Evaluation of Two New Commercial Tests for the Diagnosis of Acute Dengue Virus Infection Using NS1 Antigen Detection in Human Serum

Dengue is a viral disease transmitted by mosquitoes that is endemic in more than 100 countries in tropical areas, threatening over 2.5 billion people. It causes a wide range of symptoms and has severe forms. In reference laboratories, dengue disease is confirmed by virus isolation or genome detection during the acute phase, and by serological methods during the early convalescent phase. The viral NS1 protein circulates in the sera of infected patients throughout the clinical phase of the disease. Novel diagnostic tests based on NS1 detection have been recently developed and marketed. We compared the performance of two tests for detecting dengue NS1 protein during the clinical phase of dengue infection (an immunochromatographic test (ICT) from Bio-Rad allowing rapid detection of the NS1 antigen and a two-step sandwich-format ELISA from Panbio) with the one-step sandwich-format microplate ELISA (Bio-Rad). The ICT test performed better than the ELISA test from Panbio. This study confirms that diagnostic tests based on NS1 could be used in routine clinical practice in poorly equipped laboratories and that dengue diagnosis could therefore be confirmed without the need for testing in reference laboratories. This represents a crucial step towards the control of dengue disease in the human population

Cross-reactivities between human IgMs and the four serotypes of dengue virus as probed with artificial homodimers of domain-III from the envelope proteins

International audienceBackgroundDengue fever is the most important vector-borne viral disease. Four serotypes of dengue virus, DENV1 to DENV4, coexist. Infection by one serotype elicits long-lasting immunity to that serotype but not the other three. Subsequent infection by a different serotype is a risk factor for severe dengue. Domain III (ED3) of the viral envelope protein interacts with cell receptors and contains epitopes recognized by neutralizing antibodies. We determined the serotype specificity and cross-reactivity of human IgMs directed against ED3 by using a well-characterized collection of 90 DENV-infected and 89 DENV-uninfected human serums.MethodsThe recognitions between the four serotypes of ED3 and the serums were assayed with an IgM antibody-capture ELISA (MAC-ELISA) and artificial homodimeric antigens. The results were analyzed with Receiving Operator Characteristic (ROC) curves.ResultsThe DENV-infected serums contained IgMs that reacted with one or several ED3 serotypes. The discrimination by ED3 between serums infected by the homotypic DENV and uninfected serums varied with the serotype in the decreasing order DENV1 > DENV2 > DENV3 > DENV4. The ED3 domain of DENV1 gave the highest discrimination between DENV-infected and DENV-uninfected serums, whatever the infecting serotype, and thus behaved like a universal ED3 domain for the detection of IgMs against DENV. Some ED3 serotypes discriminated between IgMs directed against the homotypic and heterotypic DENVs. The patterns of cross-reactivities and discriminations varied with the serotype.ConclusionsThe results should help better understand the IgM immune response and protection against DENV since ED3 is widely used as an antigen in diagnostic assays and an immunogen in vaccine candidates

Thermodynamic stability of domain III from the envelope protein of flaviviruses and its improvement by molecular design

International audienceThe Flavivirus genus includes widespread and severe human pathogens like the four serotypes of dengue virus (DENV1 to DENV4), yellow fever virus, Japanese encephalitis virus and West Nile virus. Domain III (ED3) of the viral envelope protein interacts with cell receptors and contains epitopes recognized by virus neutralizing antibodies. Its structural, antigenic and immunogenic properties have been thoroughly studied contrary to its physico-chemical properties. Here, the ED3 domains of the above pathogenic flaviviruses were produced in the periplasm of Escherichia coli. Their thermodynamic stabilities were measured and compared in experiments of unfolding equilibriums, induced with chemicals or heat and monitored through protein fluorescence. A designed ED3 domain, with the consensus sequence of DENV strains from all serotypes, was highly stable. The low stability of the ED3 domain from DENV3 was increased by three changes of residues in the protein core without affecting its reactivity towards DENV-infected human serums. Additional changes showed that the stability of ED3 varied with the DENV3 genotype. The T(m) of ED3 was higher than 69°C for all the tested viruses and reached 86°C for the consensus ED3. The latter, deprived of its disulfide bond by mutations, was predominantly unfolded at 20°C. These results will help better understand and design the properties of ED3 for its use as diagnostic, vaccine or therapeutic tools

Techniques de routine et approches innovantes pour le diagnostic biologique de la dengue

International audienceDengue fever is a major public health problem in intertropical regions due to the concomitant increases in the frequencies of epidemics and of severe forms of the disease. In the absence of a vaccine, control methods are limited to vector-targeting approaches and early rapid differential diagnosis to improve disease surveillance in populations. Various techniques can be used to diagnose dengue virus infection, depending on the clinical phase during which samples are taken. However, some of these techniques have major disadvantages, ranging from the need to collect venous blood samples from children to serological cross-reactivity between the dengue virus and related viruses, making it difficult to interpret the results. Innovative diagnostic approaches have been developed to overcome these problems and to enlarge the range of available technical tools available: early, rapid detection of the viral NS1 antigen by immunocapture, capillary blood sampling as an alternative to venous blood sampling for diagnosis, the use of IgG avidity to discriminate between primary infection and re-infection and the use of recombinant proteins derived from the viral envelope protein to improve the sensitivity and specificity of immunoenzymatic techniques.La dengue constitue un problème majeur de santé publique dans les régions intertropicales du fait de l’augmentation concomitante des épidémies et des formes sévères de la maladie. Dans l’attente d’un vaccin, les moyens de contrôle restent la lutte anti-vectorielle et la mise en œuvre d’un diagnostic d’infection précoce, rapide et différenciel pour un meilleur suivi des populations. À ce titre, différentes techniques, dépendant de la phase clinique au cours de laquelle le patient est prélevé, peuvent être réalisées pour poser un diagnostic d’infection par le virus de la dengue. Cependant, certaines d’entres elles présentent des inconvénients majeurs allant de la simple réalisation d’un prélèvement veineux chez un enfant à l’existence de réactions sérologiques croisées entre le virus de la dengue et d’autres virus apparentés qui compromettent l’interprétation des résultats. Pour pallier à ces contraintes et élargir par là même l’éventail des outils techniques, de nouvelles approches diagnostiques ont été mises en œuvre : la détection précoce et rapide d’un antigène viral NS1 par immunocapture, le prélèvement de sang capillaire comme alternative au prélèvement veineux pour le diagnostic, l’avidité des IgG pour discriminer une infection primaire d’une réinfection par DENV ou encore l’utilisation de protéines recombinantes issues de la protéine d’enveloppe du virus pour une meilleure sensibilité et spécificité des techniques immuno-enzymatiques

Propriétés virologiques et sérologiques de l’infection à virus Chikungunya : bilan de l’épidémie dans les DFA

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The use of serum spotted onto filter paper for diagnosing and monitoring Chikungunya virus infection

AbstractBackgroundThe recent emergence of Chikungunya Virus (CHIKV) in the Americas constitutes a major public health problem on this continent, where the mosquito vector is widespread. The rapid diagnosis of suspected cases is essential for the monitoring and control of this ongoing outbreak. However, this requires reliable tools that are difficult to establish in areas without specialized laboratories.ObjectivesThe aim was to evaluate the performances of serum samples spotted onto filter paper for molecular and serological diagnosis of Chikungunya infection.Study designAnalyses were performed from frozen sera and serum spotted onto filter paper provided from 121 Chikungunya suspected cases collected at a biological laboratory on Saint-Martin Island.ResultsThis approach performed well in comparisons with standard methods, with a sensitivity of 100% and a specificity of 93.6% for the combined technical approaches (RT-PCR and serological results). Comparisons of serum samples spotted onto filter paper and frozen samples showed a concordance rate of 94.8% in molecular tests and 98.2% in serological tests.ConclusionsThis simple sampling technique could overcome the problems of the lack of efficient CHIKV diagnosis tools in remote regions, providing good results regardless of the molecular or serological approach used. This simple filter paper-based method can be used to diagnose both chikungunya and dengue infections, as previously demonstrated following transport at ambient temperature to specialized laboratories. Given the set-up costs and high performance of this method, it could be recommended for the monitoring and control of Chikungunya virus expansion in the Americas and in other affected regions

Hantavirus Pulmonary Syndrome Caused by Maripa Virus in French Guiana, 2008–2016

International audienceWe report 5 human cases of hantavirus pulmonary syndrome found during surveillance in French Guiana in 2008-2016; of the 5 patients, 4 died. This pathogen should continue to be monitored in humans and rodents in effort to reduce the occurrence of these lethal infections in humans stemming from ecosystem disturbances

Maripa Virus RNA Load and Antibody Response in Hantavirus Pulmonary Syndrome, French Guiana

We report viral RNA loads and antibody responses in 6 severe human cases of Maripa virus infection (2 favorable outcomes) and monitored both measures during the 6-week course of disease in 1 nonfatal case. Further research is needed to determine prevalence of this virus and its effect on other hantaviruses