The IRIDICA BAC BSI Assay: Rapid, Sensitive and Culture-Independent Identification of Bacteria and Candida in Blood.

Unlabelled: Bloodstream infection (BSI) and sepsis are rising in incidence throughout the developed world. The spread of multi-drug resistant organisms presents increasing challenges to treatment. Surviving BSI is dependent on rapid and accurate identification of causal organisms, and timely application of appropriate antibiotics.

Analytical characterization of an assay designed to detect and identify diverse agents of disseminated viral infection.

Background: Diverse viruses often reactivate in or infect cancer patients, patients with immunocompromising infections or genetic conditions, and transplant recipients undergoing immunosuppressive therapy. These infections can disseminate, leading to death, transplant rejection, and other severe outcomes.

Objectives: To develop and characterize an assay capable of inclusive and accurate identification of diverse potentially disseminating viruses directly from plasma specimens.

Comparison of base composition analysis and Sanger sequencing of mitochondrial DNA for four U.S. population groups.

A set of 711 samples from four U.S. population groups was analyzed using a novel mass spectrometry based method for mitochondrial DNA (mtDNA) base composition profiling.

Broad-spectrum biosensor capable of detecting and identifying diverse bacterial and Candida species in blood.

We describe an assay which uses broad-spectrum, conserved-site PCR paired with mass spectrometry analysis of amplicons (PCR/electrospray ionization-mass spectrometry [ESI-MS]) to detect and identify diverse bacterial and Candida species in uncultured specimens. The performance of the assay was characterized using whole-blood samples spiked with low titers of 64 bacterial species and 6 Candida species representing the breadth of coverage of the assay. The assay had an average limit of detection of 100 CFU of bacteria or Candida per milliliter of blood, and all species tested yielded limits of detection between 20 and 500 CFU per milliliter.

PCR followed by electrospray ionization mass spectrometry for broad-range identification of fungal pathogens.

Invasive fungal infections are a significant cause of morbidity and mortality among immunocompromised patients. Early and accurate identification of these pathogens is central to direct therapy and to improve overall outcome. PCR coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) was evaluated as a novel means for identification of fungal pathogens.

Comprehensive biothreat cluster identification by PCR/electrospray-ionization mass spectrometry.

Technology for comprehensive identification of biothreats in environmental and clinical specimens is needed to protect citizens in the case of a biological attack. This is a challenge because there are dozens of bacterial and viral species that might be used in a biological attack and many have closely related near-neighbor organisms that are harmless. The biothreat agent, along with its near neighbors, can be thought of as a biothreat cluster or a biocluster for short.

Automated analysis of sequence polymorphism in STR alleles by PCR and direct electrospray ionization mass spectrometry.

Short tandem repeats (STRs) are the primary genetic markers used for the analysis of biological samples in forensic and human identity testing. The discrimination power of a combination of STRs is sufficient in many human identity testing comparisons unless the evidence is substantially compromised and/or there are insufficient relatives or a potential mutation may have arisen in kinship analyses. An automated STR assay system that is based on electrospray ionization mass spectrometry (ESI-MS) has been developed that can increase the discrimination power of some of the CODIS core STR loci and thus provide more information in typical and challenged samples and cases.

Evaluation of repetitive sequence PCR and PCR-mass spectrometry for the identification of clinically relevant Candida species.

The application of molecular diagnostic methods may improve the timeliness and accuracy with which fungi are identified. A total of 76 well-characterized reference strains of clinically relevant Candida species and 61 clinical Candida isolates were tested by repetitive sequence PCR (rep-PCR) and PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) and results compared against internal transcribed spacer (ITS) ribosomal RNA gene sequencing as a reference standard. Both rep-PCR and PCR/ESI-MS correctly identified 51 isolates to the species level.

New technology for rapid molecular diagnosis of bloodstream infections.

Technologies for the correct and timely diagnosis of bloodstream infections are urgently needed. Molecular diagnostic methods have yet to have a major impact on the diagnosis of bloodstream infections; however, new methods are being developed that are beginning to address key issues. In this article, we discuss the key needs and objectives of molecular diagnostics for bloodstream infections and review some of the currently available methods and how these techniques meet key needs.

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.

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.

Base composition profiling of human mitochondrial DNA using polymerase chain reaction and direct automated electrospray ionization mass spectrometry.

We describe an automated system for high-resolution profiling of human mitochondrial DNA (mtDNA) based upon multiplexed polymerase chain reaction (PCR) followed by desolvation and direct analysis using electrospray ionization mass spectrometry (PCR/ESI-MS). The assay utilizes 24 primer pairs that amplify targets in the mtDNA control region, including the hypervariable regions typically sequenced in a forensic analysis. Profiles consisting of product base compositions can be stored in a database, compared to each other, and compared to sequencing results.

Rapid and high-throughput pan-Orthopoxvirus detection and identification using PCR and mass spectrometry.

The genus Orthopoxvirus contains several species of related viruses, including the causative agent of smallpox (Variola virus). In addition to smallpox, several other members of the genus are capable of causing human infection, including monkeypox, cowpox, and other zoonotic rodent-borne poxviruses. Therefore, a single assay that can accurately identify all orthopoxviruses could provide a valuable tool for rapid broad orthopovirus identification.

Molecular genotyping of microbes by multilocus PCR and mass spectrometry: a new tool for hospital infection control and public health surveillance.

We describe a new technology for the molecular genotyping of microbes using a platform known commercially as the Ibis T5000. The technology couples multilocus polymerase chain reaction (PCR) to electrospray ionization/mass spectrometry (PCR/ESI-MS) and was developed to provide rapid, high-throughput, and precise digital analysis of either isolated colonies or original patient specimens on a platform suitable for use in hospital or reference diagnostic laboratories or public health settings. The PCR/ESI-MS method measures digital molecular signatures from microbes, enabling real-time epidemiological surveillance and outbreak investigation.

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.

Usefulness of multilocus polymerase chain reaction followed by electrospray ionization mass spectrometry to identify a diverse panel of bacterial isolates.

Polymerase chain reaction electrospray ionization mass spectrometry (PCR/ESI-MS) was tested for its ability to accurately identify a blinded panel of 156 diverse bacterial isolates, mostly human and/or animal pathogens. Here, 142/156 (91%) isolates were correctly identified to the genus level and 115/156 (74%) were correctly identified to the species level. Only 9% were misidentified.

Ibis T5000: a universal biosensor approach for microbiology.

We describe a new technology, the Ibis T5000, for the identification of pathogens in clinical and environmental samples. The Ibis T5000 couples nucleic acid amplification to high-performance electrospray ionization mass spectrometry and base-composition analysis. The system enables the identification and quantification of a broad set of pathogens, including all known bacteria, all major groups of pathogenic fungi and the major families of viruses that cause disease in humans and animals, along with the detection of virulence factors and antibiotic resistance markers.

High-resolution genotyping of Campylobacter species by use of PCR and high-throughput mass spectrometry.

In this work we report on a high-throughput mass spectrometry-based technique for the rapid high-resolution identification of Campylobacter jejuni strain types. This method readily distinguishes C. jejuni from C.

Rapid detection and molecular serotyping of adenovirus by use of PCR followed by electrospray ionization mass spectrometry.

We have developed a PCR/electrospray ionization mass spectrometry (PCR/ESI-MS) assay for the rapid detection, identification, and serotyping of human adenoviruses. The assay employs a high-performance mass spectrometer to "weigh" the amplicons obtained from PCR using primers designed to amplify known human adenoviruses. Masses are converted to base compositions and, by comparison against a database of the genetic sequences, the serotype present in a sample is determined.

Direct broad-range detection of alphaviruses in mosquito extracts.

Members of the genus Alphavirus are a diverse group of principally mosquito-borne RNA viruses. There are at least 29 species and many more subtypes of alphaviruses and some are considered potential bioweapons. We have developed a multi-locus RT-PCR followed by electrospray ionization mass spectrometry (RT-PCR/ESI-MS) assay that uses the amplicon base compositions to detect and identify alphaviruses.

Global surveillance of emerging Influenza virus genotypes by mass spectrometry.

Background: 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.

Methods And Principal Findings: 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.

Rapid identification of emerging infectious agents using PCR and electrospray ionization mass spectrometry.

Newly emergent infectious diseases are a global public health problem. The population dense regions of Southeast Asia are the epicenter of many emerging diseases, as evidenced by the outbreak of Nipah, SARS, avian influenza (H5N1), Dengue, and enterovirus 71 in this region in the past decade. Rapid identification, epidemiologic surveillance, and mitigation of transmission are major challenges in ensuring public health safety.

Mitochondrial DNA mutation detection by electrospray mass spectrometry.

Background: Mitochondrial DNA (mtDNA) mutations cause a large spectrum of clinically important neurodegenerative, neuromuscular, cardiovascular, and endocrine disorders. We describe the novel application of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS) to the rapid and accurate identification of pathogenic mtDNA variants.

Methods: In a blinded study, we used ESI-FTICR MS to analyze 24 unrelated samples of total cellular DNA containing 12 mtDNA variants and compared the results with those obtained by conventional PCR-restriction fragment length polymorphism (PCR-RFLP) analysis and gel electrophoresis.

Chemical process based reconstruction of exposures for an epidemiological study. III. Analysis of industrial hygiene samples.

As part of an historical cohort study to investigate the mortality experience of industrial workers exposed to chloroprene (beta-CD) and other substances, all available industrial hygiene exposure monitoring data were collected and summarized. From discussions with on-site industrial hygiene personnel, it was apparent that these data were not collected for epidemiological purposes and, therefore, their use in characterization of exposures was problematic as the data mostly pertained to samples collected to investigate the performance of specific tasks. These data were, however, informative for validating the exposure modeling process used to estimate historical exposures.