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Find video protocols related to scientific articles indexed in Pubmed.
Improved sensitivity for molecular detection of bacterial and Candida infections in blood.
J. Clin. Microbiol.
PUBLISHED: 06-20-2014
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The rapid identification of bacteria and fungi directly from the blood of patients with suspected bloodstream infections aids in diagnosis and guides treatment decisions. The development of an automated, rapid, and sensitive molecular technology capable of detecting the diverse agents of such infections at low titers has been challenging, due in part to the high background of genomic DNA in blood. PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) allows for the rapid and accurate identification of microorganisms but with a sensitivity of about 50% compared to that of culture when using 1-ml whole-blood specimens. Here, we describe a new integrated specimen preparation technology that substantially improves the sensitivity of PCR/ESI-MS analysis. An efficient lysis method and automated DNA purification system were designed for processing 5 ml of whole blood. In addition, PCR amplification formulations were optimized to tolerate high levels of human DNA. An analysis of 331 specimens collected from patients with suspected bloodstream infections resulted in 35 PCR/ESI-MS-positive specimens (10.6%) compared to 18 positive by culture (5.4%). PCR/ESI-MS was 83% sensitive and 94% specific compared to culture. Replicate PCR/ESI-MS testing from a second aliquot of the PCR/ESI-MS-positive/culture-negative specimens corroborated the initial findings in most cases, resulting in increased sensitivity (91%) and specificity (99%) when confirmed detections were considered true positives. The integrated solution described here has the potential to provide rapid detection and identification of organisms responsible for bloodstream infections.
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Analytical characterization of an assay designed to detect and identify diverse agents of disseminated viral infection.
J. Clin. Virol.
PUBLISHED: 01-21-2014
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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.
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Rapid PCR/ESI-MS-based molecular genotyping of Staphylococcus aureus from nasal swabs of emergency department patients.
BMC Infect. Dis.
PUBLISHED: 01-06-2014
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A limitation of both culture-based and molecular methods of screening for staphylococcal infection is that current tests determine only the presence or absence of colonization with no information on the colonizing strain type. A technique that couples polymerase chain reaction to mass spectrometry (PCR/ESI-MS) has recently been developed and an assay validated to identify and genotype S. aureus and coagulase-negative staphylococci (CoNS).
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Enhanced diagnostic yields of bacteremia and candidemia in blood specimens by PCR-electrospray ionization mass spectrometry.
J. Clin. Microbiol.
PUBLISHED: 08-21-2013
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A prospective study was performed to determine the value of direct molecular testing of whole blood for detecting the presence of culturable and unculturable bacteria and yeasts in patients with suspected bloodstream infections. A total of 464 adult and pediatric patients with positive blood cultures matched with 442 patients with negative blood cultures collected during the same period were recruited during a 10-month study. PCR amplification coupled with electrospray ionization mass spectrometry (PCR-ESI-MS) plus blood culture reached an overall agreement of 78.6% in the detection and species-level identification of bacterial and candidal pathogens. Of 33 culture-negative/PCR-ESI-MS-positive specimens, 31 (93.9%) were judged to be truly bacteremic and/or candidemic based on a medical chart review and analytical metrics. Among the 15 culture-positive specimens in which PCR-ESI-MS detected additional bacterial or yeast species, 66.7% and 20.0% of the additional positive specimens by PCR-ESI-MS were judged to be truly or possibly bacteremic and/or candidemic, respectively. Direct analysis of blood samples by PCR-ESI-MS rapidly detects bacterial and yeast pathogens in patients with bloodstream infections. When used in conjunction with blood culture, PCR-ESI-MS enhances the diagnostics of septicemia by shortening test turnaround time and improving yields.
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Survey of culture, goldengate assay, universal biosensor assay, and 16S rRNA Gene sequencing as alternative methods of bacterial pathogen detection.
J. Clin. Microbiol.
PUBLISHED: 07-24-2013
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Cultivation-based assays combined with PCR or enzyme-linked immunosorbent assay (ELISA)-based methods for finding virulence factors are standard methods for detecting bacterial pathogens in stools; however, with emerging molecular technologies, new methods have become available. The aim of this study was to compare four distinct detection technologies for the identification of pathogens in stools from children under 5 years of age in The Gambia, Mali, Kenya, and Bangladesh. The children were identified, using currently accepted clinical protocols, as either controls or cases with moderate to severe diarrhea. A total of 3,610 stool samples were tested by established clinical culture techniques: 3,179 DNA samples by the Universal Biosensor assay (Ibis Biosciences, Inc.), 1,466 DNA samples by the GoldenGate assay (Illumina), and 1,006 DNA samples by sequencing of 16S rRNA genes. Each method detected different proportions of samples testing positive for each of seven enteric pathogens, enteroaggregative Escherichia coli (EAEC), enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), Shigella spp., Campylobacter jejuni, Salmonella enterica, and Aeromonas spp. The comparisons among detection methods included the frequency of positive stool samples and kappa values for making pairwise comparisons. Overall, the standard culture methods detected Shigella spp., EPEC, ETEC, and EAEC in smaller proportions of the samples than either of the methods based on detection of the virulence genes from DNA in whole stools. The GoldenGate method revealed the greatest agreement with the other methods. The agreement among methods was higher in cases than in controls. The new molecular technologies have a high potential for highly sensitive identification of bacterial diarrheal pathogens.
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Broad-spectrum biosensor capable of detecting and identifying diverse bacterial and Candida species in blood.
J. Clin. Microbiol.
PUBLISHED: 06-12-2013
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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. Over 99% of all detections yielded correct identifications, whether they were obtained at concentrations well above the limit of detection or at the lowest detectable concentrations. This study demonstrates the ability of broad-spectrum PCR/ESI-MS assays to detect and identify diverse organisms in complex natural matrices that contain high levels of background DNA.
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PCR followed by electrospray ionization mass spectrometry for broad-range identification of fungal pathogens.
J. Clin. Microbiol.
PUBLISHED: 01-09-2013
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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. Using a database grounded by 60 ATCC reference strains, a total of 394 clinical fungal isolates (264 molds and 130 yeasts) were analyzed by PCR/ESI-MS; results were compared to phenotypic identification, and discrepant results were sequence confirmed. PCR/ESI-MS identified 81.4% of molds to either the genus or species level, with concordance rates of 89.7% and 87.4%, respectively, to phenotypic identification. Likewise, PCR/ESI-MS was able to identify 98.4% of yeasts to either the genus or species level, agreeing with 100% of phenotypic results at both the genus and species level. PCR/ESI-MS performed best with Aspergillus and Candida isolates, generating species-level identification in 94.4% and 99.2% of isolates, respectively. PCR/ESI-MS is a promising new technology for broad-range detection and identification of medically important fungal pathogens that cause invasive mycoses.
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Rapid diagnosis of bloodstream infections with PCR followed by mass spectrometry.
PLoS ONE
PUBLISHED: 01-01-2013
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Achieving a rapid microbiological diagnosis is crucial for decreasing morbidity and mortality of patients with a bloodstream infection, as it leads to the administration of an appropriate empiric antimicrobial therapy. Molecular methods may offer a rapid alternative to conventional microbiological diagnosis involving blood culture. In this study, the performance of a new technology that uses broad-spectrum PCR coupled with mass spectrometry (PCR/ESI-MS) was evaluated for the detection of microorganisms directly from whole blood. A total of 247 whole blood samples and paired blood cultures were prospectively obtained from 175 patients with a suspicion of sepsis. Both sample types were analyzed using the PCR/ESI-MS technology, and the results were compared with those obtained by conventional identification methods. The overall agreement between conventional methods and PCR/ESI-MS performed in blood culture aliquots was 94.2% with 96.8% sensitivity and 98.5% specificity for the molecular method. When comparing conventional methods with PCR/ESI-MS performed in whole blood specimens, the overall agreement was 77.1% with 50% sensitivity and 93.8% specificity for the molecular method. Interestingly, the PCR/ESI-MS technology led to the additional identification of 13 pathogens that were not found by conventional methods. Using the PCR/ESI-MS technology the microbiological diagnosis of bloodstream infections could be anticipated in about half of the patients in our setting, including a small but significant proportion of patients newly diagnosed. Thus, this promising technology could be very useful for the rapid diagnosis of sepsis in combination with traditional methods.
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Molecular characterization of drug-resistant Mycobacterium tuberculosis isolates circulating in China by multilocus PCR and electrospray ionization mass spectrometry.
J. Clin. Microbiol.
PUBLISHED: 04-27-2011
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We used multilocus PCR and electrospray ionization mass spectrometry (PCR/ESI-MS) to determine the genotype and drug resistance profiles for 96 Mycobacterium tuberculosis isolates circulating in regions of high and low tuberculosis (TB) endemicity in China. The dominant principal genetic group (PGG) circulating in China was PGG1, and drug-resistant gene mutations were more diversified in the region of low rather than high TB endemicity.
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Reverse transcription polymerase chain reaction and electrospray ionization mass spectrometry for identifying acute viral upper respiratory tract infections.
Diagn. Microbiol. Infect. Dis.
PUBLISHED: 01-22-2011
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Diagnosis of respiratory viruses traditionally relies on culture or antigen detection. We aimed to demonstrate capacity of the reverse transcription polymerase chain reaction/electrospray ionization mass spectrometry (RT-PCR/ESI-MS) platform to identify clinical relevant respiratory viruses in nasopharyngeal aspirate (NPA) samples and compare the diagnostic performance characteristics relative to conventional culture- and antigen-based methods. An RT-PCR/ESI-MS respiratory virus surveillance kit designed to detect respiratory syncytial virus, influenza A and B, parainfluenza types 1-4, Adenoviridae types A-F, Coronaviridae, human bocavirus, and human metapneumovirus was evaluated using both mock-ups and frozen archived NPA (N = 280), 95 of which were positive by clinical virology methods. RT-PCR/ESI-MS detected 74/95 (77.9%) known positive samples and identified an additional 13/185 (7%) from culture-negative samples. Viruses that are nondetectable with conventional methods were also identified. Viral load was semiquantifiable and ranged from 2400 to >320?000 copies/mL. Time to results was 8 h. RT-PCR/ESI-MS showed promise in rapid detection of respiratory viruses and merits further evaluation for use in clinical settings.
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Rapid identification viruses from nasal pharyngeal aspirates in acute viral respiratory infections by RT-PCR and electrospray ionization mass spectrometry.
J. Virol. Methods
PUBLISHED: 01-06-2011
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Diagnosis of the etiologic agent of respiratory viral infection relies traditionally on culture or antigen detection. This pilot evaluation compared performance characteristics of the RT-PCR and electrospray ionization mass spectrometry (RT-PCR/ESI-MS) platform to conventional virologic methods for identifying multiple clinically relevant respiratory viruses in nasopharyngeal aspirates. The RT-PCR/ESI-MS respiratory virus surveillance kit was designed to detect respiratory syncytial virus, influenza A and B, parainfluenza types 1-4, adenoviridae types A-F, coronaviridae, human bocavirus, and human metapneumovirus. Patients (N=192) attending an emergency department during the 2007-2008 respiratory season consented, and "excess" frozen archived nasopharyngeal aspirates were analysed; 46 were positive by conventional virology and 69 by RT-PCR/ESI-MS, among which there were six samples with multiple viral pathogens detected. The sensitivity and specificity of the assay were 89.1% and 80.3%, respectively. Additional viruses that were not identified by conventional virology assays were detected (4 human bocaviruses and 7 coronaviruses). Samples in which the RT-PCR/ESI-MS results disagreed with conventional virology were sent for analysis by a third method using a commercial RT-PCR-based assay, which can identify viruses not detectable by conventional virologic procedures. Time to first result of RT-PCR/ESI-MS was 8h. RT-PCR/ESI-MS demonstrated capacity to detect respiratory viruses identifiable and unidentifiable by conventional methods rapidly.
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Simultaneous identification of mycobacterial isolates to the species level and determination of tuberculosis drug resistance by PCR followed by electrospray ionization mass spectrometry.
J. Clin. Microbiol.
PUBLISHED: 12-29-2010
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Mycobacterium tuberculosis that is resistant to both isoniazid (INH) and rifampin (RIF) is spreading. It has become a public health problem in part because the standard culture methods used to determine the appropriate treatment regimen for patients often take months following the presumptive diagnosis of tuberculosis. Furthermore, the misidentification of nontuberculosis mycobacteria (NTM) in patients presumably suffering from tuberculosis results in additional human and health care costs. The mechanisms of resistance for several drugs used to treat Mycobacterium tuberculosis are well understood and therefore should be amenable to determination by rapid molecular methods. We describe here the use of PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) in an assay that simultaneously determines INH and RIF resistance in Mycobacterium tuberculosis and identifies and determines the species of NTMs. The assay panel included 16 primer pairs in eight multiplexed reactions and was validated using a collection of 1,340 DNA samples from cultured specimens collected in the New York City area, the Republic of Georgia, and South Africa. Compared with phenotypic data, the PCR/ESI-MS assay had 89.3% sensitivity and 95.8% specificity in the determination of INH resistance and 96.3% sensitivity and 98.6% specificity in the determination of RIF resistance. Based on a set of 264 previously characterized liquid culture specimens, the PCR/ESI-MS method had 97.0% sensitivity and 99.9% specificity for determination of NTM identity. The assay also provides information on ethambutol, fluoroquinolone, and diarylquinoline resistance and lineage-specific polymorphisms, to yield highly discriminative digital signatures potentially suitable for epidemiology tracking.
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Pyoderma gangrenosum-like ulcer in a patient with X-linked agammaglobulinemia: identification of Helicobacter bilis by mass spectrometry analysis.
Arch Dermatol
PUBLISHED: 05-19-2010
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Pyoderma gangrenosum-like ulcers and cellulitis of the lower extremities associated with recurrent fevers in patients with X-linked (Bruton) agammaglobulinemia have been reported to be caused by Helicobacter bilis (formerly classified as Flexispira rappini and then Helicobacter strain flexispira taxon 8). Consistent themes in these reports are the difficulty in recovering this organism in blood and wound cultures and in maintaining isolates in vitro. We confirmed the presence of this organism in a patients culture by using a novel application of gene amplification polymerase chain reaction and electrospray ionization time-of-flight mass spectrometry.
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New technology for rapid molecular diagnosis of bloodstream infections.
Expert Rev. Mol. Diagn.
PUBLISHED: 05-15-2010
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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. We then focus on a new method that combines nucleic acid amplification with mass spectrometry in a novel approach to molecular diagnosis of bloodstream infections.
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Rapid molecular assays for microbial contaminant monitoring in the bioprocess industry.
PDA J Pharm Sci Technol
PUBLISHED: 04-09-2010
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Adventitious contaminations with bacterial, viral, or fungal infectious agents represent a major risk associated with the manufacture and release of pharmaceutical products for human use, including vaccines, protein-based therapeutics, and antibodies. Early detection of contaminants in the biologicals production process might allow immediate action to correct such events without a significant interruption in the rate of production. Among the methods currently used for testing are cell culture, animal inoculation, electron microscopy, and in vitro molecular and antibody assays. Bacteria such as mycoplasma and mycobacterial species and most of the viral and fungal agents can take several days to weeks or even months to grow in culture. We have developed a broad-range microbial detection assay that uses the innovative Ibis biosensor platform, a rapid and high-throughput biosensor that is based on polymerase chain reaction (PCR) and electrospray ionization mass spectrometry to identify and quantify microbial contaminants. By combining the sensitivity of PCR with the accuracy of mass spectrometric detection, this technology generates a fingerprint that uniquely identifies an organism without a priori assumptions of the sample identity. This approach is capable of detecting known and unknown pathogens, as well as providing high-resolution genotyping and strain typing, and drug resistance and virulence information. Representative case studies are discussed here showing detection of minute virus of mice, mycoplasma, and an unknown virus that was identified as bluetongue virus.
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Application of the Ibis-T5000 pan-Orthopoxvirus assay to quantitatively detect monkeypox viral loads in clinical specimens from macaques experimentally infected with aerosolized monkeypox virus.
Am. J. Trop. Med. Hyg.
PUBLISHED: 02-06-2010
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Monkeypox virus (MPXV), a member of the family Poxviridae and genus Orthopoxvirus, causes a smallpox-like disease in humans. A previously described pan-Orthopoxvirus assay, based on a broad-range polymerase chain reaction (PCR) coupled with electrospray ionization mass spectrometry (PCR/ESI-MS), was evaluated for its ability to detect MPXV from spiked human and aerosol-infected cynomolgous macaque (Macaca fascicularis) samples. Detection of MPXV DNA from macaque tissue, blood, and spiked human blood by the PCR/ESI-MS pan-Orthopoxvirus assay was comparable, albeit at slightly higher levels, to the current gold standard method of real-time PCR with the pan-Orthopoxvirus assay and had a limit of detection of 200 plaque-forming units. Furthermore, the platform was able to distinguish MPXV and vaccinia viruses that were spiked into macaque blood samples at various concentrations. This platform provides a new tool for the diagnosis and monitoring of orthopoxviral loads during vaccine or antiviral studies, but also could provide rapid identification during natural outbreaks or bioterrorism attacks.
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Rapid identification of vector-borne flaviviruses by mass spectrometry.
Mol. Cell. Probes
PUBLISHED: 02-05-2010
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Flaviviruses are a highly diverse group of RNA viruses classified within the genus Flavivirus, family Flaviviridae. Most flaviviruses are arthropod-borne, requiring a mosquito or tick vector. Several flaviviruses are highly pathogenic to humans; however, their high genetic diversity and immunological relatedness makes them extremely challenging to diagnose. In this study, we developed and evaluated a broad-range Flavivirus assay designed to detect both tick- and mosquito-borne flaviviruses by using RT-PCR/electrospray ionization mass spectrometry (RT-PCR/ESI-MS) on the Ibis T5000 platform. The assay was evaluated with a panel of 13 different flaviviruses. All samples were correctly identified to the species level. To determine the limit of detection for the mosquito-borne primer sets, serial dilutions of RNA from West Nile virus (WNV) were assayed and could be detected down to an equivalent viral titer of 0.2 plaque-forming units/mL. Analysis of flaviviruses in their natural biological background included testing Aedes aegypti mosquitoes that were laboratory-infected with dengue-1 virus. The assay accurately identified the virus within infected mosquitoes, and we determined the average viral genome per mosquito to be 2.0 x 10(6). Using human blood, serum, and urine spiked with WNV and mouse blood and brain tissues from Karshi virus-infected mice, we showed that these clinical matrices did not inhibit the detection of these viruses. Finally, we used the assay to test field-collected Ixodes scapularis ticks collected from sites in New York and Connecticut. We found 16/322 (5% infection rate) ticks positive for deer tick virus, a subtype of Powassan virus. In summary, we developed a single high-throughput Flavivirus assay that could detect multiple tick- and mosquito-borne flaviviruses and thus provides a new analytical tool for their medical diagnosis and epidemiological surveillance.
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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.
Appl. Environ. Microbiol.
PUBLISHED: 01-29-2010
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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.
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Detection and identification of Ehrlichia species in blood by use of PCR and electrospray ionization mass spectrometry.
J. Clin. Microbiol.
PUBLISHED: 12-02-2009
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Rapid detection and identification of Ehrlichia species improves clinical outcome for patients suspected of ehrlichiosis. We describe an assay that employs multilocus PCR and electrospray ionization mass spectrometry (PCR/ESI-MS) to detect and identify Ehrlichia species directly from blood specimens. The results were compared to those of a colorimetric microtiter PCR enzyme immunoassay (PCR-EIA) used as a diagnostic assay. Among 213 whole-blood samples collected from patients who were clinically suspected of ehrlichiosis from 1 May to 1 August 2008 at Vanderbilt University Hospital, 40 were positive for an Ehrlichia species by PCR/ESI-MS, giving a positive rate of 18.8%. In comparison to the PCR-EIA, PCR/ESI-MS possessed a sensitivity, a specificity, and positive and negative predictive values of 95.0%, 98.8%, 95.0%, and 98.8%, respectively. The 38 specimens that were positive for Ehrlichia by both PCR/ESI-MS and the PCR-EIA were further characterized to the species level, with 100% agreement between the two assays. In addition, Rickettsia rickettsii was detected by PCR/ESI-MS from four specimens that were confirmed retrospectively by serology and PCR-EIA. In three specimens, the PCR/ESI-MS assay identified Pseudomonas aeruginosa, Neisseria meningitidis, and Staphylococcus aureus; these were confirmed by culture and/or clinical diagnosis as being clinically relevant. From specimen processing to result reporting, the PCR/ESI-MS assay can be completed within 6 h, providing another laboratory tool for the diagnosis of ehrlichiosis. Moreover, this system may provide rapid detection and identification of additional pathogens directly from blood specimens.
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Pathogen profiling: rapid molecular characterization of Staphylococcus aureus by PCR/electrospray ionization-mass spectrometry and correlation with phenotype.
J. Clin. Microbiol.
PUBLISHED: 08-26-2009
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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.
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Molecular genotyping of microbes by multilocus PCR and mass spectrometry: a new tool for hospital infection control and public health surveillance.
Methods Mol. Biol.
PUBLISHED: 06-13-2009
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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. This technology will facilitate understanding of the pathways by which infectious organisms spread and will enable appropriate interventions on a time frame not previously achievable.
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Rapid molecular genotyping and clonal complex assignment of Staphylococcus aureus isolates by PCR coupled to electrospray ionization-mass spectrometry.
J. Clin. Microbiol.
PUBLISHED: 03-18-2009
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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.
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Rapid determination of quinolone resistance in Acinetobacter spp.
J. Clin. Microbiol.
PUBLISHED: 03-18-2009
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In the treatment of serious bacterial infections, the rapid institution of appropriate antimicrobial chemotherapy may be lifesaving. Choosing the correct antibiotic or combination of antibiotics is becoming very important, as multidrug resistance is found in many pathogens. Using a collection of 75 well-characterized multidrug-resistant (MDR) Acinetobacter sp. isolates, we show that PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) and base composition analysis of PCR amplification products can quickly and accurately identify quinolone resistance mediated by mutations in the quinolone resistance-determining regions of gyrA and parC, two essential housekeeping genes. Single point mutations detected by PCR/ESI-MS in parC (found in 55/75 of the isolates) and in gyrA (found in 66/75 of the isolates) correlated with susceptibility testing and sequencing. By targeting resistance determinants that are encoded by genes with highly conserved DNA sequences (e.g., gyrA and parC), we demonstrate that PCR/ESI-MS can provide critical information for resistance determinant identification and can inform therapeutic decision making in the treatment of Acinetobacter sp. infections.
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Rapid and high-throughput pan-Orthopoxvirus detection and identification using PCR and mass spectrometry.
PLoS ONE
PUBLISHED: 02-11-2009
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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. We have developed a pan-Orthopoxvirus assay for identification of all members of the genus based on four PCR reactions targeting Orthopoxvirus DNA and RNA helicase and polymerase genes. The amplicons are detected using electrospray ionization-mass spectrometry (PCR/ESI-MS) on the Ibis T5000 system. We demonstrate that the assay can detect and identify a diverse collection of orthopoxviruses, provide sub-species information and characterize viruses from the blood of rabbitpox infected rabbits. The assay is sensitive at the stochastic limit of PCR and detected virus in blood containing approximately six plaque-forming units per milliliter from a rabbitpox virus-infected rabbit.
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Occurrence, distribution, and origins of Streptococcus pneumoniae Serotype 6C, a recently recognized serotype.
J. Clin. Microbiol.
PUBLISHED: 02-03-2009
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The prevalence of Streptococcus pneumoniae serotype 6C, a recently recognized serotype that cross-reacts serologically with serotype 6A, was investigated. Isolates of serotype 6A in various collections were recovered, and serotype 6C was differentiated from 6A by multiplex PCR of DNA extracts by using appropriate primers. Antimicrobial susceptibility was performed by Clinical and Laboratory Standards Institute broth microdilution, and selected isolates were typed by pulsed-field gel electrophoresis, repetitive sequence-based PCR typing, and rapid multilocus sequence typing (MLST) by electrospray ionization mass spectrometry of PCR products. A total of 60 serotype 6C isolates were found: 30 of 122 Cleveland isolates collected from 1979 to 2007, 19 of 39 pediatric isolates collected nationwide in 2005 and 2006, and 11 pediatric isolates from Massachusetts collected in 2006 and 2007. Only four isolates were recovered prior to introduction of the conjugate pneumococcal vaccine in 2000; the earliest isolate was recovered in 1989. The sources of the isolates included blood (n = 5), the lower respiratory tract (n = 27), the sinus (n = 5), the ear (n = 2), and the nasopharynx (n = 18); isolates were recovered from 49 children and 11 adults. Pediatric isolates were found in all six major U.S. geographic regions. Antimicrobial susceptibility showed that 22 isolates were nonsusceptible to penicillin, macrolides, and trimethoprim-sulfamethoxazole, 8 had other resistance patterns, and 30 were fully susceptible. The three typing methods used showed similar clusters of up to eight isolates per cluster. MLST showed five clusters related to serotype 6A, two clusters related to serotype 6B, one cluster related to serotype 3, and one cluster related to serotype 34. This study documents the occurrence, nationwide distribution, diversity, likely origins, and increasing incidence after 2001 of this recently recognized serotype. Serotype 6C warrants consideration for addition to future conjugate pneumococcal vaccines.
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Comparative analysis of two broad-range PCR assays for pathogen detection in positive-blood-culture bottles: PCR-high-resolution melting analysis versus PCR-mass spectrometry.
J. Clin. Microbiol.
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Detection of pathogens in bloodstream infections is important for directing antimicrobial treatment, but current culture-based approaches can be problematic. Broad-range PCR assays which target conserved genomic motifs for postamplification amplicon analysis permit detection of sepsis-causing pathogens. Comparison of different broad-range assays is important for informing future implementation strategies. In this study, we compared positive-blood-culture bottles processed by PCR coupled to high-resolution melting curve analysis (PCR/HRMA) and PCR coupled to electrospray ionization-mass spectrometry (PCR/ESI-MS) to microbiology culture results. Genus-level concordance was 90% (confidence interval [CI], 80 to 96%) for PCR/HRMA and 94% (CI, 85 to 98%) for PCR/ESI-MS. Species-level concordance was 90% (CI, 80 to 96%) for PCR/HRMA and 86% (CI, 75 to 93%) for PCR/ESI-MS. Unlike PCR/HRMA, PCR/ESI-MS was able to resolve polymicrobial samples. Our results demonstrated that the two assays have similar overall concordance rates but may have different roles as potential adjunctive tests with standard blood culture, since each method has different capabilities, advantages, and disadvantages.
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Comprehensive biothreat cluster identification by PCR/electrospray-ionization mass spectrometry.
PLoS ONE
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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. The ability to comprehensively detect the important biothreat clusters with resolution sufficient to distinguish the near neighbors with an extremely low false positive rate is required. A technological solution to this problem can be achieved by coupling biothreat group-specific PCR with electrospray ionization mass spectrometry (PCR/ESI-MS). The biothreat assay described here detects ten bacterial and four viral biothreat clusters on the NIAID priority pathogen and HHS/USDA select agent lists. Detection of each of the biothreat clusters was validated by analysis of a broad collection of biothreat organisms and near neighbors prepared by spiking biothreat nucleic acids into nucleic acids extracted from filtered environmental air. Analytical experiments were carried out to determine breadth of coverage, limits of detection, linearity, sensitivity, and specificity. Further, the assay breadth was demonstrated by testing a diverse collection of organisms from each biothreat cluster. The biothreat assay as configured was able to detect all the target organism clusters and did not misidentify any of the near-neighbor organisms as threats. Coupling biothreat cluster-specific PCR to electrospray ionization mass spectrometry simultaneously provides the breadth of coverage, discrimination of near neighbors, and an extremely low false positive rate due to the requirement that an amplicon with a precise base composition of a biothreat agent be detected by mass spectrometry.
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Monitoring seasonal influenza A evolution: rapid 2009 pandemic H1N1 surveillance with an reverse transcription-polymerase chain reaction/electro-spray ionization mass spectrometry assay.
J. Clin. Virol.
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The emergence of the pandemic H1N1 influenza strain in 2009 reinforced the need for improved influenza surveillance efforts. A previously described influenza typing assay that utilizes RT-PCR coupled to electro-spray ionization mass spectrometry (ESI-MS) played an early role in the discovery of the pandemic H1N1 influenza strain, and has potential application for monitoring viral genetic diversity in ongoing influenza surveillance efforts.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.