ABSTRACT
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).
ABSTRACT
Zika virus (ZIKV) constitutes an increasing public health problem. Previous studies have shown that CD8+ T cells play an important role in ZIKV-specific protective immunity. We have previously defined antigenic targets of the ZIKV-specific CD8+ T cell response in humans. In this study, we characterized the quality and phenotypes of these responses by a combined use of flow cytometry and transcriptomic methods, using PBMCs from donors deriving from different geographical locations collected in the convalescent phase of infection. We show that ZIKV-specific CD8+ T cells are characterized by a polyfunctional IFN-γ signature with upregulation of TNF-α, TNF receptors, and related activation markers, such as CD69, as well as a cytotoxic signature characterized by strong upregulation of GZMB and CRTAM. The signature is stable and not influenced by previous dengue virus exposure, geographical location, or time of sample collection postinfection. To our knowledge, this work elucidates the first in-depth characterization of human CD8+ T cells responding to ZIKV infection.
Subject(s)
CD8-Positive T-Lymphocytes/physiology , Zika Virus Infection/immunology , Zika Virus/physiology , Antigens, Viral/immunology , Cells, Cultured , Cytotoxicity, Immunologic , Epitopes, T-Lymphocyte/immunology , Gene Expression Profiling , Granzymes/genetics , Humans , Immunoglobulins/genetics , Immunophenotyping , Interferon-gamma/genetics , Receptors, Tumor Necrosis Factor/genetics , Tumor Necrosis Factor-alpha/geneticsABSTRACT
INTRODUCTION: Since the methodologies used to calculate Stegomyia indices have been shown to be inadequate for assessing the risk of dengue virus transmission and targeting Aedes aegypti control strategies, new surveillance methods are needed. OBJECTIVE: To evaluate the water-surface sweeping method in combination with calibration factors to estimate the total number of Ae. aegypti late larval stages (L3/L4) in large water-storage containers at different temperatures at which transmission of dengue virus occurs. MATERIALS AND METHODS: Calibration factors were derived based on the proportion of L3/L4 recovered from a predetermined number of larvae using a net of specific dimensions and water-storage containers of different capacities and water levels in semi-field conditions and at four different altitudes (14, 358, 998 and 1,630 meters above sea level). The calibration factors obtained at 14 masl were then fully validated in a field study site at this altitude. RESULTS: Four calibration factors were derived at 14 masl (28-30°C) that were used to estimate the total L3/L4 numbers in large water storage containers greater than 20 L (n=478) at 1/3, 2/3 and full water-levels. This methodology was accurate and robust within and between the 10 pairs of field workers who applied it. Different calibration factors were, however, derived to accurately estimate the total L3/L4 numbers at each of the study sites located at 358, 998 and 1,630 masl, where average temperatures were 19°C, 24°C, and 26°C respectively. CONCLUSIONS: The accurate estimates of L3/L4 numbers calculated using the water surface sweeping method can be useful for evaluating intervention strategies directed against the larval stages.
Subject(s)
Aedes , Aedes/growth & development , Animals , Entomology/methods , Larva , Pupa , Reproducibility of Results , Time Factors , WaterABSTRACT
Introduction. Since the methodologies used to calculate Stegomyia indices have been shown to be inadequate for assessing the risk of dengue virus transmission and targeting Aedes aegypti control strategies, new surveillance methods are needed. Objective. To evaluate the water-surface sweeping method in combination with calibration factors to estimate the total number of Ae. aegypti late larval stages (L3/L4) in large water-storage containers at different temperatures at which transmission of dengue virus occurs. Materials and methods. Calibration factors were derived based on the proportion of L3/L4 recovered from a predetermined number of larvae using a net of specific dimensions and water-storage containers of different capacities and water levels in semi-field conditions and at four different altitudes (14, 358, 998 and 1,630 meters above sea level). The calibration factors obtained at 14 masl were then fully validated in a field study site at this altitude. Results. Four calibration factors were derived at 14 masl (28-30°C) that were used to estimate the total L3/L4 numbers in large water storage containers greater than 20 L (n=478) at 1/3, 2/3 and full water-levels. This methodology was accurate and robust within and between the 10 pairs of field workers who applied it. Different calibration factors were, however, derived to accurately estimate the total L3/L4 numbers at each of the study sites located at 358, 998 and 1,630 masl, where average temperatures were 19°C, 24°C, and 26°C respectively. Conclusions. The accurate estimates of L3/L4 numbers calculated using the water surface sweeping method can be useful for evaluating intervention strategies directed against the larval stages.
Introducción. Las metodologías usadas para calcular los índices de Stegomyia son inadecuadas para evaluar el riesgo de transmisión del virus del dengue y, tampoco, permiten enfocar estrategias de control de Aedes aegypti, por lo cual se requiere desarrollar nuevos métodos para la vigilancia. Objetivo. Evaluar el método de barrido del agua superficial combinado con factores de calibración para estimar el número total estadios larvarios tardíos (L3/L4) de Ae. aegypti en depósitos de grandes capacidades a diferentes temperaturas de transmisión del virus del dengue. Materiales y métodos. Los factores de calibración se derivaron de la proporción de L3/L4 recolectadas con una malla de dimensiones específicas y a partir de un número conocido de larvas, en depósitos de diferentes capacidades y niveles de agua, en condiciones de campo simuladas y a cuatro altitudes diferentes (14, 358, 998 y 1.630 metros sobre el nivel del mar). Los factores de calibración obtenidos a 14 msnm fueron plenamente validados en el campo a esa altitud. Resultados. Se derivaron cuatro factores de calibración a 14 msnm (28°C-30°C) los cuales se emplearon para estimar el número total de L3/L4 en depósitos con capacidades mayores a 20 L (n=478) y a niveles de agua de un tercio, dos tercios y lleno. Esta metodología fue precisa y sólida en los 10 pares de trabajadores que aplicaron el método y entre ellos. Sin embargo, diferentes factores de calibración fueron derivados para estimar con precisión los números totales de L3/L4 en cada uno de los sitios de estudio localizados a 358, 998 y 1.630 msnm, donde las temperaturas promedio fueron de 19°C, 24°C y 26°C, respectivamente. Conclusión. La estimación precisa del número total de L3/L4 usando el barrido descrito permite proponer el uso de este método para evaluar estrategias de control dirigido a contra estados larvarios.
Subject(s)
Dengue , Dengue Virus , Statistics as Topic , Virus CultivationABSTRACT
BACKGROUND: More than 12,000 simple sequence repeats (SSRs) have been identified in the genome of Burkholderia mallei ATCC 23344. As a demonstrated mechanism of phase variation in other pathogenic bacteria, these may function as mutable loci leading to altered protein expression or structure variation. To determine if such alterations are occurring in vivo, the genomes of various single-colony passaged B. mallei ATCC 23344 isolates, one from each source, were sequenced from culture, a mouse, a horse, and two isolates from a single human patient, and the sequence compared to the published B. mallei ATCC 23344 genome sequence. RESULTS: Forty-nine insertions and deletions (indels) were detected at SSRs in the five passaged strains, a majority of which (67.3%) were located within noncoding areas, suggesting that such regions are more tolerant of sequence alterations. Expression profiling of the two human passaged isolates compared to the strain before passage revealed alterations in the mRNA levels of multiple genes when grown in culture. CONCLUSION: These data support the notion that genome variability upon passage is a feature of B. mallei ATCC23344, and that within a host B. mallei generates a diverse population of clones that accumulate genome sequence variation at SSR and other loci.
Subject(s)
Burkholderia mallei/genetics , Glanders/microbiology , Mutation/genetics , Animals , Burkholderia mallei/growth & development , Gene Deletion , Gene Expression Profiling , Genome, Bacterial/genetics , Horses , Humans , Mice , Mice, Inbred BALB C , Minisatellite Repeats/genetics , Oligonucleotide Array Sequence Analysis , Polymorphism, Single Nucleotide/genetics , Sequence Analysis, DNA/methodsABSTRACT
The complete genome sequence of Burkholderia mallei ATCC 23344 provides insight into this highly infectious bacterium's pathogenicity and evolutionary history. B. mallei, the etiologic agent of glanders, has come under renewed scientific investigation as a result of recent concerns about its past and potential future use as a biological weapon. Genome analysis identified a number of putative virulence factors whose function was supported by comparative genome hybridization and expression profiling of the bacterium in hamster liver in vivo. The genome contains numerous insertion sequence elements that have mediated extensive deletions and rearrangements of the genome relative to Burkholderia pseudomallei. The genome also contains a vast number (>12,000) of simple sequence repeats. Variation in simple sequence repeats in key genes can provide a mechanism for generating antigenic variation that may account for the mammalian host's inability to mount a durable adaptive immune response to a B. mallei infection.
