Multiplex lateral flow assay development for snake venom detection in biological matrices.

Bothrops and Lachesis are two of Brazil's medically most relevant snake genera, causing tens of thousands of bites annually. Fortunately, Brazil has good accessibility to high-quality antivenoms at the genus and inter-genus level, enabling the treatment of many of these envenomings. However, the optimal use of these treatments requires that the snake species responsible for the bite is determined. Currently, physicians use a syndromic approach to diagnose snakebite, which can be difficult for medical personnel with limited training in clinical snakebite management. In this work, we have developed a novel monoclonal antibody-based multiplex lateral flow assay for differentiating Bothrops and Lachesis venoms within 15 min. The test can be read by the naked eye or (semi)-quantitatively by a smartphone supported by a 3D-printed attachment for controlling lighting conditions. The LFA can detect Bothrops and Lachesis venoms in spiked plasma and urine matrices at concentrations spanning six orders of magnitude. The LFA has detection limits of 10-50 ng/mL in spiked plasma and urine, and 50-500 ng/mL in spiked sera, for B. atrox and L. muta venoms. This test could potentially support medical personnel in correctly diagnosing snakebite envenomings at the point-of-care in Brazil, which may help improve patient outcomes and save lives.

Prototyping of a lateral flow assay based on monoclonal antibodies for detection of Bothrops venoms.

BACKGROUND: Brazil is home to a multitude of venomous snakes; perhaps the most medically relevant of which belong to the Bothrops genus. Bothrops spp. are responsible for roughly 70% of all snakebites in Brazil, and envenomings caused by their bites can be treated with three types of antivenom: bothropic antivenom, bothro-lachetic antivenom, and bothro-crotalic antivenom. The choice to administer antivenom depends on the severity of the envenoming, while the choice of antivenom depends on availability and on how certain the treating physician is that the patient was bitten by a bothropic snake. The diagnosis of a bothropic envenoming can be made based on expert identification of the dead snake or a photo thereof or based on a syndromic approach wherein the clinician examines the patient for characteristic manifestations of envenoming. This approach can be very effective but requires staff that has been trained in clinical snakebite management, which, unfortunately, far from all relevant staff has. RESULTS: In this article, we describe a prototype of the first lateral flow assay (LFA) capable of detecting venoms from Brazilian Bothrops spp. The monoclonal antibodies for the assay were generated using hybridoma technology and screened in sandwich enzyme-linked immunosorbent assays (ELISAs) to identify Bothrops spp.-specific antibody sandwich pairs. The prototype LFA is able to detect venom from several Bothrops spp. The LFA has a limit of detection (LoD) of 9.5 ng/mL in urine, when read with a commercial reader, and a visual LoD of approximately 25 ng/mL. SIGNIFICANCE: The work presented here serves as a proof of concept for a genus-specific venom detection kit that could support physicians in diagnosing Bothrops envenomings. Although further optimisation and testing is needed before the LFA can find clinical use, such a device could aid in decentralising antivenoms in the Brazilian Amazon and help ensure optimal snakebite management for even more victims of this highly neglected disease.

Snakebite Envenoming Diagnosis and Diagnostics.

Snakebite envenoming is predominantly an occupational disease of the rural tropics, causing death or permanent disability to hundreds of thousands of victims annually. The diagnosis of snakebite envenoming is commonly based on a combination of patient history and a syndromic approach. However, the availability of auxiliary diagnostic tests at the disposal of the clinicians vary from country to country, and the level of experience within snakebite diagnosis and intervention may be quite different for clinicians from different hospitals. As such, achieving timely diagnosis, and thus treatment, is a challenge faced by treating personnel around the globe. For years, much effort has gone into developing novel diagnostics to support diagnosis of snakebite victims, especially in rural areas of the tropics. Gaining access to affordable and rapid diagnostics could potentially facilitate more favorable patient outcomes due to early and appropriate treatment. This review aims to highlight regional differences in epidemiology and clinical snakebite management on a global scale, including an overview of the past and ongoing research efforts within snakebite diagnostics. Finally, the review is rounded off with a discussion on design considerations and potential benefits of novel snakebite diagnostics.

Snake Venomics Display: An online toolbox for visualization of snake venomics data.

With the introduction of powerful mass spectrometry equipment into the field of snake venom proteomics, a large body of venomics data is accumulating. To allow for better comparison between venom compositions from different snake species and to provide an online database containing this data, we devised the Snake Venomics Display toolbox for visualization of snake venomics data on linear scales. This toolbox is freely available to be used online at https://tropicalpharmacology.com/tools/snake-venomics-display/ and allows researchers to visualize venomics data in a Relative Abundance (%) visualization mode and in an Absolute Abundance (mg) visualization mode, the latter taking venom yields into account. The curated venomics data for all snake species included in this database is also made available in a downloadable Excel file format. The Snake Venomics Display toolbox represents a simple way of handling snake venomics data, which is better suited for large data sets of venom compositions from multiple snake species.