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      2 nd International External Quality Control Assessment for the Molecular Diagnosis of Dengue Infections

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          Abstract

          Background

          Currently dengue viruses (DENV) pose an increasing threat to over 2.5 billion people in over 100 tropical and sub-tropical countries worldwide. International air travel is facilitating rapid global movement of DENV, increasing the risk of severe dengue epidemics by introducing different serotypes. Accurate diagnosis is critical for early initiation of preventive measures. Different reverse transcriptase PCR (RT-PCR) methods are available, which should be evaluated and standardized. Epidemiological and laboratory-based surveillance is required to monitor and guide dengue prevention and control programmes, i.e., by mosquito control or possible vaccination (as soon as an effective and safe vaccine becomes available).

          Objective

          The purpose of the external quality assurance (EQA) study described is to assess the efficiency and accuracy of dengue molecular diagnosis methods applied by expert laboratories.

          Study Design

          A panel of 12 human plasma samples was distributed and tested for DENV-specific RNA. The panel comprised 9 samples spiked with different DENV serotypes (DENV-1 to DENV-4), including 10-fold dilution series of DENV-1 and DENV-3. Two specificity controls consisted of a sample with a pool of 4 other flaviviruses and a sample with chikungunya virus. A negative control sample was also included.

          Results

          Thirty-seven laboratories (from Europe, Middle East Asia, Asia, the Americas/Caribbean, and Africa) participated in this EQA study, and reports including 46 sets of results were returned. Performance among laboratories varied according to methodologies used. Only 5 (10.9%) data sets met all criteria with optimal performance, and 4 (8.7%) with acceptable performance, while 37 (80.4%) reported results showed the need for improvement regarding accomplishment of dengue molecular diagnosis. Failures were mainly due to lack of sensitivity and the presence of false positives.

          Conclusions

          The EQA provides information on each laboratory's efficacy of RT-PCR techniques for dengue diagnosis and indicates for most laboratories an urgent need to improve sensitivity and specificity.

          Author Summary

          Dengue viruses (DENV) are the most widespread arthropod-borne viruses which have shown an unexpected geographic expansion, as well as an increase in the number and severity of outbreaks in the last decades. In this context, the accurate diagnosis and reliable surveillance of dengue infections are essential. The laboratory diagnosis of dengue relies on the use of several methods detecting markers of DENV infection present in patient serum. Molecular diagnosis methods are usually rapid, sensitive, and simple when correctly standardized. Moreover, PCR-based diagnosis techniques are able to readily detect DENV during the acute phase of the disease and may assume an important role in dengue diagnosis and surveillance. Different reverse transcriptase PCR (RT-PCR) methods have been developed and are currently available and should be standardized in each laboratory to maintain high quality performance. In this work an External quality assessment (EQA) activity has been carried out to evaluate the accuracy and quality of laboratory data for the molecular diagnosis and surveillance of dengue, which involved worldwide dengue reference laboratories. In conclusion, RT-PCR techniques for dengue diagnosis applied by the participating laboratories demonstrated the need of further improvement in most laboratories.

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          Most cited references 27

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          Dengue viremia titer, antibody response pattern, and virus serotype correlate with disease severity.

          Viremia titers in serial plasma samples from 168 children with acute dengue virus infection who were enrolled in a prospective study at 2 hospitals in Thailand were examined to determine the role of virus load in the pathogenesis of dengue hemorrhagic fever (DHF). The infecting virus serotype was identified for 165 patients (DEN-1, 46 patients; DEN-2, 47 patients; DEN-3, 47 patients, DEN-4, 25 patients). Patients with DEN-2 infections experienced more severe disease than those infected with other serotypes. Eighty-one percent of patients experienced a secondary dengue virus infection that was associated with more severe disease. Viremia titers were determined for 41 DEN-1 and 46 DEN-2 patients. Higher peak titers were associated with increased disease severity for the 31 patients with a peak titer identified (mean titer of 107.6 for those with dengue fever vs. 108.5 for patients with DHF, P=.01). Increased dengue disease severity correlated with high viremia titer, secondary dengue virus infection, and DEN-2 virus type.
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            Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction.

            We report on the development and application of a rapid assay for detecting and typing dengue viruses. Oligonucleotide consensus primers were designed to anneal to any of the four dengue virus types and amplify a 511-bp product in a reverse transcriptase-polymerase chain reaction (PCR). First, we produced a cDNA copy of a portion of the viral genome in a reverse transcriptase reaction in the presence of primer D2 and then carried out a standard PCR (35 cycles of heat denaturation, annealing, and primer extension) with the addition of primer D1. The resulting double-stranded DNA product of the RT-PCR was typed by two methods: dot blot hybridization of the 511-bp amplified product to dengue virus type-specific probes or a second round of PCR amplification (nested PCR) with type-specific primers, yielding DNA products the unique sizes of which were diagnostic for each dengue virus serotype. The accumulated data demonstrated that dengue viruses can be accurately detected and typed from viremic human serum samples.
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              Rapid detection and quantification of RNA of Ebola and Marburg viruses, Lassa virus, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, dengue virus, and yellow fever virus by real-time reverse transcription-PCR.

              Viral hemorrhagic fevers (VHFs) are acute infections with high case fatality rates. Important VHF agents are Ebola and Marburg viruses (MBGV/EBOV), Lassa virus (LASV), Crimean-Congo hemorrhagic fever virus (CCHFV), Rift Valley fever virus (RVFV), dengue virus (DENV), and yellow fever virus (YFV). VHFs are clinically difficult to diagnose and to distinguish; a rapid and reliable laboratory diagnosis is required in suspected cases. We have established six one-step, real-time reverse transcription-PCR assays for these pathogens based on the Superscript reverse transcriptase-Platinum Taq polymerase enzyme mixture. Novel primers and/or 5'-nuclease detection probes were designed for RVFV, DENV, YFV, and CCHFV by using the latest DNA database entries. PCR products were detected in real time on a LightCycler instrument by using 5'-nuclease technology (RVFV, DENV, and YFV) or SybrGreen dye intercalation (MBGV/EBOV, LASV, and CCHFV). The inhibitory effect of SybrGreen on reverse transcription was overcome by initial immobilization of the dye in the reaction capillaries. Universal cycling conditions for SybrGreen and 5'-nuclease probe detection were established. Thus, up to three assays could be performed in parallel, facilitating rapid testing for several pathogens. All assays were thoroughly optimized and validated in terms of analytical sensitivity by using in vitro-transcribed RNA. The >or=95% detection limits as determined by probit regression analysis ranged from 1,545 to 2,835 viral genome equivalents/ml of serum (8.6 to 16 RNA copies per assay). The suitability of the assays was exemplified by detection and quantification of viral RNA in serum samples of VHF patients.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Negl Trop Dis
                plos
                plosntds
                PLoS Neglected Tropical Diseases
                Public Library of Science (San Francisco, USA )
                1935-2727
                1935-2735
                October 2010
                5 October 2010
                : 4
                : 10
                Affiliations
                [1 ]Robert Koch Institute, Berlin, Germany
                [2 ]United States Army Medical Component of the Armed Forces Research Institute of the Medical Sciences (USAMC-AFRIMS), Bangkok, Thailand
                University of California, Berkeley, United States of America
                Author notes

                Conceived and designed the experiments: CD MN ODM. Performed the experiments: CD AT MK. Analyzed the data: CD MN LR ODM. Contributed reagents/materials/analysis tools: MK RGJ. Wrote the paper: CD. Critically revised the manuscript: ODM MK LR. Critically reviewed the manuscript: MN RGJ.

                Article
                10-PNTD-RA-0924R3
                10.1371/journal.pntd.0000833
                2950135
                20957194
                This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
                Page count
                Pages: 9
                Categories
                Research Article
                Infectious Diseases/Neglected Tropical Diseases
                Infectious Diseases/Tropical and Travel-Associated Diseases
                Virology/Diagnosis
                Virology/Emerging Viral Diseases

                Infectious disease & Microbiology

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