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Background: Dengue and chikungunya are co-circulating vector-borne diseases with substantial overlap in clinical presentations. It is important to differentiate between them during first presentation as their management, especially for dengue hemorrhagic fever (DHF), is different. This study compares their clinical presentation in Singapore adults to derive predictors to assist doctors in diagnostic decision-making. Methods: We compared 117 patients with chikungunya infection diagnosed with reverse transcription-polymerase chain reaction (RT-PCR) with 917 dengue RT-PCR-positive adult patients (including 55 with DHF). We compared dengue fever (DF), DHF, and chikungunya infections by evaluating clinical characteristics of dengue and chikungunya; developing classification tools via multivariate logistic regression models and classification trees of disease etiology using clinical and laboratory factors; and assessing the time course of several clinical variables. Findings: At first presentation to hospital, significantly more chikungunya patients had myalgia or arthralgia, and fewer had a sore throat, cough (for DF), nausea, vomiting, diarrhea, abdominal pain, anorexia or tachycardia than DF or DHF patients. From the decision trees, platelets <118×109/L was the only distinguishing feature for DF versus chikungunya with an overall correct classification of 89%. For DHF versus chikungunya using platelets <100×109/L and the presence of bleeding, the overall correct classification was 98%. The time course analysis supported platelet count as the key distinguishing variable. Interpretation: There is substantial overlap in clinical presentation between dengue and chikungunya infections, but simple clinical and laboratory variables can predict these infections at presentation for appropriate management. Author Summary: Dengue and chikungunya are mosquito-borne diseases that are found in similar geographical areas and present with similar symptoms. As their treatment is different, especially for dengue haemorraghic fever (DHF) which is a more severe form of dengue, it is important for healthcare workers to differentiate between them. We studied 117 chikungunya and 917 dengue adult patients (including 55 with DHF) by comparing their clinical presentation and developed decision trees to classify them using simple symptoms and laboratory tests. From the study, we found that at their first appearance in hospital, more chikungunya patients had muscle or joint pains, and fewer had a sore throat, cough, nausea, vomiting, diarrhea, stomach pain, loss of appetite or fast heart beat than DF or DHF patients. From the decision trees, of DF versus chikungunya using only platelet level cut-offs, we could correctly classify 89% of the cases. For DHF versus chikungunya using platelet level cut-offs and the presence of bleeding, the correct classification was 98%. The use of these simple decision trees can therefore predict the subsequent development of these infections for appropriate treatment. [ABSTRACT FROM AUTHOR]

Background. Chikungunya virus (CHIKV) and related arboviruses have been responsible for large epidemic outbreaks with serious economic and social impact. Although infected individuals clear the virus from the blood, some develop debilitating and prolonged arthralgia. Methods. We investigated specificity and strength of antibody responses in a longitudinal study on CHIKV-infected patients and analyzed their association with viral load, cytokine profile, and severity. Results. We found that CHIKV-specific response is dominated by immunoglobulin G3 (IgG3) antibodies. The antibodies were neutralizing, and patients with high viremia rapidly developed high levels of anti-CHIKV antibodies of this specific isotype. Although these patients endured a more severe disease progression during the acute viremic phase, they cleared the virus faster and did not experience persistent arthralgia. However, significant persistent arthralgia was observed in patients with low viremia who developed IgG3 at a later stage. Conclusions. Absence of early CHIKV-specific IgG3 may therefore serve as a specific marker of patients with increased risk of disease. [ABSTRACT FROM AUTHOR]

Background. Chikungunya virus (CHIKV) infection induces arthralgia. The involvement of inflammatory cytokines and chemokines has been suggested, but very little is known about their secretion profile in CHIKV-infected patients. Methods. A case-control longitudinal study was performed that involved 30 adult patients with laboratory-confirmed Chikungunya fever. Their profiles of clinical disease, viral load, and immune mediators were investigated. Results. When patients were segregated into high viral load and low viral load groups during the acute phase, those with high viremia had lymphopenia, lower levels of monocytes, neutrophilia, and signs of inflammation. The high viral load group was also characterized by a higher production of pro-inflammatory cytokines, such as interferon-a and interleukin (IL)-6, during the acute phase. As the disease progressed to the chronic phase, IL-17 became detectable. However, persistent arthralgia was associated with higher levels of IL-6 and granulocyte macrophage colony-stimulating factor, whereas patients who recovered fully had high levels of Eotaxin and hepatocyte growth factor. Conclusions. The level of CHIKV viremia during the acute phase determined specific patterns of pro-inflammatory cytokines, which were associated with disease severity. At the chronic phase, levels of IL-6, and granulocyte macrophage colony-stimulating factor found to be associated with persistent arthralgia provide a possible explanation for the etiology of arthralgia that plagues numerous CHIKV-infected patients. [ABSTRACT FROM AUTHOR]

Background: The sensitivity and specificity of two in-house MAC-ELISA assays were tested and compared with the performance of commercially-available CTK lateral flow rapid test and EUROIMMUN IFA assays for the detection of anti-Chikungunya virus (CHIKV) IgM. Each MAC-ELISA assay used a whole virus-based antigen derived from genetically distinct CHIKV strains involved in two chikungunya disease outbreaks in Singapore (2008); a January outbreak strain with alanine at amino acid residue 226 of the E1 glycoprotein (CHIKV-A226) and a May-to-September outbreak strain that possessed valine at the same residue (CHIKV-226V). We report differences in IgM detection efficacy of different assays between the two outbreaks. The sensitivities of two PCR protocols were also tested. Methods and Findings: For sera from January outbreak, the average detection threshold of CTK lateral flow test, MAC-ELISAs and EUROIMMUN IFA assays was 3.75, 4.38 and 4.88 days post fever onset respectively. In contrast, IgM detection using CTK lateral flow test was delayed to more than 7 days after fever onset in the second outbreak sera. However, MAC-ELISA using CHIKV-226V detected IgM in the second outbreak sera 3.96 days after fever onset, which was approximately one day earlier compared to the same assay using CHIKV-A226 (4.86 days). Specificity was 100% for both commercial assays, and 95.6% for the in-house MAC-ELISAs. For sensitivity determination of the PCR protocols, the probe-based real time RT-PCR method was found to be 10 times more sensitive than one based on SYBR Green. Conclusion: Our findings suggested that the two strains of CHIKV using variants A226 and 226V resulted in variation in sensitivities of the assays evaluated. We postulated that the observed difference in antigen efficacy could be due to the amino acid substitution differences in viral E1 and E2 envelope proteins, especially the E1-A226V substitution. This evaluation demonstrates the importance of appraisal of different diagnostic assays before their application in clinical and operational settings. Author Summary: Chikungunya is a mounting public health concern in many parts of the world. Definitive diagnosis is critical in differentiating the diseases, especially in dengue endemic areas. There are some commercial chikungunya kits and published molecular protocols available, but no comprehensive comparative evaluation of them was performed. Using sera collected in outbreaks caused by two variants of Chikungunya virus (A226 and 226V), we tested 2 commercial IgM tests (CTK lateral flow rapid test and EUROIMMUN IFA) alongside our in-house IgM assays (using both variants of the virus). Sensitivities of 2 published PCR protocols were also evaluated based on RNA standards derived from cell-cultured viruses. The commercial assays had different performances in each outbreak, with CTK's lateral flow test having the best performance in the first outbreak and EUROIMMUN IFA being more sensitive in the second outbreak. Use of the current circulating virus in a test assay improves sensitivity of the MAC-ELISAs. For PCR, a probe-based real time RT-PCR method was found to be 10 times more sensitive than the SYBR Green method. Despite this, the latter protocol is found to be more suitable and cost-effective for our diagnostic laboratory. This evaluation demonstrates the importance of appraisal of commercial kits and published protocols before application of a diagnostic tool in the clinical and operational setting. [ABSTRACT FROM AUTHOR]

Background: Little is known about the immunopathogenesis of Chikungunya virus. Circulating levels of immune mediators and growth factors were analyzed from patients infected during the first Singaporean Chikungunya fever outbreak in early 2008 to establish biomarkers associated with infection and/or disease severity. Methods and Findings: Adult patients with laboratory-confirmed Chikungunya fever infection, who were referred to the Communicable Disease Centre/Tan Tock Seng Hospital during the period from January to February 2008, were included in this retrospective study. Plasma fractions were analyzed using a multiplex-microbead immunoassay. Among the patients, the most common clinical features were fever (100%), arthralgia (90%), rash (50%) and conjunctivitis (40%). Profiles of 30 cytokines, chemokines, and growth factors were able to discriminate the clinical forms of Chikungunya from healthy controls, with patients classified as non-severe and severe disease. Levels of 8 plasma cytokines and 4 growth factors were significantly elevated. Statistical analysis showed that an increase in IL-1b, IL-6 and a decrease in RANTES were associated with disease severity. Conclusions: This is the first comprehensive report on the production of cytokines, chemokines, and growth factors during acute Chikungunya virus infection. Using these biomarkers, we were able to distinguish between mild disease and more severe forms of Chikungunya fever, thus enabling the identification of patients with poor prognosis and monitoring of the disease. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented in response to the article "Interleukin 6, RANKL and osteoprotegerin expression by chikungunya virus infected human osteoblast" by M. Noret, L. Herrero and M. Rolph in the previous issue.

The article presents a letter to the editor on Chikungunya outbreak in Asia in 2008.

Population-based serosurveillance studies provide critical estimates on community-level immunity and the potential for future outbreaks. Currently, serological assays, such as IgG enzyme-linked immunosorbent assays (ELISAs) and indirect immunofluorescence tests (IIFT) based on the inactivated whole virus are used to determine past Chikungunya virus (CHIKV) infection. However, these commercially available tests have variable sensitivities. To develop and evaluate recombinant based CHIKV-specific IgG antibody capture ELISAs (GAC-ELISAs), baculoviruses carrying wild-type (E1-A226, named WT) or mutant (E1-A226V, named MUT) E1 envelope protein genes of CHIKV were generated. The seroreactivity of recombinant CHIKV WT and MUT envelope proteins were determined using residual blood, collected from CHIKV-confirmed patients. The sensitivities of both recombinant CHIKV envelope proteins were 83.0% as measured by GAC-ELISAs. The specificities of both recombinant proteins were 87.8%. These GAC-ELISAs were also able to detect the persistence of anti-CHIKV IgG antibodies up to 6 months after the disease onset, together with rise in sensitivities with increasing time. These results suggest that the baculovirus purified recombinant CHIKV envelope proteins react with anti-CHIKV IgG antibodies and may be useful in population-based seroprevalence surveys. In addition, these GAC-ELISAs offer good diagnostic value to determine the recent/past CHIKV infection status in non-endemic populations. [ABSTRACT FROM AUTHOR]

Chikungunya virus (CHIKV) is a mosquito-borne arthralgia arbovirus that is reemergent in sub-Saharan Africa and Southeast Asia. CHIKV infection has been shown to be self-limiting, but the molecular mechanisms of the innate immune response that control CHIKV replication remain undefined. Here, longitudinal transcriptional analyses of PBMCs from a cohort of CHIKV-infected patients revealed that type I IFNs controlled CHIKV infection via RSAD2 (which encodes viperin), an enigmatic multifunctional IFN-stimulated gene (ISG). Viperin was highly induced in monocytes, the major target cell of CHIKV in blood. Anti-CHIKV functions of viperin were dependent on its localization in the ER, and the N-terminal amphipathic a-helical domain was crucial for its antiviral activity in controlling CHIKV replication. Furthermore, mice lacking Rsad2 had higher viremia and severe joint inflammation compared with wild-type mice. Our data demonstrate that viperin is a critical antiviral host protein that controls CHIKV infection and provide a preclinical basis for the design of effective control strategies against CHIKV and other reemerging arthrogenic alphaviruses. [ABSTRACT FROM AUTHOR]

Chikungunya virus (CHIKV) is an alphavirus which causes chronic and incapacitating arthralgia in humans. Although previous studies have shown that antibodies against the virus are produced during and after infection, the fine specificity of the antibody response against CHIKV is not known. Here, using plasma from patients at different times postinfection, we characterized the antibody response against various proteins of the virus. We have shown that the E2 and E3 glycoproteins and the capsid and nsP3 proteins are targets of the anti-CHIKV antibody response. Moreover, we have identified the different regions in these proteins which contain the linear epitopes recognized by the anti-CHIKV antibodies and determined their structural localization. Data also illustrated the effect of a single K252Q amino acid change at the E2 glycoprotein that was able to influence antibody binding and interaction between the antibodies and epitope because of the changes of epitope-antibody binding capacity. This study provides important knowledge that will not only aid in the understanding of the immune response to CHIKV infection but also provide new knowledge in the design of modern vaccine development. Furthermore, these pathogen-specific epitopes could be used for future seroepidemiological studies that will unravel the molecular mechanisms of human immunity and protection from CHIKV disease. [ABSTRACT FROM AUTHOR]