Functional and transcriptional heterogeneity within the massively expanding HLADR+CD38+ CD8 T cell population in acute febrile dengue patients.

IMPORTANCE: CD8 T cells play a crucial role in protecting against intracellular pathogens such as viruses by eliminating infected cells and releasing anti-viral cytokines such as interferon gamma (IFNγ). Consequently, there is significant interest in comprehensively characterizing CD8 T cell responses in acute dengue febrile patients. Previous studies, including our own, have demonstrated that a discrete population of CD8 T cells with HLADR+ CD38+ phenotype undergoes massive expansion during the acute febrile phase of natural dengue virus infection. Although about a third of these massively expanding HLADR+ CD38+ CD8 T cells were also CD69high when examined ex vivo, only a small fraction of them produced IFNγ upon in vitro peptide stimulation. Therefore, to better understand such functional diversity of CD8 T cells responding to dengue virus infection, it is important to know the cytokines/chemokines expressed by these peptide-stimulated HLADR+CD38+ CD8 T cells and the transcriptional profiles that distinguish the CD69+IFNγ+, CD69+IFNγ-, and CD69-IFNγ- subsets.

Investigating the aggregation perspective of Dengue virus proteome.

Dengue viruses are human pathogens that are transmitted through mosquitoes. Apart from the typical symptoms associated with viral fevers, DENV infections are known to cause several neurological complications such as meningitis, encephalitis, intracranial haemorrhage, retinopathies along with the more severe, and sometimes fatal, vascular leakage and dengue shock syndrome. This study was designed to investigate, in detail, the predicted viral protein aggregation prone regions among all serotypes. Further, in order to understand the cross-talk between viral protein aggregation and aggregation of cellular proteins, cross-seeding experiments between the DENV NS1 (1-30), corresponding to the ß-roll domain and the diabetes hallmark protein, amylin, were performed. Various techniques such as fluorescence spectroscopy, circular dichroism, atomic force microscopy and immunoblotting have been employed for this. We observe that the DENV proteomes have many predicted APRs and the NS1 (1-30) of DENV1-3, 2K and capsid anchor of DENV2 and DENV4 are capable of forming amyloids, in vitro. Further, the DENV NS1 (1-30), aggregates are also able to cross-seed and enhance amylin aggregation and vice-versa. This knowledge may lead to an opportunity for designing suitable inhibitors of protein aggregation that may be beneficial for viral infections and comorbidities.

Zika virus infection of retinal cells and the developing mouse eye induces host responses that contrasts to the brain and dengue virus infection.

Zika virus (ZIKV) infection causes ocular and neurological pathologies with ZIKV-induction of developmental abnormalities following in utero infection a major concern. The study here has compared ZIKV and the related dengue virus (DENV) infection in the eye and brain. In vitro, both ZIKV and DENV could infect cell lines representing the retinal pigmented epithelium, endothelial cells, and Mueller cells, with distinct innate responses in each cell type. In a 1-day old mouse challenge model, both ZIKV and DENV infected the brain and eye by day 6 post-infection (pi). ZIKV was present at comparable levels in both tissues, with RNA increasing with time post-infection. DENV infected the brain, but RNA was detected in the eye of less than half of the mice challenged. NanoString analysis demonstrated comparable host responses in the brain for both viruses, including induction of mRNA for myosin light chain-2 (Mly2), and numerous antiviral and inflammatory genes. Notably, mRNA for multiple complement proteins were induced, but C2 and C4a were uniquely induced by ZIKV but not DENV. Consistent with the viral infection in the eye, DENV induced few responses while ZIKV induced substantial inflammatory and antiviral responses. Compared to the brain, ZIKV in the eye did not induce mRNAs such as C3, downregulated Retnla, and upregulated CSF-1. Morphologically, the ZIKV-infected retina demonstrated reduced formation of specific retinal layers. Thus, although ZIKV and DENV can both infect the eye and brain, there are distinct differences in host cell and tissue inflammatory responses that may be relevant to ZIKV replication and disease.

Differential expression of interferon inducible protein: Guanylate binding protein (GBP1 & GBP2) in severe dengue.

Dengue virus is reported to activate endothelial cells (EC), but the precise cause for severe dengue (SD) is not known. Guanylate binding proteins (GBPs) are IFN-inducible proteins secreted by ECs and are involved in the anti-oxidant and anti-viral response. The involvement of GBPs in the pathogenesis of dengue remains under explored. In the present study, we quantified the mRNA and protein levels of GBP1 and 2 during acute, defervescence and convalescent phase in SD-10, dengue without warning sign-15 and dengue with warning sign-25 compared to other febrile illnesses-10 and healthy controls-8 using RT-PCR and ELISA respectively. Lipid peroxidation in plasma samples were measured using the Kei Satoh method. Protein and DNA oxidation were determined by ELISA. The efficacy of the proteins in predicting disease severity was done by Support Vector Machine (SVM) model. A significant (P ≤ 0.01) decrease in the levels of mRNA and protein of both GBP1 and GBP2 was observed during defervescence in both SD and DWW cases. The levels were significantly (P ≤ 0.05) tapered off in SD cases from acute till critical phases compared to other study groups. DNA, protein and lipid oxidation markers showed an increasing trend in SD (P ≤ 0.01). Both GBP1 & 2 were found to be negatively associated plasma leakage and oxidative stress markers. EC's activated with SD serum showed a reduced expression of GBP 1 and 2. Nevertheless, the SVM model revealed that plasma levels of proteins along with clinical symptoms could predict the disease outcomes with higher precision. This is the first study reporting a downregulated expression of GBP1 & 2 and their association with oxidative stress and plasma leakage in dengue cases. This suggests the importance of GBPs in regulating disease manifestation. However, further investigations are required to ascertain its role as a biomarker or therapeutic target in dengue infection.

Defective Mitochondrial Quality Control during Dengue Infection Contributes to Disease Pathogenesis.

Mitochondrial fitness is governed by mitochondrial quality control pathways comprising mitochondrial dynamics and mitochondrial-selective autophagy (mitophagy). Disruption of these processes has been implicated in many human diseases, including viral infections. Here, we report a comprehensive analysis of the effect of dengue infection on host mitochondrial homeostasis and its significance in dengue disease pathogenesis. Despite severe mitochondrial stress and injury, we observed that the pathways of mitochondrial quality control and mitochondrial biogenesis are paradoxically downregulated in dengue-infected human liver cells. This leads to the disruption of mitochondrial homeostasis and the onset of cellular injury and necrotic death in the infected cells. Interestingly, dengue promotes global autophagy but selectively disrupts mitochondrial-selective autophagy (mitophagy). Dengue downregulates the expression of PINK1 and Parkin, the two major proteins involved in tagging the damaged mitochondria for elimination through mitophagy. Mitophagy flux assays also suggest that Parkin-independent pathways of mitophagy are also inactive during dengue infection. Dengue infection also disrupts mitochondrial biogenesis by downregulating the master regulators PPARγ and PGC1α. Dengue-infected cells release mitochondrial damage-associated molecular patterns (mtDAMPs) such as mitochondrial DNA into the cytosol and extracellular milieu. Furthermore, the challenge of naive immune cells with culture supernatants from dengue-infected liver cells was sufficient to trigger proinflammatory signaling. In correlation with our in vitro observations, dengue patients have high levels of cell-free mitochondrial DNA in their blood in proportion to the degree of thrombocytopenia. Overall, our study shows how defective mitochondrial homeostasis in dengue-infected liver cells can drive dengue disease pathogenesis. IMPORTANCE Many viruses target host cell mitochondria to create a microenvironment conducive to viral dissemination. Dengue virus also exploits host cell mitochondria to facilitate its viral life cycle. Dengue infection of liver cells leads to severe mitochondrial injury and inhibition of proteins that regulate mitochondrial quality control and biogenesis, thereby disrupting mitochondrial homeostasis. A defect in mitochondrial quality control leads to the accumulation of damaged mitochondria and promotes cellular injury. This leads to the release of mitochondrial damage-associated molecular patterns (mt-DAMPs) into the cell cytoplasm and extracellular milieu. These mt-DAMPs activate the naive immune cells and trigger proinflammatory signaling, leading to the release of cytokines and chemokines, which may trigger systemic inflammation and contribute to dengue disease pathogenesis. In correlation with this, we observed high levels of cell-free mitochondrial DNA in dengue patient blood. This study provides insight into how the disruption of mitochondrial quality control in dengue-infected cells can trigger inflammation and drive dengue disease pathogenesis.

Neurological Complications of Dengue Fever.

PURPOSE OF REVIEW: To discuss the neurological complications of dengue virus (DENV) infection and their pathogenesis. RECENT FINDINGS: Include recognition of the four different serotypes of DENV and their epidemiology as well as recognition of the expanded dengue syndrome encompassing multisystem involvement in the severe form of the disease including involvement of the central nervous system (CNS). DENV is a neurotropic virus with the ability to infect the supporting cells of the CNS. Neural injury during the acute stage of the infection results from direct neuro-invasion and/or the phenomenon of antibody-dependent enhancement, resulting in plasma leakage and coagulopathy. Immune mechanisms have been implicated in the development of the delayed neurological sequelae through molecular mimicry. A myriad of neurological syndromes has been described as a result of the involvement of the CNS, the peripheral nervous system (PNS), or both. Neurological manifestations in DENV infection are increasingly being recognized, some of which are potentially fatal if not treated promptly. DENV encephalopathy and encephalitis should be considered in the differential diagnosis of other acute febrile encephalopathies, autoimmune encephalitides, and in cases of encephalopathy/encephalitis related to SARS-CoV2 infection, especially in dengue-endemic areas. Acute disseminated encephalomyelitis (ADEM) may be occasionally encountered. Clinicians should be knowledgeable of the expanded dengue syndrome characterized by the concurrent compromise of cardiac, neurological, gastrointestinal, renal, and hematopopoietic systems. Isolated cranial nerve palsies occur rather uncommonly and are often steroid responsive. These neuropathies may result from the direct involvement of cranial nerve nuclei or nerve involvement or may be immune-mediated. Even if the diagnosis of dengue is confirmed, it is absolutely imperative to exclude other well-known causes of isolated cranial nerve palsies. Ischemic and hemorrhagic strokes may occur following dengue fever. The pathogenesis may be beyond the commonly observed thrombocytopenia and include cerebral vasculitis. Involvement of ocular blood vessels may cause maculopathy or retinal hemorrhages. Posterior reversible encephalopathy syndrome (PRES) is uncommon and possibly related to dysregulated cytokine release phenomena. Lastly, any patient developing acute neuromuscular weakness during the course or within a fortnight of remission from dengue fever must be screened for acute inflammatory demyelinating polyneuropathy (AIDP), hypokalemic paralysis, or acute myositis. Rarely, a Miller-Fisher-like syndrome with negative anti-GQ1b antibody may develop.

Awareness and perception of malaria and dengue at school and college level in the district of Multan.

The purpose of this study is to examine the awareness and perception of malaria and dengue fever in Multan Punjab, Pakistan while taking into account the important role of government policies and other variables. The goal of this study is to examine the awareness of students in Multan, Pakistan on malaria and dengue. This study is based on a quantitative approach of secondary evidence from scientific journals and questionnaire surveys. It is also based on observational evidence gathered in Multan Punjab Pakistan, in a field study. The survey with school children, teachers and healthcare professionals were both formal and semi-structuralize. Studies have found that malaria and dengue mainly affect children's schooling through their absence, but can also induce brain loss and cognitive disability. In questionnaires, students were seen to have different understanding of the illness, but also to be able to serve as agents of health reform only through teachers. A sample size of 500 respondents has been selected from different colleges of district Multan Punjab, Pakistan. Correlation technique is used for the data analysis. According to our results it is concluded that the students at college level are aware of malaria and dengue diseases, but they are not capable of engaging and serving as agents for health reform. On the basis of results it is recommended that students must teach about epidemics diseases regarding how to handle these diseases.

Phytochemical Compound Screening to Identify Novel Small Molecules against Dengue Virus: A Docking and Dynamics Study.

The spread of the Dengue virus over the world, as well as multiple outbreaks of different serotypes, has resulted in a large number of deaths and a medical emergency, as no viable medications to treat Dengue virus patients have yet been found. In this paper, we provide an in silico virtual screening and molecular dynamics-based analysis to uncover efficient Dengue infection inhibitors. Based on a Google search and literature mining, a large phytochemical library was generated and employed as ligand molecules. In this investigation, the protein target NS2B/NS3 from Dengue was employed, and around 27 compounds were evaluated in a docking study. Phellodendroside (-63 kcal/mole), quercimeritrin (-59.5 kcal/mole), and quercetin-7-O-rutinoside (-54.1 kcal/mole) were chosen based on their binding free energy in MM-GBSA. The tested compounds generated numerous interactions at Lys74, Asn152, and Gln167 residues in the active regions of NS2B/NS3, which is needed for the protein's inhibition. As a result, the stable mode of docked complexes is defined by various descriptors from molecular dynamics simulations, such as RMSD, SASA, Rg, RMSF, and hydrogen bond. The pharmacological properties of the compounds were also investigated, and no toxicity was found in computational ADMET properties calculations. As a result, this computational analysis may aid fellow researchers in developing innovative Dengue virus inhibitors.

A hospital based cross-sectional study on platelet parameters in adult patients with dengue, its serological subgroups, and controls.

Our objective was to analyze if there was a significant relationship between platelet parameters (PLT, MPV, PDW, P-LCR, PCT) among dengue, its serological subgroups and controls. Serologically proven adult patients with dengue {(n = 238) (NS1 positive = 218, IgM positive = 14, NS1 & IgM positive = 6)} and age- and gender-matched controls (n = 254) were included. The MPV, PDW and P-LCR were significantly higher, and PLT and PCT were significantly lower in cases compared with controls. Cases as well as controls showed a positive correlation between PLT and PCT, both parameters individually showed negative correlation with MPV, PDW, P-LCR. MPV, PDW and P-LCR showed positive correlation with each other. The results were similar in the serological subgroups. Comparison of our results with other studies points toward an overall hyperdestructive etiology for thrombocytopenia in dengue. There were two subgroups of cases based on the severity of thrombocytopenia. The mean/median value of all the platelet parameters was lesser in the ≤20k group than the >20k group, except for PDW, which was high although not statistically significant. Suppression of megakaryopoiesis with concomitant immune destruction of platelets in severe dengue could explain low MPV and P-LCR with a high PDW in view of the presence of microthrombocytes as a result of immune destruction. Although an overall hyperdestructive mechanism contributes to thrombocytopenia in dengue, regular monitoring of the platelet indices could reflect the status of megakaryopoiesis and thrombokinetic axis, thus aiding easy determination of pathophysiology and treatment.

Dengue virus infection induces inflammation and oxidative stress on the heart.

OBJECTIVE: Dengue fever is one of the most important arboviral diseases in the world, and its severe forms are characterised by a broad spectrum of systemic and cardiovascular hallmarks. However, much remains to be elucidated regarding the pathogenesis triggered by Dengue virus (DENV) in the heart. Herein, we evaluated the cardiac outcomes unleashed by DENV infection and the possible mechanisms associated with these effects. METHODS: A model of an adapted DENV-3 strain was used to infect male BALB/c mice to assess haemodynamic measurements and the functional, electrophysiological, inflammatory and oxidative parameters in the heart. RESULTS: DENV-3 infection resulted in increased systemic inflammation and vascular permeability with consequent reduction of systolic blood pressure and increase in heart rate. These changes were accompanied by a decrease in the cardiac output and stroke volume, with a reduction trend in the left ventricular end-systolic and end-diastolic diameters and volumes. Also, there was a reduction trend in the calcium current density in the ventricular cardiomyocytes of DENV-3 infected mice. Indeed, DENV-3 infection led to leucocyte infiltration and production of inflammatory mediators in the heart, causing pericarditis and myocarditis. Moreover, increased reactive oxygen species generation and lipoperoxidation were also verified in the cardiac tissue of DENV-3 infected mice. CONCLUSIONS: DENV-3 infection induced a marked cardiac dysfunction, which may be associated with inflammation, oxidative stress and electrophysiological changes in the heart. These findings provide new cardiac insights into the mechanisms involved in the pathogenesis triggered by DENV, contributing to the research of new therapeutic targets for clinical practice.

Transcriptome analysis and connectivity mapping of Cissampelos pareira L. provides molecular links of ESR1 modulation to viral inhibition.

Bioactive fractions obtained from medicinal plants which have been used for the treatment of multiple diseases could exert their effects by targeting common pathways. Prior knowledge of their usage could allow us to identify novel molecular links. In this study, we explored the molecular basis of action of one such herbal formulation Cissampelos pareira L. (Cipa), used for the treatment of female hormone disorders and fever. Transcriptomic studies on MCF7 cell lines treated with Cipa extract carried out using Affymetrix arrays revealed a downregulation of signatures of estrogen response potentially modulated through estrogen receptor α (ERα). Molecular docking analysis identified 38 Cipa constituents that potentially bind (ΔG < - 7.5) with ERα at the same site as estrogen. The expression signatures in the connectivity map ( https://clue.io/; ) revealed high positive scores with translation inhibitors such as emetine (score: 99.61) and knockdown signatures of genes linked to the antiviral response such as ribosomal protein RPL7 (score: 99.92), which is a reported ERα coactivator. Further, gene knockdown experiments revealed that Cipa exhibits antiviral activity in dengue infected MCF7 cells potentially modulated through estrogen receptor 1. This approach reveals a novel pathway involving the ESR1-RPL7 axis which could be a potential target in dengue viral infection.

Differentiating coronavirus disease 2019 (COVID-19) from influenza and dengue.

The novel coronavirus disease 2019 (COVID-19) presents with non-specific clinical features. This may result in misdiagnosis or delayed diagnosis, and lead to further transmission in the community. We aimed to derive early predictors to differentiate COVID-19 from influenza and dengue. The study comprised 126 patients with COVID-19, 171 with influenza and 180 with dengue, who presented within 5 days of symptom onset. All cases were confirmed by reverse transcriptase polymerase chain reaction tests. We used logistic regression models to identify demographics, clinical characteristics and laboratory markers in classifying COVID-19 versus influenza, and COVID-19 versus dengue. The performance of each model was evaluated using receiver operating characteristic (ROC) curves. Shortness of breath was the strongest predictor in the models for differentiating between COVID-19 and influenza, followed by diarrhoea. Higher lymphocyte count was predictive of COVID-19 versus influenza and versus dengue. In the model for differentiating between COVID-19 and dengue, patients with cough and higher platelet count were at increased odds of COVID-19, while headache, joint pain, skin rash and vomiting/nausea were indicative of dengue. The cross-validated area under the ROC curve for all four models was above 0.85. Clinical features and simple laboratory markers for differentiating COVID-19 from influenza and dengue are identified in this study which can be used by primary care physicians in resource limited settings to determine if further investigations or referrals would be required.

Virus-like particles with FLAG-tagged envelope protein as a tetravalent dengue vaccine candidate.

The global incidence of dengue, which is caused by dengue virus (DENV) infection, has grown dramatically in recent decades and secondary infection with heterologous serotype of the virus may cause severe symptoms. Efficacious dengue vaccines should be able to provide long-lasting immunity against all four DENV serotypes simultaneously. In this study, we constructed a novel vaccine platform based on tetravalent dengue virus-like particles (DENV-LPs) in which envelope (E) protein carried a FLAG tag sequence at the position located not only in the exterior loop on the protruding domain but outside of dimerization interface of the protein. We demonstrated an effective strategy to produce the DENV-LPs by transient transfection with expression plasmids for pre-membrane and E proteins of DENV-1 to DENV-4 in mammalian cells and to concentrate and purify them with one-step affinity chromatography. Characteristic features of VLPs such as particle size, shape and density were comparable to flavivirus-like particles reported. The neutralizing activity against all four DENV serotypes was successfully induced by immunization with the purified tetravalent VLPs in mice. Simple, one-step purification systems for VLP vaccine platforms using epitope-tagging strategy should be advantageous for vaccine development not only for dengue but for emerging pandemics in the future.

The relationship between changes in IL-2 / IL-18 and liver enzyme with dengue severity.

A synergistic impact of virulence capacity of dengue viruses and host immune response contributes to the pathogenesis of dengue virus infection (DENV). Elevation of hepatic enzyme and cytokine storm is the major contributory factor producing severity. IL-2 and IL-18 both play a critical role in generating immunogenicity during viral infection. The goal of the study was to evaluate the correlation between alterations in IL-2 and IL-18 levels with alterations in hepatic enzymes in dengue-infected patients. Accordingly, a total of 91 NS1/IgM confirmed DENV infected patients were selected for the IL-2 and IL-18 evaluation using a standard ELISA assay. Biochemical, haematological parameters were recorded at the time of admission. Interestingly, raised levels of IL-2 (p < 0.0001) and IL-18 (p < 0.0001) was obtained in severe dengue as compared to non-severe dengue patients. A significant positive correlation was observed between IL and 2 levels and SGOT (r = 0.3168 with p = 0.002), SGPT (r = 0.569 with p < 0.0001). Similarly IL-18 was also highly associated with SGOT (r = 0.387 with p = 0.0002) and SGPT (r = 0.407 with p < 0.0001) in dengue infected patient.Our data reveal that a rising amount of IL-2 and IL-18 in initial stage of the disease could be predictors for developing severe form of dengue infection.

In Silico Analysis of Dengue Virus Serotype 2 Mutations Detected at the Intrahost Level in Patients with Different Clinical Outcomes.

Intrahost genetic diversity is thought to facilitate arbovirus adaptation to changing environments and hosts, and it may also be linked to viral pathogenesis. Intending to shed light on the viral determinants for severe dengue pathogenesis, we previously analyzed the DENV-2 intrahost genetic diversity in 68 patients clinically classified as dengue fever (n = 31), dengue with warning signs (n = 19), and severe dengue (n = 18), performing viral whole-genome deep sequencing from clinical samples with an amplicon-free approach. From it, we identified a set of 141 relevant mutations distributed throughout the viral genome that deserved further attention. Therefore, we employed molecular modeling to recreate three-dimensional models of the viral proteins and secondary RNA structures to map the mutations and assess their potential effects. Results showed that, in general lines, disruptive variants were identified primarily among dengue fever cases. In contrast, potential immune-escape variants were associated mainly with warning signs and severe cases, in line with the latter's longer intrahost evolution times. Furthermore, several mutations were located on protein-surface regions, with no associated function. They could represent sites of further investigation, as the interaction of viral and host proteins is critical for both host immunomodulation and virus hijacking of the cellular machinery. The present analysis provides new information about the implications of the intrahost genetic diversity of DENV-2, contributing to the knowledge about the viral factors possibly involved in its pathogenesis within the human host. Strengthening our results with functional studies could allow many of these variants to be considered in the design of therapeutic or prophylactic compounds and the improvement of diagnostic assays. IMPORTANCE Previous evidence showed that intrahost genetic diversity in arboviruses may be linked to viral pathogenesis and that one or a few amino acid replacements within a single protein are enough to modify a biological feature of an RNA virus. To assess dengue virus serotype 2 determinants potentially involved in pathogenesis, we previously analyzed the intrahost genetic diversity of the virus in patients with different clinical outcomes and identified a set of 141 mutations that deserved further study. Thus, through a molecular modeling approach, we showed that disruptive variants were identified primarily among cases with mild dengue fever, while potential immune-escape variants were mainly associated with cases of greater severity. We believe that some of the variants pointed out in this study were attractive enough to be potentially considered in future intelligent designs of therapeutic or prophylactic compounds or the improvement of diagnostic tools. The present analysis provides new information about DENV-2 viral factors possibly involved in its pathogenesis within the human host.

Tree Shrew as an Emerging Small Animal Model for Human Viral Infection: A Recent Overview.

Viral infection is a global public health threat causing millions of deaths. A suitable small animal model is essential for viral pathogenesis and host response studies that could be used in antiviral and vaccine development. The tree shrew (Tupaia belangeri or Tupaia belangeri chinenesis), a squirrel-like non-primate small mammal in the Tupaiidae family, has been reported to be susceptible to important human viral pathogens, including hepatitis viruses (e.g., HBV, HCV), respiratory viruses (influenza viruses, SARS-CoV-2, human adenovirus B), arboviruses (Zika virus and dengue virus), and other viruses (e.g., herpes simplex virus, etc.). The pathogenesis of these viruses is not fully understood due to the lack of an economically feasible suitable small animal model mimicking natural infection of human diseases. The tree shrew model significantly contributes towards a better understanding of the infection and pathogenesis of these important human pathogens, highlighting its potential to be used as a viable viral infection model of human viruses. Therefore, in this review, we summarize updates regarding human viral infection in the tree shrew model, which highlights the potential of the tree shrew to be utilized for human viral infection and pathogenesis studies.

A unique double tango: Construct validation and reliability analysis of risk perception, attitude and practice (RPAP) questionnaire on dengue infection.

INTRODUCTION/BACKGROUND: Dengue fever remains a public health threat despite being preventable. A solution to the constant problem of dengue infection will require active intervention and a paradigm shift. Assessing perceived risk and correlating it with the attitude and practice of the community will help in designing appropriate measures. However, possible instruments for these assessments come with limitations. OBJECTIVE: The aim is to develop and validate a new scoring-based questionnaire, using dual statistical approaches to measure risk perception, attitude, and practices (RPAP) related to dengue in the community. METHODS: The RPAP questionnaire was developed bilingually using the International Society for Pharmacoeconomics and Outcome Research (ISPOR) guidelines. Content analysis was reviewed scrupulously by four expert panels. The initial 35-item scale was tested among 253 Malaysian respondents recruited non-probabilistically via multiple online platforms. Two statistical methods were employed to measure the construct validity: Exploratory Factor Analysis (EFA) as part of the Classical Test Theory (CTT) measurement, while Rasch Measurement Analysis (Rasch) was performed for the Item Response Theory (IRT) measurement. All results were cross-validated with their counterpart to ensure stability. Confirmatory Factor Analysis (CFA) was used to obtain a model fit index. RESULTS: 29 questions were retained after the final analysis. Both EFA and Rasch analysis detect multidimensionality. Nine latent factors were extracted from EFA, while only eight factors remained in the final model following CFA: 1) perceived susceptibility; 2) perceived severity; 3) perceived barrier; 4) perceived benefit; 5) cues to action; 6) self-efficacy; 7) attitude; and 8) practice. All items had adequate factor loadings and showed good internal consistency. The final model after CFA achieved a good fit with an RMSEA value of 0.061, SRMR of 0.068, PNFI of 0.649, and GFI of 0.996. CONCLUSION: The RPAP questionnaire contains 29 items and is a reliable and accurate psychometric instrument for measuring the risk perception of dengue fever, attitude, and practice of the community in dengue prevention. The Rasch measurement provides additional rigour to complement the CTT analysis. This RPAP questionnaire is suitable for use in studies related to dengue prevention in the community.

Clinical and Epidemiological Features of Acute Zika Virus Infections in León, Nicaragua.

The American Zika virus (ZIKV) epidemic has highlighted the need to gain a better understanding of this emerging virus. The goal of this study was to describe the clinical symptoms, laboratory findings, and risk factors for symptomatic ZIKV infection in an area with ongoing transmission of other arboviral infections. We recruited patients at least 2 years of age seeking care at public health centers in León, Nicaragua, between January 2016 and August 2017, for fever, maculopapular rash, and/or nonsuppurative conjunctivitis with a duration of less than 1 week. A laboratory diagnosis of ZIKV was established using a combination of molecular and serological tests. Clinical and laboratory findings and potential risk factors were compared between participants with and without acute ZIKV infection. Fifty-eight (26%) of the 225 participants included in the analysis were found to have acute ZIKV infection. Pregnancy and reports of previous arboviral infection were associated with a higher risk of ZIKV infection. Rash, conjunctivitis, sore throat, and lower absolute neutrophil counts were associated with acute ZIKV infection. The clinical characteristics and risk factors identified were consistent with those identified by previous studies; however, we found sore throat to be a feature of ZIKV infection. We also found that neutrophil counts were lower in ZIKV-infected subjects. These clinical symptoms and laboratory data may help clinicians suspect ZIKV infection during future outbreaks.