Jose Gomez-Marquez

Global Health Technology 2.0

Collaborative approach for global health technology is reported. The Global Health Initiative (GHI) at the Center for Integration of Medicine and Innovative Technology, the Center for Global Health at Massachusetts General Hospital (MGH), and Innovations in International Health at Massachusetts Institute of Technology (IIH@MIT) have formed a collaboration that puts our research and development model, Global Health Technology 2.0, to work and advances a growing global health portfolio.

Rapid antigen tests for dengue virus serotypes and Zika virus in patient serum

A low-cost, equipment-free rapid antigen test distinguishes dengue virus serotypes and Zika virus in patient sera without detectable cross-reactivity. Distinguishing dengue from Zika More than mere summer pests, mosquitoes can transmit viruses, such as dengue and Zika. Diagnosing infections of these related flaviviruses can be difficult because of cross-reactivity in diagnostic tests. Bosch et al. developed monoclonal antibodies to detect viral nonstructural 1 (NS1) protein antigens specific to dengue and Zika. Incorporating the antibodies into an immunochromatography format yielded a rapid diagnostic assay that produces a visual readout in the presence of NS1. The assay identified the four dengue serotypes and Zika viral infections without cross-reaction when testing human serum samples from endemic areas in Central and South America and India. This approach could be useful for developing rapid diagnostics for other emerging pathogens. The recent Zika virus (ZIKV) outbreak demonstrates that cost-effective clinical diagnostics are urgently needed to detect and distinguish viral infections to improve patient care. Unlike dengue virus (DENV), ZIKV infections during pregnancy correlate with severe birth defects, including microcephaly and neurological disorders. Because ZIKV and DENV are related flaviviruses, their homologous proteins and nucleic acids can cause cross-reactions and false-positive results in molecular, antigenic, and serologic diagnostics. We report the characterization of monoclonal antibody pairs that have been translated into rapid immunochromatography tests to specifically detect the viral nonstructural 1 (NS1) protein antigen and distinguish the four DENV serotypes (DENV1–4) and ZIKV without cross-reaction. To complement visual test analysis and remove user subjectivity in reading test results, we used image processing and data analysis for data capture and test result quantification. Using a 30-μl serum sample, the sensitivity and specificity values of the DENV1–4 tests and the pan-DENV test, which detects all four dengue serotypes, ranged from 0.76 to 1.00. Sensitivity/specificity for the ZIKV rapid test was 0.81/0.86, respectively, using a 150-μl serum input. Serum ZIKV NS1 protein concentrations were about 10-fold lower than corresponding DENV NS1 concentrations in infected patients; moreover, ZIKV NS1 protein was not detected in polymerase chain reaction–positive patient urine samples. Our rapid immunochromatography approach and reagents have immediate application in differential clinical diagnosis of acute ZIKV and DENV cases, and the platform can be applied toward developing rapid antigen diagnostics for emerging viruses.

A comparison of nanoparticle-antibody conjugation strategies in sandwich immunoassays

ABSTRACT Point-of-care (POC) diagnostics such as lateral flow and dipstick immunoassays use gold nanoparticle (NP)-antibody conjugates for visual readout. We investigated the effects of NP conjugation, surface chemistries, and antibody immobilization methods on dipstick performance. We compared orientational, covalent conjugation, electrostatic adsorption, and a commercial conjugation kit for dipstick assays to detect dengue virus NS1 protein. Assay performance depended significantly on their conjugate properties. We also tested arrangements of multiple test lines within strips. Results show that orientational, covalent conjugation with PEG shield could improve NS1 detection. These approaches can be used to optimize immunochromatographic detection for a range of biomarkers.

Portable digital lock-in instrument to determine chemical constituents with single-color absorption measurements for Global Health Initiatives.

Innovations in international health require the use of state-of-the-art technology to enable clinical chemistry for diagnostics of bodily fluids. We propose the implementation of a portable and affordable lock-in amplifier-based instrument that employs digital technology to perform biochemical diagnostics on blood, urine, and other fluids. The digital instrument is composed of light source and optoelectronic sensor, lock-in detection electronics, microcontroller unit, and user interface components working with either power supply or batteries. The instrument performs lock-in detection provided that three conditions are met. First, the optoelectronic signal of interest needs be encoded in the envelope of an amplitude-modulated waveform. Second, the reference signal required in the demodulation channel has to be frequency and phase locked with respect to the optoelectronic carrier signal. Third, the reference signal should be conditioned appropriately. We present three approaches to condition the signal appropriately: high-pass filtering the reference signal, precise offset tuning the reference level by low-pass filtering, and by using a voltage divider network. We assess the performance of the lock-in instrument by comparing it to a benchmark device and by determining protein concentration with single-color absorption measurements. We validate the concentration values obtained with the proposed instrument using chemical concentration measurements. Finally, we demonstrate that accurate retrieval of phase information can be achieved by using the same instrument.

Multicolor rapid diagnostics for infectious disease

Recent epidemic outbreaks have highlighted the need for a rapid point-of-care assay that can provide a diagnosis to enable treatment, proper quarantining, and disease surveillance. One promising diagnostic is the lateral flow test, i.e., the same type of assay used in pregnancy tests.1 It is simply a paper strip to which you add a biological fluid. Two colored stripes show up if it is positive, one if it is negative. These assays are attractive for diagnostics because they are inexpensive, easy to use, and do not require special reagents or experts to run them. Sample-toanswer can be reached in minutes, and can be read out by eye. Because the sample wicks through the strip by capillary action, it does not need power to run, which makes the assays highly portable and deployable. One shortcoming of these assays is that they can test only for a single disease at a time. However, at point of care, it is critical to be able to test for multiple diseases all at once to enable proper quarantining and treatment. For example, with the current Zika outbreak, Zika virus co-circulates with other diseases such as dengue and chikungunya because they are spread by the same mosquitoes. In addition, the initial symptoms of all three diseases are highly similar and nonspecific. Thus, a point-of-care assay that can screen for all of these simultaneously would enable a more rapid response for proper treatment. We describe a multiplexed assay developed in the lab by using silver nanoparticles of different colors.2 Here, the assay shows a different color test line depending on the biomarker that is present: see Figure 1(a). We synthesized silver nanoparticles to be of different sizes in the 30–50nm size range: see Figure 1(b).3 The different sizes have different colors that are distinguishable by eye both in solution and on paper: see Figure 1(c). We attached the silver nanoparticles to different antibodies. We attached orange nanoparticles to antibodies that can bind to the Figure 1. (a) Multiplexed diagnosis of yellow fever (YFV), Ebola (ZEBOV), and dengue (DENV) by silver nanoparticles of different colors used in sandwich immunoassays. (b) Silver nanoparticles synthesized (transmission electron microscopy images). Left to right: Yellow nanoparticle ‘seeds’ (used to grow the larger nanoparticles), orange nanoparticles, red nanoparticles, green nanoparticles. (c) Vials of the silver nanoparticles synthesized and solutions of the samples dried down on paper. (d) Assay strip for a multicolor multiplexed immunoassay. Control line: Positive control band that confirms flow.