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Point of sampling detection of Zika virus within a multiplexed kit capable of detecting dengue and chikungunya.

ABSTRACT: Zika, 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.Procedures 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.The 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.A 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.

SUBMITTER: Yaren O

PROVIDER: S-EPMC5399334 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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Point of sampling detection of Zika virus within a multiplexed kit capable of detecting dengue and chikungunya.

Yaren Ozlem O Alto Barry W BW Gangodkar Priyanka V PV Ranade Shatakshi R SR Patil Kunal N KN Bradley Kevin M KM Yang Zunyi Z Phadke Nikhil N Benner Steven A SA

BMC infectious diseases 20170420 1

<h4>Background</h4>Zika, 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 ...[more]

PMID: 28427352

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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.

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.

Project description:Dengue, chikungunya, and Zika virus epidemics transmitted by Aedes aegypti mosquitoes have recently (re)emerged and spread throughout the Americas, Southeast Asia, the Pacific Islands, and elsewhere. Understanding how environmental conditions affect epidemic dynamics is critical for predicting and responding to the geographic and seasonal spread of disease. Specifically, we lack a mechanistic understanding of how seasonal variation in temperature affects epidemic magnitude and duration. Here, we develop a dynamic disease transmission model for dengue virus and Aedes aegypti mosquitoes that integrates mechanistic, empirically parameterized, and independently validated mosquito and virus trait thermal responses under seasonally varying temperatures. We examine the influence of seasonal temperature mean, variation, and temperature at the start of the epidemic on disease dynamics. We find that at both constant and seasonally varying temperatures, warmer temperatures at the start of epidemics promote more rapid epidemics due to faster burnout of the susceptible population. By contrast, intermediate temperatures (24-25°C) at epidemic onset produced the largest epidemics in both constant and seasonally varying temperature regimes. When seasonal temperature variation was low, 25-35°C annual average temperatures produced the largest epidemics, but this range shifted to cooler temperatures as seasonal temperature variation increased (analogous to previous results for diurnal temperature variation). Tropical and sub-tropical cities such as Rio de Janeiro, Fortaleza, and Salvador, Brazil; Cali, Cartagena, and Barranquilla, Colombia; Delhi, India; Guangzhou, China; and Manila, Philippines have mean annual temperatures and seasonal temperature ranges that produced the largest epidemics. However, more temperate cities like Shanghai, China had high epidemic suitability because large seasonal variation offset moderate annual average temperatures. By accounting for seasonal variation in temperature, the model provides a baseline for mechanistically understanding environmental suitability for virus transmission by Aedes aegypti. Overlaying the impact of human activities and socioeconomic factors onto this mechanistic temperature-dependent framework is critical for understanding likelihood and magnitude of outbreaks.

Dataset Information

Point of sampling detection of Zika virus within a multiplexed kit capable of detecting dengue and chikungunya.

Publications

Point of sampling detection of Zika virus within a multiplexed kit capable of detecting dengue and chikungunya.

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