Instrument-Free Point-of-Care Molecular Detection of Zika Virus.
ABSTRACT: The recent outbreak of Zika virus (ZIKV) infection in the Americas and its devastating impact on fetal development have prompted the World Health Organization (WHO) to declare the ZIKV pandemic as a Public Health Emergency of International Concern. Rapid and reliable diagnostics for ZIKV are vital because ZIKV-infected individuals display no symptoms or nonspecific symptoms similar to other viral infections. Because immunoassays lack adequate sensitivity and selectivity and are unable to identify active state of infection, molecular diagnostics are an effective means to detect ZIKV soon after infection and throughout pregnancy. We report on a highly sensitive reverse-transcription loop-mediated, isothermal amplification (RT-LAMP) assay for rapid detection of ZIKV and its implementation in a simple, easy-to-use, inexpensive, point-of-care (POC) disposable cassette that carries out all the unit operations from sample introduction to detection. For thermal control of the cassette, we use a chemically heated cup without a need for electrical power. Amplification products are detected with leuco crystal violet (LCV) dye by eye without a need for instrumentation. We demonstrated the utility of our POC diagnostic system by detecting ZIKV in oral samples with sensitivity of 5 plaque-forming units (PFU) in less than 40 min. Our system is particularly suitable for resource-poor settings, where centralized laboratory facilities, funds, and trained personnel are in short supply, and for use in doctors' offices, clinics, and at home.
Project description:The recent outbreak of Zika virus (ZIKV) in the Americas and its devastating developmental and neurological manifestations has prompted the development of field-based diagnostics that are rapid, reliable, handheld, specific, sensitive, and inexpensive. The gold standard molecular method for lab-based diagnosis of ZIKV, from either patient samples or insect vectors, is reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The method, however, is costly and requires lab-based equipment and expertise, which severely limits its use as a point-of-care (POC) tool in resource-poor settings. Moreover, given the lack of antivirals or approved vaccines for ZIKV infection, a POC diagnostic test is urgently needed for the early detection of new outbreaks and to adequately manage patients. Loop-mediated isothermal amplification (LAMP) is a compelling alternative to RT-qPCR for ZIKV and other arboviruses. This low-cost molecular system can be freeze-dried for distribution and exhibits high specificity, sensitivity, and efficiency. A growing body of evidence suggests that LAMP assays can provide greater accessibility to much-needed diagnostics for ZIKV infections, especially in developing countries where the ZIKV is now endemic. This review summarizes the different LAMP methods that have been developed for the virus and summarizes their features, advantages, and limitations.
Project description:Zika virus (ZIKV) is a reemerging flavivirus causing an ongoing pandemic and public health emergency worldwide. There are currently no effective vaccines or specific therapy for Zika infection. Rapid, low-cost diagnostics for mass screening and early detection are of paramount importance in timely management of the infection at the point-of-care (POC). The current Zika diagnostics are laboratory-based and cannot be implemented at the POC particularly in resource-limited settings. Here, we develop a nanoparticle-enhanced viral lysate electrical sensing assay for Zika virus detection on paper microchips with printed electrodes. The virus is isolated from biological samples using antibodies and labeled with platinum nanoparticles (PtNPs) to enhance the electrical signal. The captured ZIKV-PtNP complexes are lysed using a detergent to release the electrically charged molecules associated with the intact virus and the PtNPs on the captured viruses. The released charged molecules and PtNPs change the electrical conductivity of the solution, which can be measured on a cellulose paper microchip with screen-printed microelectrodes. The results confirmed a highly specific detection of ZIKV in the presence of other non-targeted viruses, including closely related flaviviruses such as dengue virus-1 and dengue virus-2 with a detection limit down to 101 virus particles per ?l. The developed assay is simple, rapid, and cost-effective and has the potential for POC diagnosis of viral infections and treatment monitoring.
Project description:The rapid spread of Zika virus (ZIKV) represents a global public health problem, especially in areas that harbor several mosquito species responsible for virus transmission, such as Brazil. In these areas, improvement in mosquito control needs to be a top priority, but mosquito viral surveillance occurs inefficiently in ZIKV-endemic countries. Quantitative reverse transcription PCR (qRT-PCR) is the gold standard for molecular diagnostic of ZIKV in both human and mosquito samples. However, the technique presents high cost and limitations for Point-of-care (POC) diagnostics, which hampers its application for a large number of samples in entomological surveillance programs. Here, we developed and validated a one-step reverse transcription LAMP (RT-LAMP) platform for detection of ZIKV in mosquito samples. The RT-LAMP assay was highly specific for ZIKV and up to 10,000 times more sensitive than qRT-PCR. Assay validation was performed using 60 samples from Aedes aegypti and Culex quinquefasciatus mosquitoes collected in Pernambuco State, Brazil, which is at the epicenter of the Zika epidemic. The RT-LAMP had a sensitivity of 100%, specificity of 91.18%, and overall accuracy of 95.24%. Thus, our POC diagnostics is a powerful and inexpensive tool to monitor ZIKV in mosquito populations and will allow developing countries to establish better control strategies for this devastating pathogen.
Project description:BACKGROUND: To date, the use of traditional nucleic acid amplification tests (NAAT) for detection of HIV-1 DNA or RNA has been restricted to laboratory settings due to time, equipment, and technical expertise requirements. The availability of a rapid NAAT with applicability for resource-limited or point-of-care (POC) settings would fill a great need in HIV diagnostics, allowing for timely diagnosis or confirmation of infection status, as well as facilitating the diagnosis of acute infection, screening and evaluation of infants born to HIV-infected mothers. Isothermal amplification methods, such as reverse-transcription, loop-mediated isothermal amplification (RT-LAMP), exhibit characteristics that are ideal for POC settings, since they are typically quicker, easier to perform, and allow for integration into low-tech, portable heating devices. METHODOLOGY/SIGNIFICANT FINDINGS: In this study, we evaluated the HIV-1 RT-LAMP assay using portable, non-instrumented nucleic acid amplification (NINA) heating devices that generate heat from the exothermic reaction of calcium oxide and water. The NINA heating devices exhibited stable temperatures throughout the amplification reaction and consistent amplification results between three separate devices and a thermalcycler. The performance of the NINA heaters was validated using whole blood specimens from HIV-1 infected patients. CONCLUSION: The RT-LAMP isothermal amplification method used in conjunction with a chemical heating device provides a portable, rapid and robust NAAT platform that has the potential to facilitate HIV-1 testing in resource-limited settings and POC.
Project description:We have developed a generalizable "smart molecular diagnostic" capable of accurate point-of-care (POC) detection of variable nucleic acid targets. Our isothermal assay relies on multiplex execution of four loop-mediated isothermal amplification reactions, with primers that are degenerate and redundant, thereby increasing the breadth of targets while reducing the probability of amplification failure. An easy-to-read visual answer is computed directly by a multi-input Boolean OR logic gate (gate output is true if either one or more gate inputs is true) signal transducer that uses degenerate strand exchange probes to assess any combination of amplicons. We demonstrate our methodology by using the same assay to detect divergent Asian and African lineages of the evolving Zika virus (ZIKV), while maintaining selectivity against non-target viruses. Direct analysis of biological specimens proved possible, with crudely macerated ZIKV-infected Aedes aegypti mosquitoes being identified with 100% specificity and sensitivity. The ease-of-use with minimal instrumentation, broad programmability, and built-in fail-safe reliability make our smart molecular diagnostic attractive for POC use.
Project description:Quantitative detection of RNA is important in molecular biology and clinical diagnostics. Nucleic acid sequence-based amplification (NASBA), a single-step method to amplify single-stranded RNA, is attractive for use in point-of-care (POC) diagnostics because it is an isothermal technique that is as sensitive as RT-PCR with a shorter reaction time. However, NASBA is limited in its ability to provide accurate quantitative information, such as viral load or RNA copy number. Here we test a digital format of NASBA (dNASBA) using a self-digitization (SD) chip platform, and apply it to quantifying HIV-1 RNA. We demonstrate that dNASBA is more sensitive and accurate than the real-time quantitative NASBA, and can be used to quantify HIV-1 RNA in plasma samples. Digital NASBA is thus a promising POC diagnostics tool for use in resource-limited settings.
Project description:The re-emergence of Zika virus (ZIKV) and its associated neonatal microcephaly and Guillain-Barré syndrome have led the World Health Organization to declare a global health emergency. Until today, many related studies have successively reported the role of various viral proteins of ZIKV in the process of ZIKV infection and pathogenicity. These studies have provided significant insights for the treatment and prevention of ZIKV infection. Here we review the current research advances in the functional characterization of the interactions between each ZIKV viral protein and its host factors.
Project description:Advances in nucleic acid amplification technologies have revolutionized diagnostics for systemic, inherited, and infectious diseases. Current assays and platforms, however, often require lengthy experimental procedures and multiple instruments to remove contaminants and inhibitors from clinically-relevant, complex samples. This requirement of sample preparation has been a bottleneck for using nucleic acid amplification tests (NAATs) at the point of care (POC), though advances in "lab-on-chip" platforms that integrate sample preparation and NAATs have made great strides in this space. Alternatively, direct NAATs-techniques that minimize or even bypass sample preparation-present promising strategies for developing POC diagnostic tools for analyzing real-world samples. In this review, we discuss the current status of direct NAATs. Specifically, we surveyed potential testing systems published from 1989 to 2017, and analyzed their performances in terms of robustness, sensitivity, clinical relevance, and suitability for POC diagnostics. We introduce bubble plots to facilitate our analysis, as bubble plots enable effective visualization of the performances of these direct NAATs. Through our review, we hope to initiate an in-depth examination of direct NAATs and their potential for realizing POC diagnostics, and ultimately transformative technologies that can further enhance healthcare.
Project description:: Despite major advances in HIV testing, early detection of infection at the point of care (PoC) remains a key challenge. Although rapid antibody PoC and laboratory-based nucleic acid amplification tests dominate the diagnostics market, the viral capsid protein p24 is recognized as an alternative early virological biomarker of infection. However, the detection of ultra-low levels of p24 at the PoC has proven challenging. Here we review the landscape of p24 diagnostics to identify knowledge gaps and barriers and help shape future research agendas. Five hundred and seventy-four research articles to May 2018 that propose or evaluate diagnostic assays for p24 were identified and reviewed. We give a brief history of diagnostic development, and the utility of p24 as a biomarker in different populations such as infants, the newly infected, those on preexposure prophylaxis and self-testers. We review the performance of commercial p24 assays and consider elements such as immune complex disruption, resource-poor settings, prevalence, and assay antibodies. Emerging and ultrasensitive assays are reviewed and show a number of promising approaches but further translation has been limited. We summarize studies on the health economic benefits of using antigen testing. Finally, we speculate on the future uses of high-performance p24 assays, particularly, if available in self-test format.
Project description:Zika virus (ZIKV) is a single-stranded RNA virus belonging to the family Flaviviridae. ZIKV predominantly enters cells using the TAM-family protein tyrosine kinase receptor AXL, which is expressed on a range of cell types, including neural progenitor cells, keratinocytes, dendritic cells, and osteoblasts. ZIKV infections have been associated with fetal brain damage, which prompted the World Health Organization to declare a public health emergency in 2016. ZIKV infection has also been linked to birth defects in other organs. Several studies have reported congenital heart defects (CHD) in ZIKV infected infants and cardiovascular complications in adults infected with ZIKV. To develop a better understanding of potential causes for these pathologies at a cellular level, we characterized ZIKV infection of human fetal cardiac mesenchymal stromal cells (fcMSCs), a cell type that is known to contribute to both embryological development as well as adult cardiac physiology. Total RNA, supernatants, and/or cells were collected at various time points post-infection to evaluate ZIKV replication, cell death, and antiviral responses. We found that ZIKV productively infected fcMSCs with peak (~70%) viral mRNA detected at 48 h. Use of an antibody blocking the AXL receptor decreased ZIKV infection (by ~50%), indicating that the receptor is responsible to a large extent for viral entry into the cell. ZIKV also altered protein expression of several mesenchymal cell markers, which suggests that ZIKV could affect fcMSCs' differentiation process. Gene expression analysis of fcMSCs exposed to ZIKV at 6, 12, and 24 h post-infection revealed up-regulation of genes/pathways associated with interferon-stimulated antiviral responses. Stimulation of TLR3 (using poly I:C) or TLR7 (using Imiquimod) prior to ZIKV infection suppressed viral replication in a dose-dependent manner. Overall, fcMSCs can be a target for ZIKV infection, potentially resulting in CHD during embryological development and/or cardiovascular issues in ZIKV infected adults.