Zika Virus Specific Diagnostic Epitope Discovery

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for ...

New York University College of Dentistry

The impaired host defense system in HIV infection impacts the oral and gastrointestinal microbiota and associated opportunistic infections. Antiretroviral treatment is predicted to partially restore host defenses and decrease the oral manifestation of HIV/AIDS. Well-designed longitudinal studies are needed to better understand the interactions of soluble host defense proteins with bacteria and virus in HIV/AIDS. "Crosstalk" was designed as a longitudinal study of host responses along the gastrointestinal (GI) tract and interactions between defense molecules and bacteria in HIV infection and subsequent therapy.

Design aspects of a case-control clinical investigation of the effect of HIV on oral and gastrointestinal soluble innate factors and microbes.

Malaria remains one of the most prevalent infectious diseases and results in significant mortality. Isothermal amplification (loop-mediated isothermal amplification) is used to detect malarial DNA at levels of ~1 parasite/µL blood in ≤30 minutes without the isolation of parasite nucleic acid from subject's blood or saliva. The technique targets the mitochondrial cytochrome oxidase subunit 1 gene and is capable of distinguishing Plasmodium falciparum from Plasmodium vivax. Malarial diagnosis by the gold standard microscopic examination of blood smears is generally carried out only after moderate-to-severe symptoms appear. Rapid diagnostic antigen tests are available but generally require infection levels in the range of 200-2,000 parasites/µL for a positive diagnosis and cannot distinguish if the disease has been cleared due to the persistence of circulating antigen. This study describes a rapid and simple molecular assay to detect malarial genes directly from whole blood or saliva without DNA isolation.

Rapid Point-of-Care Isothermal Amplification Assay for the Detection of Malaria without Nucleic Acid Purification.

Saliva and other types of oral samples can readily be used for noninvasive diagnosis of diseases of the oral cavity and systemic diseases. Following an introduction outlining the types of oral samples and the analytes that can be measured in these samples, a detailed description of a novel oral-based diagnostic system to detect multiple bacterial and/or viral pathogens is presented. A reasonably priced, portable, point-of-care diagnostic system should be available within five years.

Oral-based techniques for the diagnosis of infectious diseases.

Confirmatory detection of diseases, such as HIV and HIV-associated pathogens in a rapid point-of-care (POC) diagnostic remains a goal for disease control, prevention, and therapy. If a sample could be analyzed onsite with a verified result, the individual could be counseled immediately and appropriate therapy initiated. Our group is focused on developing a microfluidic "lab-on-a-chip" that will simultaneously identify antigens, antibodies, RNA, and DNA using a single oral sample. The approach has been to design individual modules for each assay that uses similar components (e.g., valves, heaters, metering chambers, mixers) installed on a polycarbonate base with a common reporter system. Assay miniaturization reduces the overall analysis time, increases accuracy by simultaneously identifying multiple targets, and enhances detector sensitivity by upconverting phosphor technology (UPT). Our microfluidic approach employs four interrelated components: (1) sample acquisition-OraSure UPlink collectors that pick-up and release bacteria, soluble analytes, and viruses from an oral sample; (2) microfluidic processing-movement of microliter volumes of analyte, target analyte extraction and amplification; (3) detection of analytes using UPT particles in a lateral flow system; and (4) software for processing the results. Ultimately, the oral-based microscale diagnostic system will detect viruses and bacteria, associated pathogen antigens and nucleic acids, and antibodies to these pathogens.

Development of a microfluidic device for detection of pathogens in oral samples using upconverting phosphor technology (UPT).

An inexpensive, hand-held, point-of-care, disposable, self-contained immunoassay cassette comprised of air pouches for pumping, a metering chamber, reagents storage chambers, a mixer, and a lateral flow strip was designed, constructed, and tested. The assay was carried out in a consecutive flow format. The detection was facilitated with up-converting phosphor (UCP) reporter particles. The automated, timely pumping of the various reagents was driven by a spring-loaded timer. The utility of the cassette was demonstrated by detecting antibodies to HIV in saliva samples and further evaluated with a non-contagious, haptenized DNA assay. The cassette has several advantages over dip sticks such as sample preprocessing, integrated storage of reagents, and automated operation that reduces operator errors and training. The cassette and actuator described herein can readily be extended to detect biomarkers of other diseases in body fluids and other fluids at the point of care. The system is particularly suitable for resource-poor countries, where funds and trained personnel are in short supply.

A timer-actuated immunoassay cassette for detecting molecular markers in oral fluids.

The building blocks for an inexpensive, disposable, luminescence-based microfluidic immunoassay cassette are described, and their integration in a point-of-care diagnostic system is demonstrated. Fluid motion in the cassette is driven by depressing finger-actuated pouches. All reagents needed for the immunoassay can be stored in the cassette in liquid form. Prior to use, the cassette consists of two separate parts. A top storage component contains pouches, sealed storage chambers, a metering chamber, and needle seats. The bottom processing component contains connection needles, a mixing chamber, and a detection chamber with immobilized proteins. Subsequent to sample introduction, the storage and processing components are mated. The needles form hydraulic connections between the two parts and, in some cases, close valves. The pouches are then actuated sequentially to induce flow of various reagents and facilitate process operations. The cassette is compatible with different detection modalities. Both a cassette with immunochromatographic-based detection and a cassette with microbead-based detection were constructed and evaluated. The immunochromatographic cassette was used to detect antibodies to HIV in saliva samples. The bead-based cassette was used to detect the proinflammatory chemokine IL-8. The experimental data demonstrates good repeatability and reasonable sensitivity.

Finger-actuated, self-contained immunoassay cassettes.

A self-contained, integrated, disposable, sample-to-answer, polycarbonate microfluidic cassette for nucleic acid-based detection of pathogens at the point of care was designed, constructed, and tested. The cassette comprises on-chip sample lysis, nucleic acid isolation, enzymatic amplification (polymerase chain reaction and, when needed, reverse transcription), amplicon labeling, and detection. On-chip pouches and valves facilitate fluid flow control. All the liquids and dry reagents needed for the various reactions are pre-stored in the cassette. The liquid reagents are stored in flexible pouches formed on the chip surface. Dry (RT-)PCR reagents are pre-stored in the thermal cycling, reaction chamber. The process operations include sample introduction; lysis of cells and viruses; solid-phase extraction, concentration, and purification of nucleic acids from the lysate; elution of the nucleic acids into a thermal cycling chamber and mixing with pre-stored (RT-)PCR dry reagents; thermal cycling; and detection. The PCR amplicons are labeled with digoxigenin and biotin and transmitted onto a lateral flow strip, where the target analytes bind to a test line consisting of immobilized avidin-D. The immobilized nucleic acids are labeled with up-converting phosphor (UCP) reporter particles. The operation of the cassette is automatically controlled by an analyzer that provides pouch and valve actuation with electrical motors and heating for the thermal cycling. The functionality of the device is demonstrated by detecting the presence of bacterial B.Cereus, viral armored RNA HIV, and HIV I virus in saliva samples. The cassette and actuator described here can be used to detect other diseases as well as the presence of bacterial and viral pathogens in the water supply and other fluids.

An integrated, self-contained microfluidic cassette for isolation, amplification, and detection of nucleic acids.

A simple, point of care, inexpensive, disposable cassette for the detection of nucleic acids extracted from pathogens was designed, constructed, and tested. The cassette utilizes a single reaction chamber for isothermal amplification of nucleic acids. The chamber is equipped with an integrated, flow-through, Flinders Technology Associates (Whatman FTA®) membrane for the isolation, concentration, and purification of DNA and/or RNA. The nucleic acids captured by the membrane are used directly as templates for amplification without elution, thus simplifying the cassette's flow control. The FTA membrane also serves another critical role-enabling the removal of inhibitors that dramatically reduce detection sensitivity. Thermal control is provided with a thin film heater external to the cassette. The amplification process was monitored in real time with a portable, compact fluorescent reader. The utility of the integrated, single-chamber cassette was demonstrated by detecting the presence of HIV-1 in oral fluids. The HIV RNA was reverse transcribed and subjected to loop-mediated, isothermal amplification (LAMP). A detection limit of less than 10 HIV particles was demonstrated. The cassette is particularly suitable for resource poor regions, where funds and trained personnel are in short supply. The cassette can be readily modified to detect nucleic acids associated with other pathogens borne in saliva, urine, and other body fluids as well as in water and food.

An isothermal amplification reactor with an integrated isolation membrane for point-of-care detection of infectious diseases.

Understanding of the human microbiome continues to grow rapidly; however, reports on changes in the microbiome after HIV infection are still limited. This review surveys the progress made in methodology associated with microbiome studies and highlights the remaining challenges to this field. Studies have shown that commensal oral, gut, vaginal, and penile bacteria are vital to the health of the human immune system. Our studies on crosstalk among oral and gastrointestinal soluble innate factors, HIV, and microbes indicated that the oral and gut microbiome was altered in the HIV-positive samples compared to the negative controls. The importance of understanding the bacterial component of HIV/AIDS, and likelihood of "crosstalk" between viral and bacterial pathogens, will help in understanding the role of the microbiome in HIV-infected individuals and facilitate identification of novel antiretroviral factors for use as novel diagnostics, microbicides, or therapeutics against HIV infection.

Human microbiome and HIV/AIDS.

&Egrave; descritto un dispositivo dual-path microfluidica prototipo (scheda Rheonix) in grado di eseguire contemporaneamente lo screening (antigene o anticorpo) e conferma (acido nucleico) rilevazione degli agenti patogeni. Il dispositivo integra completamente esempio elaborazione, rilevazione dell&#39;antigene o dell&#39;anticorpo e amplificazione dell&#39;acido nucleico e rilevamento, dimostrando la diagnosi di &quot;campione di risultato&quot;, rapido e poco costoso con prestazioni paragonabili ad analisi da banco. Per la progettazione dei chip, &egrave; stato seguito un approccio modulare permettendo l&#39;ottimizzazione delle singole fasi del processo di elaborazione di campione. Questo sistema modulare offre grande versatilit&agrave; accomodante obiettivi differenti di malattia indipendentemente dal metodo di produzione. Nel modulo di rilevamento, &egrave; stato impiegato un protocollo di flusso laterale (LF) utilizzando upconverting reporter di fosforo (UCP). Il modulo dell&#39;acido nucleico (NA) incorpora una generica microprovetta contenente i reagenti secchi. Strisce di flusso laterale e gli iniettori di PCR determinano la destinazione o la malattia che viene diagnosticata. La diagnosi di infezione da HIV &egrave; stata utilizzata come modello per indagare la rilevazione simultanea di entrambi anticorpi umani contro i virus e RNA virale. Il risultato sierologico &egrave; disponibile in meno di 30 min, e la conferma dall&#39;amplificazione di RNA prende un altro 60 min. Questo approccio combina diagnostica sierologica portatile core con una prova di conferma basato su dell&#39;acido nucleico.

Sviluppo di un dispositivo microfluidico generico per la rilevazione simultanea di anticorpi e gli acidi nucleici nei fluidi orali.

A prototype dual-path microfluidic device (Rheonix CARD) capable of performing simultaneously screening (antigen or antibody) and confirmatory (nucleic acid) detection of pathogens is described. The device fully integrates sample processing, antigen or antibody detection, and nucleic acid amplification and detection, demonstrating rapid and inexpensive "sample-to-result" diagnosis with performance comparable to benchtop analysis. For the chip design, a modular approach was followed allowing the optimization of individual steps in the sample processing process. This modular design provides great versatility accommodating different disease targets independently of the production method. In the detection module, a lateral flow (LF) protocol utilizing upconverting phosphor (UCP) reporters was employed. The nucleic acid (NA) module incorporates a generic microtube containing dry reagents. Lateral flow strips and PCR primers determine the target or disease that is diagnosed. Diagnosis of HIV infection was used as a model to investigate the simultaneous detection of both human antibodies against the virus and viral RNA. The serological result is available in less than 30â€‰min, and the confirmation by RNA amplification takes another 60â€‰min. This approach combines a core serological portable diagnostic with a nucleic acid-based confirmatory test.

Development of a generic microfluidic device for simultaneous detection of antibodies and nucleic acids in oral fluids.

Un dispositif de double voie microfluidique prototype (carte Rheonix) capable d&#39;effectuer simultan&eacute;ment d&eacute;pistage (antig&egrave;ne ou anticorps) et confirmation (acides nucl&eacute;iques) d&eacute;tection d&#39;agents pathog&egrave;nes est d&eacute;crite. Le dispositif int&egrave;gre pleinement le traitement des &eacute;chantillons, d&eacute;tection de l&#39;antig&egrave;ne ou l&#39;anticorps et amplification des acides nucl&eacute;iques et d&eacute;tection, diagnostic de &laquo; &eacute;chantillon-&agrave;-r&eacute;sultat &raquo; rapide et peu co&ucirc;teux avec des performances comparables &agrave; benchtop analyse la d&eacute;monstration. Pour la conception de la puce, une approche modulaire a &eacute;t&eacute; suivie en permettant l&#39;optimisation des diff&eacute;rentes &eacute;tapes dans le processus de traitement d&#39;&eacute;chantillon. Cette conception modulaire offre grande polyvalence pouvant accueillir des cibles diff&eacute;rentes maladie ind&eacute;pendamment de la m&eacute;thode de production. Dans le module de d&eacute;tection, un protocole de flux lat&eacute;ral (LF) utilisant la conversion ascendante reporters de phosphore (UCP) a &eacute;t&eacute; employ&eacute;. Le module d&#39;acide nucl&eacute;ique (NA) incorpore un microtube g&eacute;n&eacute;rique contenant des r&eacute;actifs secs. Bandes d&#39;&eacute;coulement lat&eacute;ral et des amorces PCR d&eacute;terminent la cible ou la maladie est diagnostiqu&eacute;e. Diagnostic d&#39;infection &agrave; VIH a &eacute;t&eacute; utilis&eacute; comme mod&egrave;le pour &eacute;tudier la d&eacute;tection simultan&eacute;e des deux anticorps humains contre le virus et l&#39;ARN viral. Le r&eacute;sultat s&eacute;rologique est disponible en moins de 30 min, et la confirmation par amplification de RNA prend un autre 60 min. Cette approche combine un diagnostic s&eacute;rologique portable de base avec un test de confirmation &agrave; base d&#39;acide nucl&eacute;ique.

D&eacute;veloppement d&#39;un dispositif microfluidique g&eacute;n&eacute;riques pour la d&eacute;tection simultan&eacute;e des anticorps et des acides nucl&eacute;iques dans des fluides par voie orale.

Gp340 is a member of the scavenger receptor cysteine-rich family of innate immune molecules and also functions as a tumor suppressor. This study describes a picogram-level assay using electrochemiluminescence technology on the MesoScale Discovery platform. Antibodies were evaluated and the best pair was used to assay whole-mouth stimulated saliva and cervical/vaginal lavage. The assay was tested using specimens obtained from healthy volunteers to determine if gp340 concentration in saliva correlates with levels in vaginal lavage fluid. Interestingly, no correlation was determined between gp340 content in these two fluids.

An electrochemiluminescence assay for gp340 (DMBT1).

Gp340 est membre de la famille des riches en cyst&eacute;ine r&eacute;cepteurs scavenger de mol&eacute;cules immunitaires inn&eacute;es et fonctions comme un suppresseur de tumeur. Cette &eacute;tude d&eacute;crit un essai au niveau du picogramme par &eacute;lectrochimiluminescence technologie sur la plateforme de d&eacute;couverte de m&eacute;so-&eacute;chelle. Les anticorps ont &eacute;t&eacute; &eacute;valu&eacute;s et la meilleure paire a &eacute;t&eacute; utilis&eacute;e pour analyser toute la bouche salive stimul&eacute;e et lavage vaginal/col de l&#39;ut&eacute;rus. L&#39;essai a &eacute;t&eacute; test&eacute; &agrave; l&#39;aide de sp&eacute;cimens obtenus &agrave; partir des volontaires sains pour d&eacute;terminer si gp340 concentration dans la salive est en corr&eacute;lation avec les concentrations dans le liquide de lavage vaginal. Fait int&eacute;ressant, aucune corr&eacute;lation n&#39;a &eacute;t&eacute; d&eacute;termin&eacute;e entre gp340 contenu dans ces deux fluides.

Un dosage par &eacute;lectrochimiluminescence pour gp340 (DMBT1).

We developed a microfluidic system to simultaneously detect host anti-HIV antibodies and viral RNA in the same specimen in order to satisfy two important diagnostic criteria, especially within resource-limited settings. First, the system can detect acute HIV infection and allow immediate confirmation of a seropositive screening result by detection of HIV RNA. It also addresses the well-known "seroconversion window" during early HIV infection when antibodies are not yet detectable and viral loads are at their highest.

William R. Abrams

Department of Basic Sciences and Craniofacial Development

New York University College of Dentistry

Zika Virus Specific Diagnostic Epitope Discovery

William R. Abrams

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Basic Sciences and Craniofacial Development

New York University College of Dentistry

Zika Virus Specific Diagnostic Epitope Discovery

Department of Basic Sciences and Craniofacial Development