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

Identification of Pathogenic Vibrio Species by Multilocus PCR-Electrospray Ionization Mass Spectrometry and Its Application to Aquatic Environments of the Former Soviet Republic of Georgia▿ †

The Ibis T5000 is a novel diagnostic platform that couples PCR and mass spectrometry. In this study, we developed an assay that can identify all known pathogenic Vibrio species and field-tested it using natural water samples from both freshwater lakes and the Georgian coastal zone of the Black Sea. Of the 278 total water samples screened, 9 different Vibrio species were detected, 114 (41%) samples were positive for V. cholerae, and 5 (0.8%) samples were positive for the cholera toxin A gene (ctxA). All ctxA-positive samples were from two freshwater lakes, and no ctxA-positive samples from any of the Black Sea sites were detected

Pathogen Profiling: Rapid Molecular Characterization of Staphylococcus aureus by PCR/Electrospray Ionization-Mass Spectrometry and Correlation with Phenotype▿

There are few diagnostic methods that readily distinguish among community-acquired methicillin (meticillin)-resistant Staphylococcus aureus strains, now frequently transmitted within hospitals. We describe a rapid and high-throughput method for bacterial profiling of staphylococcal isolates. The method couples PCR to electrospray ionization-mass spectrometry (ESI-MS) and is performed on a platform suitable for use in a diagnostic laboratory. This profiling technology produces a high-resolution genetic signature indicative of the presence of specific genetic elements that represent distinctive phenotypic features. The PCR/ESI-MS signature accurately identified genotypic determinants consistent with phenotypic traits in well-characterized reference and clinical isolates of S. aureus. Molecular identification of the antibiotic resistance genes correlated strongly with phenotypic in vitro resistance. The identification of toxin genes correlated with independent PCR analyses for the toxin genes. Finally, isolates were correctly classified into genotypic groups that correlated with genetic clonal complexes, repetitive-element-based PCR patterns, or pulsed-field gel electrophoresis types. The high-throughput PCR/ESI-MS assay should improve clinical management of staphylococcal infections

Rapid Molecular Genotyping and Clonal Complex Assignment of Staphylococcus aureus Isolates by PCR Coupled to Electrospray Ionization-Mass Spectrometry▿

We describe a high-throughput assay using PCR coupled to electrospray ionization-mass spectrometry (PCR/ESI-MS) to determine the genotypes of Staphylococcus aureus isolates. The primer sets used in the PCR/ESI-MS assay were designed to amplify the same genes analyzed in multilocus sequence typing (MLST). The method was used to identify the clonal complex and USA type of each isolate and is suitable for use in a clinical or public-health setting. The method was validated using a panel of diverse isolates from the Centers for Disease Control and Prevention that were previously characterized by MLST and pulsed-field gel electrophoresis (PFGE). Clinical isolates from two geographically distinct hospitals were characterized, and the clustering results were in agreement with those for repetitive-element PCR and PFGE. The PCR/ESI-MS method enables genotyping of over 180 samples of S. aureus per day in an automated fashion

Identification of Acinetobacter Species and Genotyping of Acinetobacter baumannii by Multilocus PCR and Mass Spectrometry

Members of the genus Acinetobacter are ubiquitous in soil and water and are an important cause of nosocomial infections. A rapid method is needed to genotype Acinetobacter isolates to determine epidemiology and clonality during infectious outbreaks. Multilocus PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) is a method that uses the amplicon base compositions to genotype bacterial species. In order to identify regions of the Acinetobacter genome useful for this method, we sequenced regions of six housekeeping genes (trpE, adk, efp, mutY, fumC, and ppa) from 267 isolates of Acinetobacter. Isolates were collected from infected and colonized soldiers and civilians involved in an outbreak in the military health care system associated with the conflict in Iraq, from previously characterized outbreaks in European hospitals, and from culture collections. Most of the isolates from the Iraqi conflict were Acinetobacter baumannii (189 of 216 isolates). Among these, 111 isolates had genotypes identical or very similar to those associated with well-characterized A. baumannii isolates from European hospitals. Twenty-seven isolates from the conflict were found to have genotypes representing different Acinetobacter species, including 8 representatives of Acinetobacter genomospecies 13TU and 13 representatives of Acinetobacter genomospecies 3. Analysis by the PCR/ESI-MS method using nine primer pairs targeting the most information-rich regions of the trpE, adk, mutY, fumC, and ppa genes distinguished 47 of the 48 A. baumannii genotypes identified by sequencing and identified at the species level at least 18 Acinetobacter species. Results obtained with our genotyping method were essentially in agreement with those obtained by pulse-field gel electrophoresis analysis. The PCR/ESI-MS genotyping method required 4 h of analysis time to first answer with additional samples subsequently analyzed every 10 min. This rapid analysis allows tracking of transmission for the implementation of appropriate infection control measures on a time scale previously not achievable

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