RESUMO
Smartphones have shown promise as an enabling technology for portable and distributed point-of-care diagnostic tests. The CMOS camera sensor can be used for detecting optical signals, including fluorescence for applications such as isothermal nucleic acid amplification tests. However, such analysis is typically limited mostly to end point detection of single targets. Here we present a smartphone-based image analysis pipeline that utilizes the CIE xyY (chromaticity-luminance) color space to measure the luminance (in lieu of RGB intensities) of fluorescent signals arising from nucleic acid amplification targets, with a discrimination sensitivity (ratio between the positive to negative signals), which is an order of magnitude more than traditional RGB intensity based analysis. Furthermore, the chromaticity part of the analysis enables reliable multiplexed detection of different targets labeled with spectrally separated fluorophores. We apply this chromaticity-luminance formulation to simultaneously detect Zika and chikungunya viral RNA via end point RT-LAMP (Reverse transcription Loop-Mediated isothermal amplification). We also show real time LAMP detection of Neisseria gonorrhoeae samples down to a copy number of 3.5 copies per 10 µL of reaction volume in our smartphone-operated portable LAMP box. Our chromaticity-luminance analysis is readily adaptable to other types of multiplexed fluorescence measurements using a smartphone camera.
Assuntos
Colorimetria , Fluorescência , Corantes Fluorescentes/química , Imagem Óptica , RNA Viral/análise , Smartphone , Vírus Chikungunya/química , Colorimetria/instrumentação , Imagem Óptica/instrumentação , Fotografação/instrumentação , Smartphone/instrumentação , Zika virus/químicaRESUMO
Reverse-transcription-loop-mediated isothermal amplification (RT-LAMP) has frequently been proposed as an enabling technology for simplified diagnostic tests for RNA viruses. However, common detection techniques used for LAMP and RT-LAMP have drawbacks, including poor discrimination capability, inability to multiplex targets, high rates of false positives, and (in some cases) the requirement of opening reaction tubes postamplification. Here, we present a simple technique that allows closed-tube, target-specific detection, based on inclusion of a dye-labeled primer that is incorporated into a target-specific amplicon if the target is present. A short, complementary quencher hybridizes to unincorporated primer upon cooling down at the end of the reaction, thereby quenching fluorescence of any unincorporated primer. Our technique, which we term QUASR (for quenching of unincorporated amplification signal reporters, read "quasar"), does not significantly reduce the amplification efficiency or sensitivity of RT-LAMP. Equipped with a simple LED excitation source and a colored plastic gel filter, the naked eye or a camera can easily discriminate between positive and negative QUASR reactions, which produce a difference in signal of approximately 10:1 without background subtraction. We demonstrate that QUASR detection is compatible with complex sample matrices such as human blood, using a novel LAMP primer set for bacteriophage MS2 (a model RNA virus particle). Furthermore, we demonstrate single-tube duplex detection of West Nile virus (WNV) and chikungunya virus (CHIKV) RNA.
Assuntos
Técnicas de Amplificação de Ácido Nucleico , Vírus de RNA/isolamento & purificação , Temperatura , Vírus de RNA/genéticaRESUMO
The development of topical anti-human immunodeficiency virus (HIV) microbicides may provide women with strategies to protect themselves against sexual HIV transmission. Pericoital drug delivery systems intended for use immediately before sex, such as microbicide gels, must deliver high drug doses for maximal effectiveness. The goal of achieving a high antiretroviral dose is complicated by the need to simultaneously retain the dose and quickly release drug compounds into the tissue. For drugs with limited solubility in vaginal gels, increasing the gel volume to increase the dose can result in leakage. While solid dosage forms like films and tablets increase retention, they often require more than 15 min to fully dissolve, potentially increasing the risk of inducing epithelial abrasions during sex. Here, we demonstrate that water-soluble electrospun fibers, with their high surface area-to-volume ratio and ability to disperse antiretrovirals, can serve as an alternative solid dosage form for microbicides requiring both high drug loading and rapid hydration. We formulated maraviroc at up to 28 wt% into electrospun solid dispersions made from either polyvinylpyrrolidone or poly(ethylene oxide) nanofibers or microfibers and investigated the role of drug loading, distribution, and crystallinity in determining drug release rates into aqueous media. We show here that water-soluble electrospun materials can rapidly release maraviroc upon contact with moisture and that drug delivery is faster (less than 6 min under sink conditions) when maraviroc is electrospun in polyvinylpyrrolidone fibers containing an excipient wetting agent. These materials offer an alternative dosage form to current pericoital microbicides.
Assuntos
Fármacos Anti-HIV/química , Anti-Infecciosos Locais/química , Cicloexanos/química , Suspensões/química , Triazóis/química , Cremes, Espumas e Géis Vaginais/química , Administração Intravaginal , Fármacos Anti-HIV/farmacologia , Anti-Infecciosos Locais/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cicloexanos/farmacologia , Técnicas Eletroquímicas , Excipientes/química , Feminino , Infecções por HIV/prevenção & controle , Humanos , Cinética , Maraviroc , Nanofibras/química , Nanofibras/ultraestrutura , Polietilenoglicóis/química , Polissorbatos/química , Povidona/química , Suspensões/farmacologia , Triazóis/farmacologia , Cremes, Espumas e Géis Vaginais/farmacologia , Molhabilidade , Agentes Molhantes/químicaRESUMO
The United States Department of Agriculture (USDA) Division of Agricultural Select Agents and Toxins (DASAT) established a list of biological agents (Select Agents List) that threaten crops of economic importance to the United States and regulates the procedures governing containment, incident response, and the security of entities working with them. Every 2 years the USDA DASAT reviews their select agent list, utilizing assessments by subject matter experts (SMEs) to rank the agents. We explored the applicability of multi-criteria decision analysis (MCDA) techniques and a decision support framework (DSF) to support the USDA DASAT biennial review process. The evaluation includes both current and non-select agents to provide a robust assessment. We initially conducted a literature review of 16 pathogens against 9 criteria for assessing plant health and bioterrorism risk and documented the findings to support this analysis. Technical review of published data and associated scoring recommendations by pathogen-specific SMEs was found to be critical for ensuring accuracy. Scoring criteria were adopted to ensure consistency. The MCDA supported the expectation that select agents would rank high on the relative risk scale when considering the agricultural consequences of a bioterrorism attack; however, application of analytical thresholds as a basis for designating select agents led to some exceptions to current designations. A second analytical approach used agent-specific data to designate key criteria in a DSF logic tree format to identify pathogens of low concern that can be ruled out for further consideration as select agents. Both the MCDA and DSF approaches arrived at similar conclusions, suggesting the value of employing the two analytical approaches to add robustness for decision making.
RESUMO
The United States Department of Agriculture (USDA), Division of Agricultural Select Agents and Toxins (DASAT) established a list of biological agents and toxins (Select Agent List) that potentially threaten agricultural health and safety, the procedures governing the transfer of those agents, and training requirements for entities working with them. Every 2 years the USDA DASAT reviews the Select Agent List, using subject matter experts (SMEs) to perform an assessment and rank the agents. To assist the USDA DASAT biennial review process, we explored the applicability of multi-criteria decision analysis (MCDA) techniques and a Decision Support Framework (DSF) in a logic tree format to identify pathogens for consideration as select agents, applying the approach broadly to include non-select agents to evaluate its robustness and generality. We conducted a literature review of 41 pathogens against 21 criteria for assessing agricultural threat, economic impact, and bioterrorism risk and documented the findings to support this assessment. The most prominent data gaps were those for aerosol stability and animal infectious dose by inhalation and ingestion routes. Technical review of published data and associated scoring recommendations by pathogen-specific SMEs was found to be critical for accuracy, particularly for pathogens with very few known cases, or where proxy data (e.g., from animal models or similar organisms) were used to address data gaps. The MCDA analysis supported the intuitive sense that select agents should rank high on the relative risk scale when considering agricultural health consequences of a bioterrorism attack. However, comparing select agents with non-select agents indicated that there was not a clean break in scores to suggest thresholds for designating select agents, requiring subject matter expertise collectively to establish which analytical results were in good agreement to support the intended purpose in designating select agents. The DSF utilized a logic tree approach to identify pathogens that are of sufficiently low concern that they can be ruled out from consideration as a select agent. In contrast to the MCDA approach, the DSF rules out a pathogen if it fails to meet even one criteria threshold. Both the MCDA and DSF approaches arrived at similar conclusions, suggesting the value of employing the two analytical approaches to add robustness for decision making.
RESUMO
Current multiplexed diagnostics for Zika, dengue, and chikungunya viruses are situated outside the intersection of affordability, high performance, and suitability for use at the point-of-care in resource-limited settings. Consequently, insufficient diagnostic capabilities are a key limitation facing current Zika outbreak management strategies. Here we demonstrate highly sensitive and specific detection of Zika, chikungunya, and dengue viruses by coupling reverse-transcription loop-mediated isothermal amplification (RT-LAMP) with our recently developed quenching of unincorporated amplification signal reporters (QUASR) technique. We conduct reactions in a simple, inexpensive and portable "LAMP box" supplemented with a consumer class smartphone. The entire assembly can be powered by a 5 V USB source such as a USB power bank or solar panel. Our smartphone employs a novel algorithm utilizing chromaticity to analyze fluorescence signals, which improves the discrimination of positive/negative signals by 5-fold when compared to detection with traditional RGB intensity sensors or the naked eye. The ability to detect ZIKV directly from crude human sample matrices (blood, urine, and saliva) demonstrates our device's utility for widespread clinical deployment. Together, these advances enable our system to host the key components necessary to expand the use of nucleic acid amplification-based detection assays towards point-of-care settings where they are needed most.
Assuntos
Vírus Chikungunya/isolamento & purificação , Vírus da Dengue/isolamento & purificação , Técnicas e Procedimentos Diagnósticos , Smartphone , Zika virus/isolamento & purificação , Animais , Bioensaio , Chlorocebus aethiops , Primers do DNA/metabolismo , Humanos , Reação em Cadeia da Polimerase , RNA Viral/genética , Sensibilidade e Especificidade , Células VeroRESUMO
Electrospun fibers show potential as a topical delivery system for vaginal microbicides. Previous reports have demonstrated delivery of anti-HIV and anti-STI (sexually transmitted infection) agents from fibers formulated using hydrophilic, hydrophobic, or pH-responsive polymers that result in rapid, prolonged, or stimuli-responsive release, respectively. However, coaxial electrospun fibers have yet to be evaluated as a highly tunable microbicide delivery vehicle. In this research, we explored the opportunities and limitations of a model coaxial electrospun fiber system to provide broad and tunable release rates for the HIV entry inhibitor maraviroc. Specifically, we prepared ethyl cellulose (EC)-shell and polyvinylpyrrolidone (PVP)-core fibers that were capable of releasing actives over a range of hours to several days. We further demonstrated simple and effective methods for combining core-shell fibers with rapid-release formulations to provide combined instantaneous and sustained maraviroc release. In addition, we investigated the effect of varying release media on maraviroc release from core-shell fibers, and found that release was strongly influenced by media surface tension and drug ionization. Finally, in vitro cell culture studies show that our fiber formulations were not cytotoxic and that electrospun maraviroc maintained similar antiviral activity compared to neat maraviroc.
Assuntos
Anti-Infecciosos/química , Cicloexanos/química , Portadores de Fármacos/química , Triazóis/química , Fármacos Anti-HIV/química , Fármacos Anti-HIV/metabolismo , Fármacos Anti-HIV/toxicidade , Anti-Infecciosos/metabolismo , Anti-Infecciosos/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Celulose/análogos & derivados , Celulose/química , Cicloexanos/metabolismo , Cicloexanos/toxicidade , Liberação Controlada de Fármacos , Humanos , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Maraviroc , Microscopia Eletrônica de Varredura , Espectroscopia Fotoeletrônica , Povidona/química , Propriedades de Superfície , Triazóis/metabolismo , Triazóis/toxicidadeRESUMO
Collection of mosquitoes and testing for vector-borne viruses is a key surveillance activity that directly influences the vector control efforts of public health agencies, including determining when and where to apply insecticides. Vector control districts in California routinely monitor for three human pathogenic viruses including West Nile virus (WNV), Western equine encephalitis virus (WEEV), and St. Louis encephalitis virus (SLEV). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) offers highly sensitive and specific detection of these three viruses in a single multiplex reaction, but this technique requires costly, specialized equipment that is generally only available in centralized public health laboratories. We report the use of reverse transcription loop-mediated isothermal amplification (RT-LAMP) to detect WNV, WEEV, and SLEV RNA extracted from pooled mosquito samples collected in California, including novel primer sets for specific detection of WEEV and SLEV, targeting the nonstructural protein 4 (nsP4) gene of WEEV and the 3' untranslated region (3'-UTR) of SLEV. Our WEEV and SLEV RT-LAMP primers allowed detection of <0.1 PFU/reaction of their respective targets in <30 minutes, and exhibited high specificity without cross reactivity when tested against a panel of alphaviruses and flaviviruses. Furthermore, the SLEV primers do not cross-react with WNV, despite both viruses being closely related members of the Japanese encephalitis virus complex. The SLEV and WEEV primers can also be combined in a single RT-LAMP reaction, with discrimination between amplicons by melt curve analysis. Although RT-qPCR is approximately one order of magnitude more sensitive than RT-LAMP for all three targets, the RT-LAMP technique is less instrumentally intensive than RT-qPCR and provides a more cost-effective method of vector-borne virus surveillance.
Assuntos
Culicidae/virologia , Vírus da Encefalite de St. Louis/isolamento & purificação , Vírus da Encefalite Equina do Oeste/isolamento & purificação , Vírus do Nilo Ocidental/isolamento & purificação , Animais , Primers do DNA , Humanos , Técnicas de Amplificação de Ácido Nucleico , Vigilância da População , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: The use of drug combinations has revolutionized the treatment of HIV but there is no equivalent combination product that exists for prevention, particularly for topical HIV prevention. Strategies to combine chemically incompatible agents may facilitate the discovery of unique drug-drug activities, particularly unexplored combination drug synergy. We fabricated two types of nanoparticles, each loaded with a single antiretroviral (ARV) that acts on a specific step of the viral replication cycle. Here we show unique combination drug activities mediated by our polymeric delivery systems when combined with free tenofovir (TFV). METHODOLOGY/PRINCIPAL FINDINGS: Biodegradable poly(lactide-co-glycolide) nanoparticles loaded with efavirenz (NP-EFV) or saquinavir (NP-SQV) were individually prepared by emulsion or nanoprecipitation techniques. Nanoparticles had reproducible size (d â¼200 nm) and zeta potential (-25 mV). The drug loading of the nanoparticles was approximately 7% (w/w). NP-EFV and NP-SQV were nontoxic to TZM-bl cells and ectocervical explants. Both NP-EFV and NP-SQV exhibited potent protection against HIV-1 BaL infection in vitro. The HIV inhibitory effect of nanoparticle formulated ARVs showed up to a 50-fold reduction in the 50% inhibitory concentration (IC50) compared to free drug. To quantify the activity arising from delivery of drug combinations, we calculated combination indices (CI) according to the median-effect principle. NP-EFV combined with free TFV demonstrated strong synergistic effects (CI50â=â0.07) at a 1â¶50 ratio of IC50 values and additive effects (CI50â=â1.05) at a 1â¶1 ratio of IC50 values. TFV combined with NP-SQV at a 1â¶1 ratio of IC50 values also showed strong synergy (CI50â=â0.07). CONCLUSIONS: ARVs with different physicochemical properties can be encapsulated individually into nanoparticles to potently inhibit HIV. Our findings demonstrate for the first time that combining TFV with either NP-EFV or NP-SQV results in pronounced combination drug effects, and emphasize the potential of nanoparticles for the realization of unique drug-drug activities.
Assuntos
Adenina/análogos & derivados , Fármacos Anti-HIV/farmacologia , Benzoxazinas/farmacologia , Infecções por HIV/prevenção & controle , Nanopartículas/química , Organofosfonatos/farmacologia , Saquinavir/farmacologia , Adenina/farmacologia , Adenina/toxicidade , Alcinos , Animais , Fármacos Anti-HIV/toxicidade , Benzoxazinas/toxicidade , Linhagem Celular , Colo do Útero/efeitos dos fármacos , Química Farmacêutica , Ciclopropanos , Preparações de Ação Retardada/farmacologia , Preparações de Ação Retardada/toxicidade , Portadores de Fármacos/farmacologia , Portadores de Fármacos/toxicidade , Sinergismo Farmacológico , Quimioterapia Combinada , Feminino , HIV-1/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Ácido Láctico/química , Macaca , Modelos Biológicos , Nanopartículas/toxicidade , Organofosfonatos/toxicidade , Tamanho da Partícula , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Saquinavir/toxicidade , Espectroscopia de Infravermelho com Transformada de Fourier , Tenofovir , Técnicas de Cultura de TecidosRESUMO
Diversity of microbicide delivery systems is essential for future success in the prevention and treatment of HIV in order to account for the varied populations of women all over the world that may benefit from use of these products. Recently, a novel dosage form for intravaginal drug delivery has been developed using drug-eluting fibers fabricated by electrospinning. There is a strong rationale to support the idea that drug-eluting fibers can be designed to realize multiple design constraints in a single product for topical HIV prevention: fibers are able to deliver a wide range of agents, incorporate multiple agents via composites, and facilitate controlled release over relevant time frames for pericoital and sustained (coitally-independent) use. It is also technologically feasible to scale-up production of fiber-based microbicides. Electrospun fibers may allow for prioritization of physical attributes that affect user perceptions without compromising biological efficacy. Challenges with using fibers as a microbicide include issues related to vehicle deployment, spreading and retention in the vaginal vault. In addition, studies will need to address the interaction of the fibers with the mucosal environment, including unknown safety and toxicity. Sustained release fiber microbicides capable of delivering multiple antiretroviral drugs while simultaneously exhibiting tunable degradation or dissolution of the fibers is also a challenge. However, electrospun fibers are a promising new platform for vaginal delivery of anti-HIV agents and future research will inform their place in the field. This article is based on a presentation at the "Product Development Workshop 2013: HIV and Multipurpose Prevention Technologies", held in Arlington, Virginia on February 20-21, 2013. It forms part of a special supplement to Antiviral Research.
Assuntos
Fármacos Anti-HIV/administração & dosagem , Sistemas de Liberação de Medicamentos/instrumentação , Infecções por HIV/prevenção & controle , HIV-1/efeitos dos fármacos , Administração Intravaginal , Sistemas de Liberação de Medicamentos/métodos , Feminino , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/fisiologia , HumanosRESUMO
Multipurpose prevention technologies (MPTs) that simultaneously prevent sexually transmitted infections (STIs) and unintended pregnancy are a global health priority. Combining chemical and physical barriers offers the greatest potential to design effective MPTs, but integrating both functional modalities into a single device has been challenging. Here we show that drug-eluting fiber meshes designed for topical drug delivery can function as a combination chemical and physical barrier MPT. Using FDA-approved polymers, we fabricated nanofiber meshes with tunable fiber size and controlled degradation kinetics that facilitate simultaneous release of multiple agents against HIV-1, HSV-2, and sperm. We observed that drug-loaded meshes inhibited HIV-1 infection in vitro and physically obstructed sperm penetration. Furthermore, we report on a previously unknown activity of glycerol monolaurate (GML) to potently inhibit sperm motility and viability. The application of drug-eluting nanofibers for HIV-1 prevention and sperm inhibition may serve as an innovative platform technology for drug delivery to the lower female reproductive tract.
Assuntos
Fármacos Anti-HIV/administração & dosagem , Anticoncepção , Anticoncepcionais/administração & dosagem , HIV-1/efeitos dos fármacos , Nanofibras/química , Animais , Fármacos Anti-HIV/química , Anticoncepcionais/química , Sistemas de Liberação de Medicamentos , Feminino , Infecções por HIV/prevenção & controle , Humanos , Lauratos/farmacologia , Macaca , Masculino , Camundongos , Monoglicerídeos/farmacologia , Nanofibras/ultraestrutura , Gravidez , Infecções Sexualmente Transmissíveis/prevenção & controle , Espermatozoides/efeitos dos fármacosRESUMO
Most laboratory assays take advantage of multi-step protocols to achieve high performance, but conventional paper-based tests (e.g., lateral flow tests) are generally limited to assays that can be carried out in a single fluidic step. We have developed two-dimensional paper networks (2DPNs) that use materials from lateral flow tests but reconfigure them to enable programming of multi-step reagent delivery sequences. The 2DPN uses multiple converging fluid inlets to control the arrival time of each fluid to a detection zone or reaction zone, and it requires a method to disconnect each fluid source in a corresponding timed sequence. Here, we present a method that allows programmed disconnection of fluid sources required for multi-step delivery. A 2DPN with legs of different lengths is inserted into a shared buffer well, and the dropping fluid surface disconnects each leg at in a programmable sequence. This approach could enable multi-step laboratory assays to be converted into simple point-of-care devices that have high performance yet remain easy to use.