T routinely observed infectious agents. We sug- 40 38 36 46 48 46 44 42 19 30 gest that the infectious disease community 48 48 should consider the impact of integrating the 28 various communities of stakeholders who 28 are concerned with infectious microorgan- 26 isms. The Ibis T5000 technology described 26 24 here is one diagnostic method that has been 24 designed to achieve this objective. 22 22 Dengue virus 1 Ibis Biosciences, 1896 Rutherford Road, Carlsbad, 20 California 92008, USA. 20 Dengue virus 2 Correspondence to D.J.E. 18 18 Dengue virus 3 e‑mail: [email protected] 16 16 Dengue virus 4 doi:10.1038/nrmicro1918 A 36 34 G Published online 3 June 2008 36 Japanese encephalitis 38 38 Tick-borne encephalitis 1. Taylor, L. H., Latham, S. M. & Woolhouse, M. E. Risk 40 40 factors for human disease emergence. Philos. Trans. 42 West Nile virus R. Soc. Lond. B 356, 983–989 (2001). 42 2. Ecker, D. J. et al. Rapid identification and strain- 44 44 Yellow fever virus typing of respiratory pathogens for epidemic 46 surveillance. Proc. Natl Acad. Sci. USA 102, 8012–8017 (2005). Figure 4 | Base-composition analysis of flaviviruses. The base composition of a variable fragment 3. Eshoo, M. W. et al. Direct broad-range detection of Nature Reviews | Microbiology alphaviruses in mosquito extracts. Virology 368, of the RNA-dependent RNA polymerase gene flanked by conserved primer target sites is depicted in 286–295 (2007). three dimensions for the flavivirus sequences in GenBank. The region depicted here corresponds to 4. Blyn, L. B. et al. Rapid detection and molecular coordinates 9,050–9,150 of Dengue virus 1 (GenBank accession code DQ285559). serotyping of adenovirus by use of PCR followed by electrospray ionization mass spectrometry. J. Clin. Microbiol. 46, 644–651 (2008). 5. Sampath, R. et al. Global surveillance of emerging provide detailed information about the Applications and conclusions influenza virus genotypes by mass spectrometry. PLoS ONE 2, e489 (2007). infectious disease agents that are present in Infectious diseases are, by definition, com- 6. Sampath, R. et al. Rapid identification of emerging a sample, these methods are currently too municable, and yet virtually all infections in pathogens: coronavirus. Emerg. Infect. Dis. 11, 373–379 (2005). complex, slow and expensive for routine individual patients are diagnosed and treated 7. Nikkari, S. et al. Broad-range bacterial detection and use in a diagnostic laboratory. The Ibis in isolation. What might be learned about the the analysis of unexplained death and critical illness. Emerg. Infect. Dis. 8, 188–194 (2002). T5000 was designed for high-throughput virulence of a microorganism or its response 8. Kroes, I., Lepp, P. W. & Relman, D. A. Bacterial operation in a clinical laboratory setting to selected therapies is not easily transferred to diversity within the human subgingival crevice. Proc. Natl Acad. Sci. USA 96, 14547–14552 (1999). with consumable costs that are comparable aid the treatment of patients who become 9. Hofstadler, S. A. et al. TIGER: the universal biosensor. to current PCR-based molecular diagnostic sequentially infected with the same clonal Int. J. Mass Spectrom. 242, 23–41 (2005). 10. Enright, M. C. & Spratt, B. G. Multilocus sequence methods. The mass spectrometer uses no strain. For example, two paediatricians who typing. Trends Microbiol. 7, 482–487 (1999). consumable products, and therefore the total work in the same medical complex are likely 11. Johansson, A. et al. Worldwide genetic relationships among Francisella tularensis isolates determined by costs per sample for Ibis T5000 analysis are to see children who have been infected by the multiple-locus variable-number tandem repeat comparable to the costs of standard molecular same pathogen, but have no way of knowing analysis. J. Bacteriol. 186, 5808–5818 (2004). 12. Hujer, K. M. et al. Analysis of antibiotic resistance diagnostic tests. Samples are analysed at a rate it. The microorganism in question might genes in multidrug-resistant Acinetobacter sp. isolates of approximately 30 seconds for each PCR be making its way across a country, having from military and civilian patients treated at the Walter Reed Army Medical Center. Antimicrob. Agents reaction, and the software interprets spectral been treated successfully or unsuccessfully by Chemother. 50, 4114–4123 (2006). results, matches signatures with a database other physicians weeks or months earlier in 13. Ecker, J. A. et al. Identification of Acinetobacter species and genotyping of Acinetobacter baumannii by and reports the bacterial, viral or fungal iden- adjacent states, provinces or countries. The multilocus PCR and mass spectrometry. J. Clin. tification, along with accessory information, technology described here has the potential Microbiol. 44, 2921–2932 (2006). such as high-resolution genotype, virulence to integrate infectious disease identification Acknowledgements factors or antibiotic resistance markers, and treatment across time and distance. As The authors acknowledge the government agencies that sup- ported the development of the Ibis T5000 universal biosen- according to the selected assay configuration. the Ibis T5000 provides digital signatures of sor technology, including the Defense Advanced Research The main drawback to the current Ibis T5000 identified microorganisms, this technology Projects Agency (DARPA), the Centers for Disease Control instrument is its high capital cost, which is allows the collection and dissemination and Prevention, the National Institutes of Health and the Department of Homeland Security. driven by the cost of a high-performance of epidemiological information in real mass spectrometer. However, equipment costs time. The microbial signatures, along with The authors declare competing financial interests: see web version for details. can be recovered over time if large numbers previous treatment information on that of samples are evaluated, which would enable pathogen, can be shared electronically. DATABASES Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query. production-scale economy. Moreover, for New signatures can be recorded in a central fcgi?db=gene high-throughput applications in a clinical database and the appearance of those signa- 9,050–9,150 of Dengue virus 1 laboratory, equipment costs are amortized tures in new locations will enable real-time FURTHER INFORMATION over large numbers of samples, making epidemiological analysis by public-health ibis Biosciences: http://www.ibisbioscien m/ Complete microbial genome sequences: http://www.ncbi. PCR reagents the dominant cost driver. In officials. Likewise, integration of biodefence nlm.nih.gov/genomes/lproks.cgi addition, mass-spectrometry technology surveillance with routine clinical diagnostics Genbank: http://www.ncbi.nlm.nih.gov/Genbank/index.html PulseNet: http://www.cdc.gov/pulsenet/ continues to evolve, with yearly performance is facilitated by the use of technology that All links are active in the online pdf increases and cost reductions. identifies any organism that infects patients,