Rapid Identification of Emerging Pathogens: Coronavirus

New surveillance approach can analyze >900 polymerase chain reactions per day

Global Surveillance of Emerging Influenza Virus Genotypes by Mass Spectrometry

Effective influenza surveillance requires new methods capable of rapid and inexpensive genomic analysis of evolving viral species for pandemic preparedness, to understand the evolution of circulating viral species, and for vaccine strain selection. We have developed one such approach based on previously described broad-range reverse transcription PCR/electrospray ionization mass spectrometry (RT-PCR/ESI-MS) technology.Analysis of base compositions of RT-PCR amplicons from influenza core gene segments (PB1, PB2, PA, M, NS, NP) are used to provide sub-species identification and infer influenza virus H and N subtypes. Using this approach, we detected and correctly identified 92 mammalian and avian influenza isolates, representing 30 different H and N types, including 29 avian H5N1 isolates. Further, direct analysis of 656 human clinical respiratory specimens collected over a seven-year period (1999-2006) showed correct identification of the viral species and subtypes with >97% sensitivity and specificity. Base composition derived clusters inferred from this analysis showed 100% concordance to previously established clades. Ongoing surveillance of samples from the recent influenza virus seasons (2005-2006) showed evidence for emergence and establishment of new genotypes of circulating H3N2 strains worldwide. Mixed viral quasispecies were found in approximately 1% of these recent samples providing a view into viral evolution.Thus, rapid RT-PCR/ESI-MS analysis can be used to simultaneously identify all species of influenza viruses with clade-level resolution, identify mixed viral populations and monitor global spread and emergence of novel viral genotypes. This high-throughput method promises to become an integral component of influenza surveillance

Mass spectrometry provides accurate characterization of two genetic marker types in Bacillus anthracis

Epidemiological and forensic analyses of bioterrorism events involving Bacillus anthracis could be improved if both variable number tandem repeats (VNTRs) and single nucleotide polymorphisms (SNPs) could be combined on a single analysis platform. Here we present the use of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) to characterize 24 alleles from 6 VNTR loci and 11 alleles from 7 SNP loci in B. anthracis. The results obtained with ESI-FTICRMS were consistent with independent results obtained from traditional approaches using electrophoretic detection of fluorescent products. However, ESI-FTICR-MS improves on the traditional approaches because it does not require fluorescent labeling of PCR products, minimizes post-PCR processing, obviates electrophoresis, and provides unambiguous base composition of both SNP and VNTR PCR products. In addition, ESI-FTICR-MS allows both marker types to be examined simultaneously and at a rate of approximately 1 sample per min. This technology represents a significant advance in our ability to rapidly characterize B. anthracis isolates using VNTR and SNP loci

The Ibis T5000 Universal Biosensor: An Automated Platform for Pathogen Identification and Strain Typing

We describe a new approach to the sensitive and specific identification of bacteria, viruses, fungi, and protozoa based on broad-range PCR and high-performance mass spectrometry. The Ibis T5000 is based on technology developed for the Department of Defense known as T.I.G.E.R. (Triangulation Identification for the Genetic Evaluation of Risks) for pathogen surveillance. The technology uses mass spectrometry—derived base composition signatures obtained from PCR amplification of broadly conserved regions of the pathogen genomes to identify most organisms present in a sample. The process of sample analysis has been automated using a combination of commercially available and custom instrumentation. A software system known as T-Track manages the sample flow, signal analysis, and data interpretation and provides simplified result reports to the user. No specialized expertise is required to use the instrumentation. In addition to pathogen surveillance, the Ibis T5000 is being applied to reducing health care—associated infections (HAIs), emerging and pandemic disease surveillance, human forensics analysis, and pharmaceutical product and food safety, and will be used eventually in human infectious disease diagnosis. In this review, we describe the automated Ibis T5000 instrument and provide examples of how it is used in HAI control