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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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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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Molecular genotyping of Acinetobacter spp. isolated in Arizona, USA, using multilocus PCR and mass spectrometry.
J. Med. Microbiol.
PUBLISHED: 06-05-2013
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Acinetobacter spp. are a diverse group of Gram-negative bacteria frequently implicated in nosocomial infections. Genotypic methods have been instrumental in studying Acinetobacter, but few offer high resolution, rapid turnaround time, technical ease and high inter-laboratory reproducibility, which has hampered understanding of disease incidence, transmission patterns and diversity within this genus. Here, we further evaluated multilocus PCR electrospray ionization/mass spectrometry (PCR/ESI-MS), a method that is simple and robust, and provides both species characterization and strain-level resolution of Acinetobacter spp. on a single platform. We examined 125 Acinetobacter isolates from 21 hospitals, laboratories and medical centres spanning four counties in Arizona, USA, using PCR/ESI-MS. We compared PCR/ESI-MS with an in-house amplified fragment length polymorphism (AFLP) genotyping scheme. PCR/ESI-MS demonstrated that Acinetobacter spp. from Arizonan hospitals had similar species and strain distributions to other US civilian hospitals. Furthermore, we showed that the PCR/ESI-MS and AFLP genotypes were highly congruent, with the former having the advantages of robust inter-laboratory reproducibility, rapid turnaround time and simple experimental set-up and data analysis. PCR/ESI-MS is an effective and high-throughput platform for strain typing of Acinetobacter baumannii and for identification of other Acinetobacter spp., including the emerging nosocomial pathogens Acinetobacter pittii and Acinetobacter nosocomialis.
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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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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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Identification of a novel Acinetobacter baumannii clone in a US hospital outbreak by multilocus polymerase chain reaction/electrospray-ionization mass spectrometry.
Diagn. Microbiol. Infect. Dis.
PUBLISHED: 04-25-2011
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We investigated the relatedness of multidrug-resistant Acinetobacter baumannii isolates from a burn intensive care unit (BICU) outbreak, control isolates, and isolates from a previous 2007 outbreak by 3 molecular typing methods (repetitive sequence-based polymerase chain reaction [rep-PCR], broad-range PCR and electrospray ionization mass spectrometry [PCR/ESI-MS], and pulsed-field gel electrophoresis [PFGE]). Partial rpoB gene sequencing confirmed all tested isolates as A. baumannii. Molecular typing analysis showed that 17 of 19 outbreak isolates were indistinguishable or closely related to each other. Three of 4 non-BICU outbreak control isolates and 5 of 6 isolates from the previous outbreak closely matched the BICU outbreak genotype. The outbreak strain represented a novel strain type, ST96, on PCR/ESI-MS with a new combination of alleles not previously seen in the United States. The ST96 strain also represented a novel rpoB Seqtype. Results of PCR/ESI-MS and PFGE genotyping were most closely correlated, while rep-PCR and PCR/ESI-MS systems generated rapid results.
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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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Genomic signature-based identification of influenza A viruses using RT-PCR/electro-spray ionization mass spectrometry (ESI-MS) technology.
PLoS ONE
PUBLISHED: 06-11-2010
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The emergence and rapid spread of the 2009 H1N1 pandemic influenza A virus (H1N1pdm) in humans highlights the importance of enhancing the capability of existing influenza surveillance systems with tools for rapid identification of emerging and re-emerging viruses. One of the new approaches is the RT-PCR electrospray ionization mass spectrometry (RT-PCR/ESI-MS) technology, which is based on analysis of base composition (BC) of RT-PCR amplicons from influenza "core" genes. Combination of the BC signatures represents a "genomic print" of an influenza A virus.
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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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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 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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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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Detection, identification, and distribution of fungi in bronchoalveolar lavage specimens by use of multilocus PCR coupled with electrospray ionization/mass spectrometry.
J. Clin. Microbiol.
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As pulmonary fungal infections continue to increase due to an increasing number of immunocompromised patients, rapid detection and accurate identification of these fungal pathogens are critical. A broad fungal assay was developed by incorporating broad-range multilocus PCR amplification and electrospray ionization/mass spectrometry (PCR/ESI-MS) to detect and identify fungal organisms directly from clinical specimens. The aims of this study were to evaluate the performance of PCR/ESI-MS for detection, identification, and determination of the distribution of fungal organisms in bronchoalveolar lavage (BAL) fluid specimens. The BAL fluid specimens submitted for fungal culture at Vanderbilt University Medical Center between May 2005 and October 2011 were included. Cultures and identification were done using standard procedures. In addition, DNA was extracted from BAL fluid specimens, and fungal DNA amplification/identification were performed by PCR/ESI-MS. The results were compared with those of the standard cultures. A total of 691 nonduplicated BAL fluid specimens with sufficient leftover volume for molecular testing were evaluated using PCR/ESI-MS. Among them, 134 specimens (19.4%) were positive for fungi by both culture and PCR/ESI-MS testing. Of the dual-positive specimens, 125 (93.3%) were positive for Candida and Aspergillus species, with concordances between culture and PCR/ESI-MS results being 84 (67.2%) at the species level and 109 (87.2%) at the genus level. In addition, 243 (35.2%) and 30 (4.3%) specimens were positive only by PCR/ESI-MS or by culture, respectively (odds ratio [OR] = 11.95, 95% confidence interval [CI] = 7.90 to 18.17, P = 0.0000). Codetection of fungal organisms was noted in 23 (3.3%) specimens by PCR/ESI-MS, which was significantly higher than the 4 (0.6%) in which they were noted by culture (OR = 5.91, 95% CI = 1.93 to 20.27, P = 0.0002). Among 53 specimens in which cultures failed because of bacterial overgrowth, at least one fungus was identified in 26 specimens (47.3%) by PCR/ESI-MS. PCR/ESI-MS provides an advanced tool for rapid and sensitive detection, identification, and determination of the distribution of fungal organisms directly from BAL fluid specimens. Moreover, it detected fungal organisms in specimens in which cultures failed because of bacterial overgrowth. The clinical relevance of the significantly higher detection rate of fungal organisms by PCR/ESI-MS merits further investigation.
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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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Rapid identification of Aspergillus terreus from bronchoalveolar lavage fluid by PCR and electrospray ionization with mass spectrometry.
J. Clin. Microbiol.
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We describe the application of PCR and electrospray-ionization with mass spectrometry (PCR/ESI-MS) to culture-negative bronchoalveolar lavage (BAL) fluid in order to identify septate hyphae noted by Gomori methenamine silver (GMS) staining of the fluid that was obtained from an immunocompromised woman with neutropenia following induction chemotherapy for treatment of acute myelogenous leukemia (AML). The patient was treated with empirical antifungal therapy, including intrathecal amphotericin B, while results of fungal cultures were pending. Ultimately, Aspergillus terreus, an amphotericin-resistant mold, was cultured from bilateral brain abscesses. PCR/ESI-MS correctly identified the mold.
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Longitudinal analysis of the temporal evolution of Acinetobacter baumannii strains in Ohio, USA, by using rapid automated typing methods.
PLoS ONE
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Genotyping methods are essential to understand the transmission dynamics of Acinetobacter baumannii. We examined the representative genotypes of A. baumannii at different time periods in select locations in Ohio, using two rapid automated typing methods: PCR coupled with electrospray ionization mass spectrometry (PCR/ESI-MS), a form of multi-locus sequence typing (MLST), and repetitive-sequence-based-PCR (rep-PCR). Our analysis included 122 isolates from 4 referral hospital systems, in 2 urban areas of Ohio. These isolates were associated with outbreaks at 3 different time periods (1996, 2000 and 2005-2007). Type assignments of PCR/ESI-MS and rep-PCR were compared to each other and to worldwide (WW) clone types. The discriminatory power of each method was determined using the Simpsons index of diversity (DI). We observed that PCR/ESI-MS sequence type (ST) 14, corresponding to WW clone 3, predominated in 1996, whereas ST 12 and 14 co-existed in the intermediate period (2000) and ST 10 and 12, belonging to WW clone 2, predominated more recently in 2007. The shift from WW clone 3 to WW clone 2 was accompanied by an increase in carbapenem resistance. The DI was approximately 0.74 for PCR/ESI-MS, 0.88 for rep-PCR and 0.90 for the combination of both typing methods. We conclude that combining rapid automated typing methods such as PCR/ESI-MS and rep-PCR serves to optimally characterize the regional molecular epidemiology of A. baumannii. Our data also sheds light on the changing sequence types in an 11 year period in Northeast Ohio.
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Concurrent serotyping and genotyping of pneumococci by use of PCR and electrospray ionization mass spectrometry.
J. Clin. Microbiol.
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A pneumococcal serotyping/genotyping system (PSGS) was developed based upon targeted PCR, followed by electrospray ionization mass spectrometry and amplicon base composition analysis. Eight multiplex PCRs, 32 targeting serotype-determining capsular biosynthetic loci, and 8 targeting multilocus sequence typing (MLST) loci were employed for each of 229 highly diverse Streptococcus pneumoniae isolates. The most powerful aspect of the PSGS system was the identification of capsular serotypes accounting for the majority of invasive and carried pneumococcal strains. Altogether, 45 different serotypes or serogroups were correctly predicted among the 196 resolvable isolates, with only 2 unexpected negative results. All 33 isolates that represented 23 serotypes not included in the PSGS yielded negative serotyping results. A genotyping database was constructed using the base compositions of 65- to 100-bp sections of MLST alleles compiled within http://www.mlst.net. From this database, one or more MLST sequence types (STs) that comprised a PSGS genotype were identified. The end result of more PSGS genotypes (163) than conventional STs actually tested (155) was primarily due to amplification failures of 1 to 3 targets. In many instances, the PSGS genotype could provide resolution of single- and double-locus variants. This molecular serotyping/genotyping scheme is well suited to rapid characterization of large sets of pneumococcal isolates.
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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.