14th ICID - Poster Abstracts - International Society for Infectious ...

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When citing these abstracts please use the following reference: Author(s) of abstract. Title of abstract [abstract]. Int J Infect Dis 2010;14S1: Abstract number. Please note that the official publication of the International Journal of Infectious Diseases 2010, Volume 14, Supplement 1 is available electronically on http://www.sciencedirect.com Final Abstract Number: 75.022 Session: Diagnostics Date: Friday, March 12, 2010 Time: 12:30-13:30 Room: Poster & Exhibition Area/Ground Level Type: Poster Presentation Detection and differentiation of Dengue infections with highly sensitive real-time PCRs M. Kaiser 1 , D. Bray 2 , K. Benlhassan-Chahour 2 , F. Bissuel 3 , P. Huc Anais 3 , W. Rozenbaum 4 , H. Ellerbrok 5 1 GenExpress, Berlin, Germany, 2 ImmunoClin Ltd, Buckinghamshire, United Kingdom, 3 Louis- Constant Fleming Hospital, Saint-Martin, France, 4 Hopital St. Louis, Paris, France, 5 Robert Koch Institute, Berlin, Germany Background: Dengue is one of the most important arboviral diseases in tropical and sub-tropical countries caused by infection with one of the four dengue virus (DENV) serotypes. The disease is endemic in more than 100 countries around the globe and an estimated 50 to 100 million cases of acute febrile disease are caused by DENV each year. For treatment rapid, early diagnosis is a prerequisit for efficient treatment of infected individuals. Routine clinical diagnosis is based on serological assays as well as on RT-PCR. Methods: We developed a real-time RT-PCR generic assay for highly sensitive detection of all four DENV serotypes. DENV-positive samples can be differentiated with a new real-time multiplex assay allowing differentiation of all 4 serotypes. Results: The assay was validated with clinical samples. Serum from patients from the island of Saint Martin was collect for routine diagnosis of DENV infection with IgM and/or IgG ELISA or NS1 antigen capture assay and in addition RT-PCR at the hospital. DENV positive samples were re-analyzed with the new generic real-time RT-PCR assay and differentiated with the multiplex assay. Comparing these real-time RT-PCR results with the clinical data demonstrated a significantly increased sensitivity and specificity for the new assay. 6 out of 36 samples positive in ELISA but previously negative in RT-PCR were tested positive with the new real-time RT-PCR assays. In addition one sample, previously characterized as DENV1-positive was shown to be DENV2 infected. This was confirmed by sequencing data. Conclusion: We could demonstrate that our new real-time PCR based approach resulted in improved detection and differentiation of Dengue viruses.
When citing these abstracts please use the following reference: Author(s) of abstract. Title of abstract [abstract]. Int J Infect Dis 2010;14S1: Abstract number. Please note that the official publication of the International Journal of Infectious Diseases 2010, Volume 14, Supplement 1 is available electronically on http://www.sciencedirect.com Final Abstract Number: 75.023 Session: Diagnostics Date: Friday, March 12, 2010 Time: 12:30-13:30 Room: Poster & Exhibition Area/Ground Level Type: Poster Presentation An automated workflow for high throughput MLVA using the BioNumerics® software, able to deal with varying experimental settings L. Del Rosario 1 , B. Pot 2 , J. Goris 2 , K. Janssens 2 , P. Vauterin 2 , L. Vauterin 2 1 Applied Maths Inc., Austin, TX, USA, 2 Applied Maths NV, Sint-Martens-Latem, Belgium Background: Multi Locus VNTR Analysis (MLVA) is a method for high -resolution typing of microbial isolates based upon Variable copy Numbers of Tandem Repeats (VNTR). The use of capillary sequencers for determining fragment lengths allows for a cost- and time-effective determination of repeat numbers, since multiple VNTRs with non-overlapping size ranges can easily be pooled and loaded in a single capillary. However, the resulting complexity of the pooling schemes and the amount of data generated require an automated workflow for acquisition and processing of fragment files. Moreover, it is well-known that fragment sizes, compared between two genotyping laboratories using capillary electrophoresis (CE), can differ considerably because different laboratories may use different CE machines and running conditions. Methods: Data processing: The software handles any pooling strategy (combination of dyes and size-compatible VNTRs) which can be parsed from the file name. Thus, each VNTR is defined by a pool, dye and expected size range, defined by the repeat length, offset and expected copy range. Using that information the software will automatically screen (in batch) all theoretical ranges for each VNTR and report on the peak(s) found (or not) in those ranges. Results: The resulting VNTR information is stored in integer-type character sets where each VNTR represents one character. For comparisons within the same laboratory or between laboratories with compatible instruments and procedures, the software allows the user to handle calculations based on an expected band size range, depending on the known offset, repeat length, expected copy number variation and a user-defined tolerance. In order to deal with a possible experimental size shift linked to the CE system used, a custom ‘mapping’ tool was developed, allowing observed sizes for a specific instrument to be mapped to real sizes and exact copy numbers. Conclusion: Data analysis: VNTR data can be analyzed as categorical characters (each different copy number is a different allele) or as quantitative characters. In the latter case, the larger the difference between copy numbers, the less related the organisms are considered. The Minimum Spanning Tree algorithm applied on VNTR data in BioNumerics has proven to be extremely useful for epidemiological study and population genetics.
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