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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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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:BackgroundZika, dengue, and chikungunya are three mosquito-borne viruses having overlapping transmission vectors. They cause diseases having similar symptoms in human patients, but requiring different immediate management steps. Therefore, rapid (< one hour) discrimination of these three viruses in patient samples and trapped mosquitoes is needed. The need for speed precludes any assay that requires complex up-front sample preparation, such as extraction of nucleic acids from the sample. Also precluded in robust point-of-sampling assays is downstream release of the amplicon mixture, as this risks contamination of future samples that will give false positives.MethodsProcedures are reported that directly test urine and plasma (for patient diagnostics) or crushed mosquito carcasses (for environmental surveillance). Carcasses are captured on paper samples carrying quaternary ammonium groups (Q-paper), which may be directly introduced into the assay. To avoid the time and instrumentation requirements of PCR, the procedure uses loop-mediated isothermal amplification (LAMP). Downstream detection is done in sealed tubes, with dTTP-dUTP mixtures in the LAMP with a thermolabile uracil DNA glycosylase (UDG); this offers a second mechanism to prevent forward contamination. Reverse transcription LAMP (RT-LAMP) reagents are distributed dry without requiring a continuous chain of refrigeration.ResultsThe tests detect viral RNA in unprocessed urine and other biological samples, distinguishing Zika, chikungunya, and dengue in urine and in mosquitoes infected with live Zika and chikungunya viruses. The limits of detection (LODs) are ~0.71 pfu equivalent viral RNAs for Zika, ~1.22 pfu equivalent viral RNAs for dengue, and ~38 copies of chikungunya viral RNA. A handheld, battery-powered device with an orange filter was constructed to visualize the output. Preliminary data showed that this architecture, working with pre-prepared tubes holding lyophilized reagent/enzyme mixtures and shipped without a chain of refrigeration, also worked with human plasma samples to detect chikungunya and dengue in Pune, India.ConclusionsA kit, complete with a visualization device, is now available for point-of-sampling detection of Zika, chikungunya, and dengue. The assay output is read in ca. 30 min by visualizing (human eye) three-color coded fluorescence signals. Assay in dried format allows it to be run in low-resource environments.

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.

Project description:Background: The Recife Metropolitan Region (RMR), north-eastern Brazil, was the epicentre of the 2015 Zika virus (ZIKV) epidemic, which was followed by a 2016 chikungunya virus (CHIKV) epidemic. It historically has amongst the highest incidence of dengue virus (DENV) infections and is the only remaining focus of lymphatic filariasis (LF) in Brazil. In early 2015, a molecular xenomonitoring surveillance project focused on Culex (Cx.) quinquefasciatus commenced to inform LF elimination activities. Aedes (Ae.) aegypti mosquitoes were also collected, concurrent with the first microcephaly cases detected in the RMR. In terms of the 2015 ZIKV epidemic, these are the earliest known field-collected mosquitoes, preserved for potential RNA virus detection, when ZIKV was known to be circulating locally. Methods: Adult mosquitoes were collected in two sites (0.4 km 2) of Sítio Novo, Olinda, RMR, from July 22 to August 21, 2015. Mosquitoes were morphologically identified, sorted by physiological status, and pooled (up to 10 mosquitoes per house per day or week). RNA was extracted, reverse transcribed and the cDNA tested by real-time PCR. Results: A total of 10,139 adult female Cx. quinquefasciatus and 939 adult female Ae. aegypti were captured. All female Ae. aegypti specimens were included within 156 pools and screened for ZIKV, DENV and CHIKV. In addition, a sub-set of 1,556 Cx. quinquefasciatus adult females in 182 pools were screened for ZIKV. No evidence of infection with any of the three arboviruses was found. Conclusions: The absence of arbovirus detection may have been expected given the extremely restricted geographic area and collection of mosquitoes during a very short time period of peak mosquito abundance (July-September), but low arbovirus circulation (November-March). However, this study demonstrates the potential to retrospectively screen for additional unexpected pathogens in situations of rapid emergence, such as occurred during the outbreak of ZIKV in the RMR.

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.

Project description:Dengue viruses are the most important arboviral pathogens in the world, which have adapted to human transmission and replication over several hundred years and were initially recognized to cause outbreaks of clinical disease in tropical and subtropical countries by Aedes aegypti mosquitoes. Subsequent global expansion of dengue infection outbreaks has occurred, with millions of cases yearly, probably from a combination of factors including proliferation of international travel and trade, possibly global climate changes, adaptation of the vectors to new environment, and emergence of a new mosquito vector, Aedes albopictus. Chikungunya virus, also transmitted by Aedes mosquitoes, causes a very similar clinical disease but with more prominent arthralgia or arthritis and was originally described in Africa in the 1960s. After a quiescent period of several decades, it reemerged in Africa in 2004 and rapidly spread across the Indian Ocean to involve Asian countries and parts of Europe. However, the past 2 years have seen the emergence of chikungunya virus in the western hemisphere with major outbreaks in the Caribbean and the Americas. Similar to dengue virus, chikungunya virus has adapted to Ae. albopictus mosquitoes which can transmit the disease. Although dengue infection is a more deadly disease especially in young children, chikungunya infection can cause prolonged severe disability and occasionally rare fatalities from encephalitis. No specific treatment is available for either diseases, but development of an effective vaccine for dengue infection is in progress. Until 2007, Zika virus [also transmitted by Aedes species] was associated with only sporadic mild infections in Africa and Asia. In 2007, Zika virus for the first time caused an outbreak beyond Africa and Asia to the Yap Island in the Federated States of Micronesia. Since then Zika virus has spread to French Polynesia, New Caledonia, Cook Islands, and Easter Island in the southeastern Pacific Ocean [Chile] in 2014 and by 2015 to Brazil. By January 2016, it became evident that Zika virus had caused an explosive outbreak in the Americas and the Caribbean with over 30 countries affected. On February 1, 2016, the World Health Organization declared Zika outbreak a global public health emergency. Zika virus infection is most commonly asymptomatic, and 20% of patients may develop a mild viral disease, but of major concern is the reported association of microcephaly in infected pregnant women in Brazil. This chapter explores the history, epidemiology, pathogenesis, clinical features, treatment, and prevention of these rapidly emerging zoonoses.

Dataset Information

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

Publications

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

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