Dataset Information

Multiplexed kit based on Luminex technology and achievements in synthetic biology discriminates Zika, chikungunya, and dengue viruses in mosquitoes.

ABSTRACT:

Background

The global expansion of dengue (DENV), chikungunya (CHIKV), and Zika viruses (ZIKV) is having a serious impact on public health. Because these arboviruses are transmitted by the same mosquito species and co-circulate in the same area, a sensitive diagnostic assay that detects them together, with discrimination, is needed.

Methods

We present here a diagnostics panel based on reverse transcription-PCR amplification of viral RNA and an xMap Luminex architecture involving direct hybridization of PCRamplicons and virus-specific probes. Two DNA innovations ("artificially expanded genetic information systems", AEGIS, and "self-avoiding molecular recognition systems", SAMRS) increase the hybridization sensitivity on Luminex microspheres and PCR specificity of the multiplex assay compared to the standard approach (standard nucleotides).

Results

The diagnostics panel detects, if they are present, these viruses with a resolution of 20 genome equivalents (DENV1), or 10 (DENV3-4, CHIKV) and 80 (DENV2, ZIKV) genome equivalents per assay. It identifies ZIKV, CHIKV and DENV RNAs in a single infected mosquito, in mosquito pools comprised of 5 to 50 individuals, and mosquito saliva (ZIKV, CHIKV, and DENV2). Infected mosquitoes and saliva were also collected on a cationic surface (Q-paper), which binds mosquito and viral nucleic acids electrostatically. All samples from infected mosquitoes displayed only target-specific signals; signals from non-infected samples were at background levels.

Conclusions

Our results provide an efficient and multiplex tool that may be used for surveillance of emerging mosquito-borne pathogens which aids targeted mosquito control in areas at high risk for transmission.

SUBMITTER: Glushakova LG

PROVIDER: S-EPMC6518713 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Publications

Multiplexed kit based on Luminex technology and achievements in synthetic biology discriminates Zika, chikungunya, and dengue viruses in mosquitoes.

Glushakova Lyudmyla G LG Alto Barry W BW Kim Myong-Sang MS Hutter Daniel D Bradley Andrea A Bradley Kevin M KM Burkett-Cadena Nathan D ND Benner Steven A SA

BMC infectious diseases 20190514 1

<h4>Background</h4>The global expansion of dengue (DENV), chikungunya (CHIKV), and Zika viruses (ZIKV) is having a serious impact on public health. Because these arboviruses are transmitted by the same mosquito species and co-circulate in the same area, a sensitive diagnostic assay that detects them together, with discrimination, is needed.<h4>Methods</h4>We present here a diagnostics panel based on reverse transcription-PCR amplification of viral RNA and an xMap Luminex architecture involving d ...[more]

PMID: 31088375

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Project description:Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different downstream patient-to-patient transmission potentials, and require very different patient treatments. Thus, recent Zika outbreaks make it urgent to develop tools that rapidly discriminate these viruses in patients and trapped mosquitoes, to select the correct patient treatment, and to understand and manage their epidemiology in real time. Unfortunately, current diagnostic tests, including those receiving 2016 emergency use authorizations and fast-track status, detect viral RNA by reverse transcription polymerase chain reaction (RT-PCR), which requires instrumentation, trained users, and considerable sample preparation. Thus, they must be sent to "approved" reference laboratories, requiring time. Indeed, in August 2016, the Center for Disease Control (CDC) was asking pregnant women who had been bitten by a mosquito and developed a Zika-indicating rash to wait an unacceptable 2 to 4 weeks before learning whether they were infected. We very much need tests that can be done on site, with few resources, and by trained but not necessarily licensed personnel. This video demonstrates an assay that meets these specifications, working with urine or serum (for patients) or crushed mosquito carcasses (for environmental surveillance), all without much sample preparation. Mosquito carcasses are captured on paper carrying quaternary ammonium groups (Q-paper) followed by ammonia treatment to manage biohazards. These are then directly, without RNA isolation, put into assay tubes containing freeze-dried reagents that need no chain of refrigeration. A modified form of reverse transcription loop-mediated isothermal amplification with target-specific fluorescently tagged displaceable probes produces readout, in 30 min, as a three-color fluorescence signal. This is visualized with a handheld, battery-powered device with an orange filter. Forward contamination is prevented with sealed tubes, and the use of thermolabile uracil DNA glycosylase (UDG) in the presence of dUTP in the amplification mixture.

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Project description:BackgroundDengue, Zika and chikungunya are arboviruses of significant public health importance that are transmitted by Aedes aegypti and Aedes albopictus mosquitoes. In Colombia, where dengue is hyperendemic, and where chikungunya and Zika were introduced in the last decade, more than half of the population lives in areas at risk. The objective of this study was to characterize Aedes spp. vectors and study their natural infection with dengue, Zika and chikungunya in Ibagué, a Colombian city and capital of the department of Tolima, with case reports of simultaneous circulation of these three arboviruses.MethodsMosquito collections were carried out monthly between June 2018 and May 2019 in neighborhoods with different levels of socioeconomic status. We used the non-parametric Friedman, Mann-Whitney and Kruskal-Wallis tests to compare mosquito density distributions. We applied logistic regression analyses to identify associations between mosquito density and absence/presence of breeding sites, and the Spearman correlation coefficient to analyze the possible relationship between climatic variables and mosquito density.ResultsWe collected Ae. aegypti in all sampled neighborhoods and found for the first time Ae. albopictus in the city of Ibagué. A greater abundance of mosquitoes was collected in neighborhoods displaying low compared to high socioeconomic status as well as in the intradomicile compared to the peridomestic space. Female mosquitoes predominated over males, and most of the test females had fed on human blood. In total, four Ae. aegypti pools (3%) were positive for dengue virus (serotype 1) and one pool for chikungunya virus (0.8%). Interestingly, infected females were only collected in neighborhoods of low socioeconomic status, and mostly in the intradomicile space.ConclusionsWe confirmed the co-circulation of dengue (serotype 1) and chikungunya viruses in the Ae. aegypti population in Ibagué. However, Zika virus was not detected in any mosquito sample, 3 years after its introduction into the country. The positivity for dengue and chikungunya viruses, predominance of mosquitoes in the intradomicile space and the high proportion of females fed on humans highlight the high risk for arbovirus transmission in Ibagué, but may also provide an opportunity for establishing effective control strategies.

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Dataset Information

Multiplexed kit based on Luminex technology and achievements in synthetic biology discriminates Zika, chikungunya, and dengue viruses in mosquitoes.

Background

Methods

Results

Conclusions

Publications

Multiplexed kit based on Luminex technology and achievements in synthetic biology discriminates Zika, chikungunya, and dengue viruses in mosquitoes.

Similar Datasets

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