Evaluation of Zika rapid tests as aids for clinical diagnosis and epidemic preparedness.

Background: Development and evaluation of diagnostics for diseases of epidemic potential are often funded during epidemics, but not afterwards, leaving countries unprepared for the next epidemic. United Nations Children's Emergency Fund (UNICEF) partnered with the United States Agency for International Development (USAID) to address this important gap by investing in an advance purchase commitment (APC) mechanism to accelerate the development and evaluation of Zika rapid diagnostic tests (RDTs) for case detection and surveillance. This paper describes the performance evaluation of five Zika RDTs eligible for procurement. Methods: A network of European Union-funded ZikaPLAN sites in Africa, Asia, Latin America with access to relevant serum specimens were selected to evaluate RDTs developed for the UNICEF APC mechanism. A standardised protocol and evaluation panels were developed and a call for specimens for the evaluation panels issued to different sites. Each site contributed specimens to the evaluation from their biobank. Data were collated, analysed and presented to the UNICEF Procurement Review Group for review. Findings: Three RDTs met the criteria for UNICEF procurement of sensitivity and specificity of 85% against a refence standard. The sensitivity/specificity of the ChemBio anti-Zika Virus (ZIKV) immunoglobulin M (IgM) test was 86.4 %/86.7% and the ChemBio ZCD system for anti-ZIKV IgM was 79.0%/97.1%, anti-dengue virus (DENV) IgM 90.0%/89.2%, anti-Chikungunya virus (CHIKV) IgM 90.6%/97.2%. The sensitivity/specificity of the SD Biosensor anti-ZIKV IgM was 96.8 %/90.8%, anti-DENV IgM 71.8%/83.5%, the DENV nonstructural protein 1 (NS1) glycoprotein 90.0%/90.2%, anti- yellow fever virus (YFV) IgM 84.6%/92.4%, anti-CHIKV IgM 86.3%/97.5%. Interpretation: Three RDTs fulfilled the performance thresholds set by WHO and were eligible for UNICEF procurement. These tests will improve the diagnosis of ZIKV and other arboviral infections as well as providing countries with better tools for surveillance and response to future epidemics. Funding: This work was supported by the USAID grant GHA-G-00-07-00007 and ZikaPLAN (European Union's Horizon 2020 Research and Innovation Programme under Grant Agreement No. 734584).

OSBPL10, RXRA and lipid metabolism confer African-ancestry protection against dengue haemorrhagic fever in admixed Cubans.

Ethnic groups can display differential genetic susceptibility to infectious diseases. The arthropod-born viral dengue disease is one such disease, with empirical and limited genetic evidence showing that African ancestry may be protective against the haemorrhagic phenotype. Global ancestry analysis based on high-throughput genotyping in admixed populations can be used to test this hypothesis, while admixture mapping can map candidate protective genes. A Cuban dengue fever cohort was genotyped using a 2.5 million SNP chip. Global ancestry was ascertained through ADMIXTURE and used in a fine-matched corrected association study, while local ancestry was inferred by the RFMix algorithm. The expression of candidate genes was evaluated by RT-PCR in a Cuban dengue patient cohort and gene set enrichment analysis was performed in a Thai dengue transcriptome. OSBPL10 and RXRA candidate genes were identified, with most significant SNPs placed in inferred weak enhancers, promoters and lncRNAs. OSBPL10 had significantly lower expression in Africans than Europeans, while for RXRA several SNPs may differentially regulate its transcription between Africans and Europeans. Their expression was confirmed to change through dengue disease progression in Cuban patients and to vary with disease severity in a Thai transcriptome dataset. These genes interact in the LXR/RXR activation pathway that integrates lipid metabolism and immune functions, being a key player in dengue virus entrance into cells, its replication therein and in cytokine production. Knockdown of OSBPL10 expression in THP-1 cells by two shRNAs followed by DENV2 infection tests led to a significant reduction in DENV replication, being a direct functional proof that the lower OSBPL10 expression profile in Africans protects this ancestry against dengue disease.

Dengue specific immunoglobulins M, A, and E in primary and secondary dengue 4 infected Salvadorian children.

El Salvador is a Central American country that has been affected by several dengue outbreaks. This study investigated the levels of IgM, IgA, and IgE anti-dengue antibodies in serum samples from children in El Salvador, with a clinical and serological diagnosis of dengue infection during the dengue 4 outbreak in 2002-2003. Seventy one serum samples were tested by ELISA and cases were classified in three groups: 13 primary dengue fever (PDF), 21 secondary dengue fever (SDF), and 37 secondary dengue hemorrhagic fever (SDHF). Also, the specificity of anti-dengue IgM for the different serotypes was tested. No significant differences in the IgM response were found between PDF and SDF, but these were detected between PDF and SDHF (P = 0.0053) and between SDF and SDHF (P = 0.0003). The IgA and IgE values showed a statistically significant difference between primary and secondary groups. The highest positivity percentage of IgA was between 95% (SDF) and 100% (SDHF) towards day 7 of onset of fever. All secondary cases were positive for IgE antibodies. The specificity of IgM was determined for DENV-4 virus in primary and secondary DF groups. This is the first study on dengue cases in Salvadorian children related to the immune response of different immunoglobulins to the type of infection and the clinical picture. Further prospective studies are needed to define if the pattern of immunoglobulins can determine early dengue infection and/or severity.

Multi-country evaluation of the sensitivity and specificity of two commercially-available NS1 ELISA assays for dengue diagnosis.

BACKGROUND: Early diagnosis of dengue can assist patient triage and management and prevent unnecessary treatments and interventions. Commercially available assays that detect the dengue virus protein NS1 in the plasma/serum of patients offers the possibility of early and rapid diagnosis. METHODOLOGY/PRINCIPAL FINDINGS: The sensitivity and specificity of the Pan-E Dengue Early ELISA and the Platelia Dengue NS1 Ag assays were compared against a reference diagnosis in 1385 patients in 6 countries in Asia and the Americas. Platelia was more sensitive (66%) than Pan-E (52%) in confirmed dengue cases. Sensitivity varied by geographic region, with both assays generally being more sensitive in patients from SE Asia than the Americas. Both kits were more sensitive for specimens collected within the first few days of illness onset relative to later time points. Pan-E and Platelia were both 100% specific in febrile patients without evidence of acute dengue. In patients with other confirmed diagnoses and healthy blood donors, Platelia was more specific (100%) than Pan-E (90%). For Platelia, when either the NS1 test or the IgM test on the acute sample was positive, the sensitivity versus the reference result was 82% in samples collected in the first four days of fever. NS1 sensitivity was not associated to disease severity (DF or DHF) in the Platelia test, whereas a trend for higher sensitivity in DHF cases was seen in the Pan-E test (however combined with lower overall sensitivity). CONCLUSIONS/SIGNIFICANCE: Collectively, this multi-country study suggests that the best performing NS1 assay (Platelia) had moderate sensitivity (median 64%, range 34-76%) and high specificity (100%) for the diagnosis of dengue. The poor sensitivity of the evaluated assays in some geographical regions suggests further assessments are needed. The combination of NS1 and IgM detection in samples collected in the first few days of fever increased the overall dengue diagnostic sensitivity.

Kinetics of dengue virus NS1 protein in dengue 4-confirmed adult patients.

In this work, the presence of NS1 protein as a possible early marker of dengue infection was studied in serum samples from confirmed adult patients with a primary and secondary dengue 4 infection. A total of 209 serum samples collected from day 2 up to day 7 of fever onset from 71 patients were tested by Platelia NS1 antigen capture ELISA kit (BioRad, Marnes-la-Coquette, France), and the results were compared with those obtained by capture antidengue virus IgM (MAC)-ELISA and ELISA inhibition method tests. The 83.3% of primary cases and 96.4% of secondary cases were NS1 positive. The kinetics of NS1 protein showed the highest values in optical density mean ratio or in percentage of positives between days 2 and 4. The results obtained in this study show the utility of the NS1 protein as a virologic early marker of dengue infection. Prospective studies should be carried out to confirm its utility as a prognostic marker of severe illness.

Immunoglobulin G antibody response in children and adults with acute dengue 3 infection.

Using a serological test, different criteria have been established for classifying a case as primary or secondary dengue virus infection. Considering the dengue epidemiological situation in Cuba, IgG antibody response to dengue virus infection in serum samples from children and adults with a dengue 3 infection, in Havana city during the 2001-2002 epidemic was evaluated. Samples were collected on days 5-7 of fever onset and tested by an ELISA inhibition. A total of 713 serum samples positive for IgM antibody, 93 from children and 620 from adult patients were studied. Serum samples collected from healthy blood donors and patients not infected with dengue were included as controls. An IgG primary infection pattern was observed in sera collected from children, with titers of < or =20 in the 89.3% of the patients, while both, a primary and secondary patterns were observed in sera collected from adult patients with titers of < or =20 (13.4%) and > or =1280 (83.9%), respectively. These results permitted the definition of a primary or secondary case of dengue virus infection in serum samples collected during the acute phase of dengue virus infection.

Evaluation of immunoglobulin M and G capture enzyme-linked immunosorbent assay Panbio kits for diagnostic dengue infections.

BACKGROUND: Serological assays are widely used to confirm dengue virus infections and to differentiate between a primary and a secondary infection. OBJECTIVE: Two commercial dengue diagnostic kits, Panbio Dengue IgM Capture and Dengue IgG Capture ELISA (Brisbane, Australia) were evaluated. STUDY DESIGN: Three hundred and seventy-three serum samples were tested. Panel sera included samples from dengue confirmed cases (representing both primary and secondary infections), from non-dengue infectious diseases, and from healthy individuals. The MAC-ELISA/Dengue IPK was used for the detection of anti-dengue virus IgM antibody in the sera and the ELISA inhibition method (EIM/Dengue IPK) was used to differentiate between primary and secondary infections. Both these reference assays, which were previously developed in the Arbovirus Laboratory at the "Pedro Kouri" Tropical Medicine Institute, were employed as the gold standard. RESULTS: High sensitivity (96.8%) and specificity (99.4%) were found with the commercial diagnostics when compared to the reference methods. Furthermore, high concordance 95.5% in classifying dengue infection types (primary or secondary infections) was observed. CONCLUSIONS: The Panbio Dengue IgM and IgG assays offer a good alternative for dengue diagnosis. They are easy to perform and results can be obtained in less than 3h.