Detection of chikungunya virus-specific IgM on laser-cut paper-based device using pseudo-particles as capture antigen.

The incidence of arbovirus infections has increased dramatically in recent decades, affecting hundreds of millions of people each year. The Togaviridae family includes the chikungunya virus (CHIKV), which is typically transmitted by Aedes mosquitoes and causes a wide range of symptoms from flu-like fever to severe arthralgia. Although conventional diagnostic tests can provide early diagnosis of CHIKV infections, access to these tests is often limited in developing countries. Consequently, there is an urgent need to develop efficient, affordable, simple, rapid, and robust diagnostic tools that can be used in point-of-care settings. Early diagnosis is crucial to improve patient management and to reduce the risk of complications. A glass-fiber laser-cut microfluidic device (paper-based analytical device [PAD]) was designed and evaluated in a proof of principle context, for the analysis of 30 µL of patient serum. Biological raw materials used for the functionalization of the PAD were first screened by MAC-ELISA (IgM capture enzyme-linked immunosorbent assay) for CHIKV Immunoglobulin M (IgM) capture and then evaluated on the PAD using various human samples. Compared with viral lysate traditionally used for chikungunya (CHIK) serology, CHIKV pseudo-particles (PPs) have proven to be powerful antigens for specific IgM capture. The PAD was able to detect CHIKV IgM in human sera in less than 10 minutes. Results obtained in patient sera showed a sensitivity of 70.6% and a specificity of around 98%. The PAD showed few cross-reactions with other tropical viral diseases. The PAD could help health workers in the early diagnosis of tropical diseases such as CHIK, which require specific management protocols in at-risk populations.

Paper-based point-of-care testing for cost-effective diagnosis of acute flavivirus infections.

Flavivirus infections are a serious healthcare concern in tropical and subtropical countries. Although well-established laboratory tests can provide early diagnosis of acute dengue or Zika infections, access to these tests is limited in developing countries, presenting an urgent need to develop simple, rapid, and robust diagnostic tools. Microfluidic Paper-based Analytical Devices (µPAD), are typically rapid, cost-effective, user-friendly, and they can be used as diagnostic tools for the diagnosis of these infections at Point of Care settings. Early and prompt diagnosis is crucial to improve patient management and reduce the risk of complications. In the present study, we developed and evaluated a wax-printed paper-based device for the detection of the dengue and Zika non-structural NS1 viral protein in blood and plasma. Experiments have been carried out to increase specificity, while maintaining the required sensitivity. As a consequence, the quality of the raw materials and the washing steps were proved to be crucial. The µPAD was able to detect specifically in 6-8 min 10 ng/mL of protein in various sample types. A prototype for the differential detection of dengue and/or Zika NS1 protein was developed. The reading of the results was simplified by using a dedicated application on a smartphone.

Differential proteomic analysis of virus-enriched fractions obtained from plasma pools of patients with dengue fever or severe dengue.

BACKGROUND: Dengue is the most widespread mosquito-borne viral disease of public health concern. In some patients, endothelial cell and platelet dysfunction lead to life-threatening hemorrhagic dengue fever or dengue shock syndrome. Prognostication of disease severity is urgently required to improve patient management. The pathogenesis of severe dengue has not been fully elucidated, and the role of host proteins associated with viral particles has received little exploration. METHODS: The proteomes of virion-enriched fractions purified from plasma pools of patients with dengue fever or severe dengue were compared. Virions were purified by ultracentrifugation combined with a water-insoluble polyelectrolyte-based technique. Following in-gel hydrolysis, peptides were analyzed by nano-liquid chromatography coupled to ion trap mass spectrometry and identified using data libraries. RESULTS: Both dengue fever and severe dengue viral-enriched fractions contained identifiable viral envelope proteins and host cellular proteins. Canonical pathway analysis revealed the identified host proteins are mainly involved in the coagulation cascade, complement pathway or acute phase response signaling pathway. Some host proteins were over- or under-represented in plasma from patients with severe dengue compared to patients with dengue fever. ELISAs were used to validate differential expression of a selection of identified host proteins in individual plasma samples of patients with dengue fever compared to patients with severe dengue. Among 22 host proteins tested, two could differentiate between dengue fever and severe dengue in two independent cohorts (olfactomedin-4: area under the curve (AUC), 0.958; and platelet factor-4: AUC, 0.836). CONCLUSION: A novel technique of virion-enrichment from plasma has allowed to identify two host proteins that have prognostic value for classifying patients with acute dengue who are more likely to develop a severe dengue. The impact of these host proteins on pathogenicity and disease outcome are discussed.

Severe dengue : from pathogenicity hypotheses to pronostic tools.

Dengue is an endemic viral disease present in inter-tropical countries. If dengue is usually benign, more severe forms (severe dengue [SD]) may lead to serious complications. The prognosis of SD is currently unreliable. To improve the prognosis, it could be necessary to know the key elements of the pathogenicity of the SD. Many hypotheses have been developed to explain a higher pathogenicity in SD patients. Numerous studies have highlighted the role of the host immune response and of the infecting virus strain. The development of these hypothesis allows to have a better understanding of the pathogenesis and consequently, to provide prognostic candidate-markers of SD, these markers being either associated with the host or with the virus. The present review proposes to paint a non-exhaustive picture of the most important hypothesis of dengue pathogenicity as well as potential prognostic markers of severe forms of dengue.

Procalcitonin detection in human plasma specimens using a fast version of proximity extension assay.

An exciting trend in clinical diagnostics is the development of easy-to-use, minimally invasive assays for screening and prevention of disease at the point of care. Proximity Extension Assay (PEA), an homogeneous, dual-recognition immunoassay, has proven to be sensitive, specific and convenient for detection or quantitation of one or multiple analytes in human plasma. In this paper, the PEA principle was applied to the detection of procalcitonin (PCT), a widely used biomarker for the identification of bacterial infection. A simple, short PEA protocol, with an assay time suitable for point-of-care diagnostics, is presented here as a proof of concept. Pairs of oligonucleotides and monoclonal antibodies were selected to generate tools specifically adapted to the development of an efficient PEA for PCT detection. The assay time was reduced by more than 13-fold compared to published versions of PEA, without significantly affecting assay performance. It was also demonstrated that T4 DNA polymerase could advantageously be replaced by other polymerases having strong 3'>5' exonuclease activity. The sensitivity of this improved assay was determined to be about 0.1 ng/mL of PCT in plasma specimen. The potential use of such an assay in an integrated system for the low-plex detection of biomarkers in human specimen at the point of care was discussed.

Nonstructural protein NS1 immunodominant epitope detected specifically in dengue virus infected material by a SELDI-TOF/MS based assay.

Dengue virus (DV) infection is the most common mosquito-born viral disease of public health significance. Though most patients only suffer from flu-like symptoms, a small group of patients experiences more severe forms of the disease. The viral nonstructural protein 1 (NS1), a secreted protein correlating with viremia, is a key element used for dengue diagnosis with potential implications in severe dengue prognosis. Capture-ELISAs for the early detection of the NS1 protein in the sera during the acute febrile stage are commonly used in routine by diagnostic laboratories. In this study, the detection of NS1 protein in DV-infected material was assessed by an alternative method combining a single NS1-directed monoclonal antibody and the SELDI-TOF/MS technology. According to the epitope mapping, the antibodies used are mainly directed against an immuno-dominant peptide located on the C-terminal part of the protein. The NS1 SELDI-TOF assay is specific, has a sensitivity level close to capture-ELISAs and is potentially useful for a coupled serotyping/detection assay or for the detection of subtle post-translational modifications on the protein.

Isotope Coded Protein Labeling analysis of plasma specimens from acute severe dengue fever patients.

BACKGROUND: Dengue fever is the most important arthropod born viral disease of public health significance. Although most patients suffer only from flu-like symptoms, a small group of patient experiences more severe forms of the disease. To contribute to a better understanding of its pathogenesis this study aims to identify proteins differentially expressed in a pool of five viremic plasma from severe dengue patients relative to a pool of five non-severe dengue patients. RESULTS: The use of Isotope Coded Protein Labeling (ICPLTM) to analyze plasma depleted of twenty high-abundance proteins allowed for the identification of 51 differentially expressed proteins, which were characterized by mass spectrometry. Using quantitative ELISA, three of these proteins (Leucine-rich glycoprotein 1, Vitamin D binding-protein and Ferritin) were confirmed as having an increased expression in a panel of severe dengue plasma. The proteins identified as overexpressed by ICPLTM in severe dengue plasma involve in clear up action after cell injury, tissue coherence and immune defense. CONCLUSION: This ICPLTM study evaluating differences between acute severe dengue plasmas and acute non-severe dengue plasmas suggests that the three proteins identified are overexpressed early in the course of the disease. Their possible use as biomarkers for the prognostic of disease severity is discussed.

Comparative study of chikungunya Virus-Like Particles and Pseudotyped-Particles used for serological detection of specific immunoglobulin M.

The incidence of chikungunya virus (CHIKV) infection has increased dramatically in recent decades. Effective diagnostic methods must be available to optimize patient management. IgM-capture Enzyme-Linked Immunosorbent Assay (MAC-ELISA) is routinely used for the detection of specific CHIKV IgM. This method requires inactivated CHIKV viral lysate (VL). The use of viral bioparticles such as Virus-Like Particles (VLPs) and Pseudotyped-Particles (PPs) could represent an alternative to VL. Bioparticles performances were established by MAC-ELISA; physico-chemical characterizations were performed by field-flow fractionation (HF5) and confirmed by electron microscopy. Non-purified PPs give a detection signal higher than for VL. Results suggested that the signal difference observed in MAC-ELISA was probably due to the intrinsic antigenic properties of particles. The use of CHIKV bioparticles such as VLPs and PPs represents an attractive alternative to VL. Compared to VL and VLPs, non-purified PPs have proven to be more powerful antigens for specific IgM capture.

HIV-1 acute infection env glycomutants designed from 3D model: effects on processing, antigenicity, and neutralization sensitivity.

The human immunodeficiency virus type 1 (HIV-1) envelope protein (Env) has evolved to limit its overall immunogenicity by extensive glycosylation. Only a few studies dealing with glycosylation sites have taken into account available 3D data in a global approach. We compared primary env sequences from patients with acute HIV-1 infection. Conserved N-glycosylation sites were placed on the gp120-3D model. Based on vicinity, we defined glycosylation clusters. According to these clusters, we engineered plasmids encoding deglycosylated gp160 mutants. We also constructed mutants corresponding to nonclustered glycans or to the full deglycosylation of the V1 or V2 loop. After in vitro expression, mutants were tested for functionality. We also compared the inhibition of pseudotyped particles infection by human-neutralizing sera. Generally, clustered and nonclustered mutants were affected similarly. Silencing of more than one glycan had deleterious effects, independently of the type of sugar removed. However, some mutants were moderately affected by glycans removal suggesting a distinct role for these N-glycans. Additionally, compared to the wild-type pseudovirus, two of these mutants were neutralized at higher sera dilutions strengthening the importance of the location of specific N-glycans in limiting the neutralizing response. These results could guide the selection of env mutants with the fewest antigenic and functional alterations but with enhanced neutralization sensitivity.