NS1-Specific Antibody Response Facilitates the Identification of Children With Dengue and Zika in Hyperendemic Areas.

BACKGROUND: Infections by dengue virus (DENV) and Zika virus (ZIKV) have some similar symptoms and a cross-reactive immune response, although with different risk populations and outcomes. Here, we evaluated the virological characteristics and the nonstructural protein 1 (NS1)-specific antibody responses to DENV and ZIKV in children suspected of dengue in different epidemiological moments in Colombia. METHODS: Viral RNA, circulating NS1 and IgM/IgG specific for DENV and ZIKV were performed by reverse transcription-quantitative polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA) in 301 children suspected of dengue enrolled in a hospital setting during the ZIKV epidemic and a primary healthcare setting during a DENV epidemic. For the detection of DENV and ZIKV-specific IgM, an NS1-based ELISA was validated using characterized pediatric samples. Clinical and laboratory parameters were also evaluated. RESULTS: DENV RNA or NS1 antigen was detected in the plasma of 62% of children, and in none, the ZIKV RNA was found. NS1-based ELISA for DENV and ZIKV IgM showed a sensitivity/specificity of 90/84% and 73/98%, respectively. Of 114 children without detectable viremia or antigenemia, 30.7%, 17.5%, 22% and 30% were IgM-DENV+, IgM-ZIKV+, IgM-DENV+ZIKV+ and IgM-DENV-ZIKV-, respectively. The ZIKV/DENV IgM-NS1 ratio allows the identification of the infecting ortho flavivirus in 88% of the children with IgM-DENV+ZIKV+, confirming a high predominance of DENV infections in the 2 pediatric settings. CONCLUSION: Overall, 88% of the children with clinical suspicion of dengue had an identifiable ortho flaviviral infection, with 80% caused by DENV, 7% by ZIKV and 0.7% classified as recent infections or coinfection, demonstrating active viral cocirculation in the pediatric population of southern Colombia. The IgM-NS1 detection improved the identification of ortho flaviviral infections in children without viremia or antigenemia, suggesting it is a helpful complementary tool for medical personnel in tropical regions with high viral cocirculation and different clinical scenes.

Influence of previous Zika virus infection on acute dengue episode.

BACKGROUND: The co-circulation of flaviviruses in tropical regions has led to the hypothesis that immunity generated by a previous dengue infection could promote severe disease outcomes in subsequent infections by heterologous serotypes. This study investigated the influence of antibodies generated by previous Zika infection on the clinical outcomes of dengue infection. METHODOLOGY/PRINCIPAL FINDINGS: We enrolled 1,043 laboratory confirmed dengue patients and investigated their prior infection to Zika or dengue. Severe forms of dengue disease were more frequent in patients with previous Zika infection, but not in those previously exposed to dengue. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that previous Zika infection may represent a risk factor for subsequent severe dengue disease, but we did not find evidence of antibody-dependent enhancement (higher viral titer or pro-inflammatory cytokine overexpression) contributing to exacerbation of the subsequent dengue infection.

Unique Immune Blood Markers Between Severe Dengue and Sepsis in Children.

BACKGROUND: Pediatric dengue and sepsis share clinical and pathophysiologic aspects. Multiple inflammatory and regulatory cytokines, decoy receptors and vascular permeability factors have been implicated in the pathogenesis of both diseases. The differential pattern and dynamic of these soluble factors, and the relationship with clinical severity between pediatric dengue and sepsis could offer new diagnosis and therapeutic strategies. METHODS: We evaluated the concentration levels of 11 soluble factors with proinflammatory, regulatory and vascular permeability involvement, in plasma from children with dengue or sepsis, both clinically ranging from mild to severe, in the early, late and convalescence phases of the disease. RESULTS: During early acute infection, children with sepsis exhibited specific higher concentration levels of IL-6, vascular endothelial growth factor (VEGF), and its soluble decoy receptor II (sVEGFR2) and lower concentration levels of IL-10 and the soluble tumor necrosis factor receptor 2 (sTNFR2), in comparison with children with severe dengue. In addition, the circulating amounts of soluble ST2, and VEGF/sVEGFR2 were widely associated with clinical and laboratory indicators of dengue severity, whereas secondary dengue virus infections were characterized by an enhanced cytokine response, relative to primary infections. In severe forms of dengue, or sepsis, the kinetics and the cytokines response during the late and convalescence phases of the disease also differentiate. CONCLUSIONS: Dengue virus infection and septic processes in children are characterized by cytokine responses of a specific magnitude, pattern and kinetics, which are implicated in the pathophysiology and clinical outcome of these diseases.

Integrative systems biology characterizes immune-mediated neurodevelopmental changes in murine Zika virus microcephaly.

Characterizing perturbation of molecular pathways in congenital Zika virus (ZIKV) infection is critical for improved therapeutic approaches. Leveraging integrative systems biology, proteomics, and RNA-seq, we analyzed embryonic brain tissues from an immunocompetent, wild-type congenital ZIKV infection mouse model. ZIKV induced a robust immune response accompanied by the downregulation of critical neurodevelopmental gene programs. We identified a negative correlation between ZIKV polyprotein abundance and host cell cycle-inducing proteins. We further captured the downregulation of genes/proteins, many of which are known to be causative for human microcephaly, including Eomesodermin/T-box Brain Protein 2 (EOMES/TBR2) and Neuronal Differentiation 2 (NEUROD2). Disturbances of distinct molecular pathways in neural progenitors and post-mitotic neurons may contribute to complex brain phenotype of congenital ZIKV infection. Overall, this report on protein- and transcript-level dynamics enhances understanding of the ZIKV immunopathological landscape through characterization of fetal immune response in the developing brain.

Multiplexed rapid antigen tests developed using multicolored nanoparticles and cross-reactive antibody pairs: Implications for pandemic preparedness.

Global public health infrastructure is unprepared for emerging pathogen epidemics, in part because diagnostic tests are not developed in advance. The recent Zika, Ebola, and SARS-CoV-2 virus epidemics are cases in point. We demonstrate here that multicolored gold nanoparticles, when coupled to cross-reactive monoclonal antibody pairs generated from a single immunization regimen, can be used to create multiple diagnostics that specifically detect and distinguish related viruses. The multiplex approach for specific detection centers on immunochromatography with pairs of antibody-conjugated red and blue gold nanoparticles, coupled with clustering algorithms to detect and distinguish related pathogens. Cross-reactive antibodies were used to develop rapid tests for i) Dengue virus serotypes 1-4, ii) Zika virus, iii) Ebola and Marburg viruses, and iv) SARS-CoV and SARS-CoV-2 viruses. Multiplexed rapid antigen tests based on multicolored nanoparticles and cross-reactive antibodies and can be developed prospectively at low cost to improve preparedness for epidemic outbreaks.

Dengue and Zika virus differential infection of human megakaryoblast MEG-01 reveals unique cellular markers.

Platelet count is widely used for the diagnosis and follow-up of patients with dengue. Despite its close viral structural and symptomatic homology, ZIKV infection does not typically induce significant thrombocytopenia. To determine the effect of DENV-2 and ZIKV infection on human platelet precursors we utilized MEG-01 cell line to evaluate the viral infection, viability, innate gene expression and release of platelet-like particles (PLPs). DENV-2 induced a higher proportion of cell death at 48-72 h post-infection than ZIKV. The median range of intracellular NS1+/E+ cells was 11.2% (3.3%-25%) and 5% (3%-8.1%) for DENV-2 and ZIKV, respectively (p = 0.03). MEG-01 cells infected with DENV-2 quickly expressed higher levels of IFN-ß, indolamine 2,3-dioxygenase and CXCL10 mRNA compared to ZIKV infected cells and DENV-2 but not ZIKV infection reduced the number PLPs from stimulated MEG-01 cells. The results shed light into mechanisms including thrombocytopenia present in patients with DENV but absent in ZIKV infections.

Combination of the Focus-Forming Assay and Digital Automated Imaging Analysis for the Detection of Dengue and Zika Viral Loads in Cultures and Acute Disease.

Optimized methods for the detection of flavivirus infections in hyperendemic areas are still needed, especially for working with patient serum as a starting material. The focus-forming assay (FFA) reveals critical aspects of virus-host interactions, as it is a quantitative assay to determine viral loads. Automated image analysis provides evaluations of relative amounts of intracellular viral protein at the single-cell level. Here, we developed an optimized FFA for the detection of infectious Zika virus (ZIKV) and dengue virus (DENV) viral particles in cell cultures and clinical serum samples, respectively. Vero-76 cells were infected with DENV-2 (16681) or ZIKV (PRVA BC59). Using a panel of anti-DENV and anti-ZIKV NS1-specific monoclonal antibodies (mAbs), the primary mAbs, concentration, and the optimal time of infection were determined. To determine whether intracellular accumulation of NS1 improved the efficiency of the FFA, brefeldin A was added to the cultures. Focus formation was identified by conventional optical microscopy combined with CellProfiler™ automated image analysis software. The FFA was used with spike assays for ZIKV and clinical specimens from natural infection by DENV-1 and DENV-2. mAb 7744-644 for ZIKV and mAb 724-323 for DENV used at a concentration of 1 µg/ml and a time of 24 hours postinfection produced the best detection of foci when combining conventional counting and automated digital analysis. Brefeldin A did not improve the assessment of FFUs or their digitally assessed intensity at single-cell level. The FFA showed 95% ZIKV recovery and achieved the detection of circulating DENV-1 and DENV-2 in the plasma of acutely ill patients. The combination of the two techniques optimized the FFA, allowing the study of DENV and ZIKV in culture supernatants and clinical specimens from natural infection in hyperendemic areas.

Role of cytokines, chemokines, C3a, and mannose-binding lectin in the evolution of the chikungunya infection.

The pathogenesis of the severity of chikungunya infection is not yet fully understood. OBJECTIVE: To assess the role of the cytokines/chemokines and system of complement in the evolution of chikungunya infection. METHODS: In both acute and chronic phases, we measured the serum levels of 12 cytokines/chemokines and two complement mediators: mannose-binding lectin (MBL) and C3a, in 83 patients with chikungunya infection and ten healthy controls. RESULTS: During the acute phase, 75.9% of the patients developed musculoskeletal disorders, and in 37.7% of them, these disorders persisted until the chronic phase. In general, patients had higher levels of cytokines than healthy controls, with significant differences for IFN-γ, IL-6, IL-8, IL-10, and MIP-1. Most cytokines exhibited a downward trend during the chronic phase. However, only IL-10, and MIP-1 levels were significantly lower in the chronic phase. Additionally, these levels never decreased to concentrations found in healthy controls. Moreover, MBL levels were significantly higher in the acute phase compared with the chronic phase. C3a levels were significantly higher in patients with musculoskeletal disorder compared with patients without it, in both acute-phase 118.2 (66.5-252.9), and chronic phase 68.5 (64.4-71.3), P < 0.001. Interestingly, C3a levels were significantly higher when patients had a severe disease version. Besides, in the acute phase, C3a levels were higher in patients that suffer arthritis as opposed to when they suffer arthralgia, 194.3 (69.5-282.2), and 70.9 (62.4-198.8), P = 0.013, respectively. CONCLUSIONS: Our results showed an immunological response that persisted until the chronic phase and the role of the complement system in the severity of the disease.

Monoclonal antibody pairs against SARS-CoV-2 for rapid antigen test development.

BACKGROUND: The focus on laboratory-based diagnosis of coronavirus disease 2019 (COVID-19) warrants alternative public health tools such as rapid antigen tests. While there are a number of commercially available antigen tests to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), all cross-react with the genetically similar SARS-CoV-1 or require an instrument for results interpretation. METHODOLOGY/PRINCIPAL FINDINGS: We developed and validated rapid antigen tests that use pairs of murine-derived monoclonal antibodies (mAbs), along with gold nanoparticles, to detect SARS-CoV-2 with or without cross-reaction to SARS-CoV-1 and other coronaviruses. In this development, we demonstrate a robust antibody screening methodology for the selection of mAb pairs that can recognize SARS-CoV-2 spike (S) and nucleocapsid (N) proteins. Linear epitope mapping of the mAbs helped elucidate SARS-CoV-2 S and N interactions in lateral flow chromatography. A candidate rapid antigen test for SARS-CoV-2 N was validated using nasal swab specimens that were confirmed positive or negative by quantitative reverse-transcription polymerase chain reaction (RT-PCR). Test results were image-captured using a mobile phone and normalized signal pixel intensities were calculated; signal intensities were inversely correlated to RT-PCR cycle threshold (Ct) value. CONCLUSION/SIGNIFICANCE: Overall, our results suggest that the rapid antigen test is optimized to detect SARS-CoV-2 N during the acute phase of COVID-19. The rapid antigen tests developed in this study are alternative tools for wide scale public health surveillance of COVID-19.

Antibodies targeting epitopes on the cell-surface form of NS1 protect against Zika virus infection during pregnancy.

There are no licensed therapeutics or vaccines available against Zika virus (ZIKV) to counteract its potential for congenital disease. Antibody-based countermeasures targeting the ZIKV envelope protein have been hampered by concerns for cross-reactive responses that induce antibody-dependent enhancement (ADE) of heterologous flavivirus infection. Nonstructural protein 1 (NS1) is a membrane-associated and secreted glycoprotein that functions in flavivirus replication and immune evasion but is absent from the virion. Although some studies suggest that antibodies against ZIKV NS1 are protective, their activity during congenital infection is unknown. Here we develop mouse and human anti-NS1 monoclonal antibodies that protect against ZIKV in both non-pregnant and pregnant mice. Avidity of antibody binding to cell-surface NS1 along with Fc effector functions engagement correlate with protection in vivo. Protective mAbs map to exposed epitopes in the wing domain and loop face of the ß-platform. Anti-NS1 antibodies provide an alternative strategy for protection against congenital ZIKV infection without causing ADE.

Importance of clinical and laboratory characteristics in the diagnosis of dengue and chikungunya co-infection: Probable case report / Importancia de las características clínicas y de laboratorio en el diagnóstico de las infecciones concomitantes por dengue y chikungunya: reporte de un caso probable.

We report the case of a 38-year-old woman who initially consulted for an undifferentiated fever. Although her clinical condition evolved with signs and symptoms compatible with dengue with alarm signs and that the anti-dengue IgM detection in a single sample indicated it was a probable case that could have happened during the previous three months, the patient kept consulting due to little improvement. On the tenth day after the onset of symptoms, she presented with painful polyarticular symmetric edema, as well as hyperpigmented lesions in the nasolabial fold. Chikungunya diagnosis was confirmed by the presence of IgM antibodies. In endemic countries for dengue and chikungunya, the possibility of co-infection exists, but it may go unnoticed. On the other hand, the co-infection may worsen the clinical course of these diseases. Therefore, physicians should evaluate the clinical and laboratory characteristics of both infections to be able to diagnose the coinfection for adequate management and to minimize complications.

Se presenta el caso de una mujer de 38 años que consultó inicialmente por fiebre indiferenciada. A pesar de que el cuadro clínico evolucionó con manifestaciones clínicas de dengue con signos de alarma y de que la detección de IgM antidengue en una sola muestra indicaba que se trataba de un caso probable que había podido ocurrir durante los tres meses anteriores, la paciente consultó de forma reiterada, pues no presentaba una mejoría significativa. En el décimo día del inicio de los síntomas, se observó edema simétrico en múltiples articulaciones acompañado de dolor, así como lesiones hiperpigmentadas en el surco nasogeniano. Se confirmó el diagnóstico de chikungunya por la presencia de anticuerpos IgM. Aunque puede pasar desapercibida, en los países endémicos para dengue y chikungunya existe la posibilidad de la infección concomitante, la cual puede agravar la evolución clínica de cada una de estas enfermedades. Por ello, es necesario que el médico considere las características clínicas y de laboratorio de ambas enfermedades para diagnosticar su presencia simultánea, garantizar un manejo adecuado y minimizar las complicaciones.

Development and Validation of a Rapid Lateral Flow E1/E2-Antigen Test and ELISA in Patients Infected with Emerging Asian Strain of Chikungunya Virus in the Americas.

Since its 2013 emergence in the Americas, Chikungunya virus (CHIKV) has posed a serious threat to public health. Early and accurate diagnosis of the disease, though currently lacking in clinics, is integral to enable timely care and epidemiological response. We developed a dual detection system: a CHIKV antigen E1/E2-based enzyme-linked immunosorbent assay (ELISA) and a lateral flow test using high-affinity anti-CHIKV antibodies. The ELISA was validated with 100 PCR-tested acute Chikungunya fever samples from Honduras. The assay had an overall sensitivity and specificity of 51% and 96.67%, respectively, with accuracy reaching 95.45% sensitivity and 92.03% specificity at a cycle threshold (Ct) cutoff of 22. As the Ct value decreased from 35 to 22, the ELISA sensitivity increased. We then developed and validated two lateral flow tests using independent antibody pairs. The sensitivity and specificity reached 100% for both lateral flow tests using 39 samples from Colombia and Honduras at Ct cutoffs of 20 and 27, respectively. For both lateral flow tests, sensitivity decreased as the Ct increased after 27. Because CHIKV E1/E2 are exposed in the virion surfaces in serum during the acute infection phase, these sensitive and specific assays demonstrate opportunities for early detection of this emerging human pathogen.

Importancia de las características clínicas y de laboratorio en el diagnóstico de las infecciones concomitantes por dengue y chikungunya: reporte de un caso probable / Importance of clinical and laboratory characteristics in the diagnosis of dengue and chikungunya co-infection: Probable case report

Se presenta el caso de una mujer de 38 años que consultó inicialmente por fiebre indiferenciada. A pesar de que el cuadro clínico evolucionó con manifestaciones clínicas de dengue con signos de alarma y de que la detección de IgM antidengue en una sola muestra indicaba que se trataba de un caso probable que había podido ocurrir durante los tres meses anteriores, la paciente consultó de forma reiterada, pues no presentaba una mejoría significativa. En el décimo día del inicio de los síntomas, se observó edema simétrico en múltiples articulaciones acompañado de dolor, así como lesiones hiperpigmentadas en el surco nasogeniano. Se confirmó el diagnóstico de chikungunya por la presencia de anticuerpos IgM. Aunque puede pasar desapercibida, en los países endémicos para dengue y chikungunya existe la posibilidad de la infección concomitante, la cual puede agravar la evolución clínica de cada una de estas enfermedades. Por ello, es necesario que el médico considere las características clínicas y de laboratorio de ambas enfermedades para diagnosticar su presencia simultánea, garantizar un manejo adecuado y minimizar las complicaciones.

We report the case of a 38-year-old woman who initially consulted for an undifferentiated fever. Although her clinical condition evolved with signs and symptoms compatible with dengue with alarm signs and that the anti-dengue IgM detection in a single sample indicated it was a probable case that could have happened during the previous three months, the patient kept consulting due to little improvement. On the tenth day after the onset of symptoms, she presented with painful polyarticular symmetric edema, as well as hyperpigmented lesions in the nasolabial fold. Chikungunya diagnosis was confirmed by the presence of IgM antibodies. In endemic countries for dengue and chikungunya, the possibility of co-infection exists, but it may go unnoticed. On the other hand, the co-infection may worsen the clinical course of these diseases. Therefore, physicians should evaluate the clinical and laboratory characteristics of both infections to be able to diagnose the coinfection for adequate management and to minimize complications.

Author Correction: AHR is a Zika virus host factor and a candidate target for antiviral therapy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

AHR is a Zika virus host factor and a candidate target for antiviral therapy.

Zika virus (ZIKV) is a flavivirus linked to multiple birth defects including microcephaly, known as congenital ZIKV syndrome. The identification of host factors involved in ZIKV replication may guide efficacious therapeutic interventions. In genome-wide transcriptional studies, we found that ZIKV infection triggers aryl hydrocarbon receptor (AHR) activation. Specifically, ZIKV infection induces kynurenine (Kyn) production, which activates AHR, limiting the production of type I interferons (IFN-I) involved in antiviral immunity. Moreover, ZIKV-triggered AHR activation suppresses intrinsic immunity driven by the promyelocytic leukemia (PML) protein, which limits ZIKV replication. AHR inhibition suppressed the replication of multiple ZIKV strains in vitro and also suppressed replication of the related flavivirus dengue. Finally, AHR inhibition with a nanoparticle-delivered AHR antagonist or an inhibitor developed for human use limited ZIKV replication and ameliorated newborn microcephaly in a murine model. In summary, we identified AHR as a host factor for ZIKV replication and PML protein as a driver of anti-ZIKV intrinsic immunity.

Multivariate time-series analysis of biomarkers from a dengue cohort offers new approaches for diagnosis and prognosis.

Dengue is a major public health problem worldwide with distinct clinical manifestations: an acute presentation (dengue fever, DF) similar to other febrile illnesses (OFI) and a more severe, life-threatening form (severe dengue, SD). Due to nonspecific clinical presentation during the early phase of dengue infection, differentiating DF from OFI has remained a challenge, and current methods to determine severity of dengue remain poor early predictors. We present a prospective clinical cohort study conducted in Caracas, Venezuela from 2001-2005, designed to determine whether clinical and hematological parameters could distinguish DF from OFI, and identify early prognostic biomarkers of SD. From 204 enrolled suspected dengue patients, there were 111 confirmed dengue cases. Piecewise mixed effects regression and nonparametric statistics were used to analyze longitudinal records. Decreased serum albumin and fibrinogen along with increased D-dimer, thrombin-antithrombin complex, activated partial thromboplastin time and thrombin time were prognostic of SD on the day of defervescence. In the febrile phase, the day-to-day rates of change in serum albumin and fibrinogen concentration, along with platelet counts, were significantly decreased in dengue patients compared to OFI, while the day-to-day rates of change of lymphocytes (%) and thrombin time were increased. In dengue patients, the absolute lymphocytes to neutrophils ratio showed specific temporal increase, enabling classification of dengue patients entering the critical phase with an area under the ROC curve of 0.79. Secondary dengue patients had elongation of Thrombin time compared to primary cases while the D-dimer formation (fibrinolysis marker) remained always lower for secondary compared to primary cases. Based on partial analysis of 31 viral complete genomes, a high frequency of C-to-T transitions located at the third codon position was observed, suggesting deamination events with five major hot spots of amino acid polymorphic sites outside in non-structural proteins. No association of severe outcome was statistically significant for any of the five major polymorphic sites found. This study offers an improved understanding of dengue hemostasis and a novel way of approaching dengue diagnosis and disease prognosis using piecewise mixed effect regression modeling. It also suggests that a better discrimination of the day of disease can improve the diagnostic and prognostic classification power of clinical variables using ROC curve analysis. The piecewise mixed effect regression model corroborated key early clinical determinants of disease, and offers a time-series approach for future vaccine and pathogenesis clinical studies.

Serotype-specific detection of dengue viruses in a nonstructural protein 1-based enzyme-linked immunosorbent assay validated with a multi-national cohort.

BACKGROUND: Dengue virus (DENV) infections pose one of the largest global barriers to human health. The four serotypes (DENV 1-4) present different symptoms and influence immune response to subsequent DENV infections, rendering surveillance, risk assessments, and disease control particularly challenging. Early diagnosis and appropriate clinical management is critical and can be achieved by detecting DENV nonstructural protein 1 (NS1) in serum during the acute phase. However, few NS1-based tests have been developed that are capable of differentiating DENV serotypes and none are currently commercially available. METHODOLOGY/PRINCIPLE FINDINGS: We developed an enzyme-linked immunosorbent assay (ELISA) to distinguish DENV-1-4 NS1 using serotype-specific pairs of monoclonal antibodies. A total of 1,046 antibodies were harvested from DENV-immunized mice and screened for antigen binding affinity. ELISA clinical performance was evaluated using 408 polymerase chain reaction-confirmed dengue samples obtained from patients in Brazil, Honduras, and India. The overall sensitivity of the test for pan-DENV was 79.66% (325/408), and the sensitivities for DENV-1-4 serotyping were 79.1% (38/48), 80.41% (78/97), 100% (45/45), and 79.6% (98/123), respectively. Specificity reached 94.07-100%. SIGNIFICANCE: Our study demonstrates a robust antibody screening strategy that enabled the development of a serotype NS1-based ELISA with maximized specific and sensitive antigen binding. This sensitive and specific assay also utilized the most expansive cohort to date, and of which about half are from Latin America, a geographic region severely underrepresented in previous similar studies. This ELISA test offers potential enhanced diagnostics during the acute phase of infection to help guide patient care and disease control. These results indicate that this ELISA is a promising aid in early DENV-1-4 diagnosis and surveillance in regions of endemicity in addition to offer convenient monitoring for future vaccine interventions.

Point-of-Care Devices to Detect Zika and Other Emerging Viruses.

Rapid diagnostic tests (point-of-care devices) are critical components of informed patient care and public health monitoring (surveillance applications). We propose that among the many rapid diagnostics platforms that have been tested or are in development, lateral flow immunoassays and synthetic biology-based diagnostics (including CRISPR-based diagnostics) represent the best overall options given their ease of use, scalability for manufacturing, sensitivity, and specificity. This review describes the identification of lateral flow immunoassay monoclonal antibody pairs that detect and distinguish between closely related pathogens and that are used in combination with functionalized multicolored nanoparticles and computational methods to deconvolute data. We also highlight the promise of synthetic biology-based diagnostic tests, which use synthetic genetic circuits that activate upon recognition of a pathogen-associated nucleic acid sequence, and discuss how the combined or parallel use of lateral flow immunoassays and synthetic biology tools may represent the future of scalable rapid diagnostics.

Clinical, laboratory and immune aspects of Zika virus-associated encephalitis in children.

OBJECTIVE: To evaluate the clinical, laboratory, and immune characteristics of Zika virus (ZIKV)-associated encephalitis in pediatric patients after the epidemic in Huila, southern Colombia. METHODS: A pediatric neuro-surveillance hospital study was conducted in a referral health center in southern Colombia, from October 2016 to October 2017. Cases of encephalitis were confirmed by nucleic acid amplification tests and serological methods in cerebrospinal fluid (CSF), plasma, and/or urine. Levels of six cytokines were evaluated by flow cytometry. Patients underwent daily clinical and laboratory follow-up. RESULTS: Twenty children with probable encephalitis were included for further studies and 16 of them were confirmed. Four cases of bacterial meningoencephalitis (Streptococcus pneumoniae, group B Streptococcus, Staphylococcus epidermidis, and Escherichia coli) and 12 cases of viral encephalitis were identified, six of them associated with ZIKV infection. Other viral encephalitis cases were caused by herpes viruses (n=3), enterovirus (n=2), and dengue virus type 2 (DENV-2; n=1) infections. ZIKV-associated encephalitis symptoms subsided faster than those of patients with encephalitis caused by other agents. CSF analysis revealed lymphocytic pleocytosis. Compared to healthy controls, children with ZIKV-associated encephalitis presented modest plasma interleukin (IL)-10 but not IL-2, IL-4, IL-6, interferon gamma (IFN-γ), or tumor necrosis factor alpha (TNF-α). Cytokine expression was differentially regulated, as dramatically elevated IL-6, IL-10, and IFN-γ levels were observed in CSF but not in paired plasma samples in one of the patients with ZIKV detectable in CSF. CONCLUSIONS: This study provides evidence that ZIKV is responsible for pediatric encephalitis in endemic areas, and the local presence of the virus may induce cephalic but not systemic expression of cytokines.