Development of rapid detection kit for stratification of snakebites

Most people will never have to worry about a deadly encounter with a venomous snake. Nonetheless, this threat is real in many parts of the tropics and subtropics, where roughly five million people are bitten every year, about 140,000 of whom die, and many more of whom suffer life-long consequences. Snakebite envenoming is an occupational disease, which disproportionately affects people in low- resource settings, especially in the rural tropics. Snake venoms are comprised of complex mixtures of toxins, capable of laying siege to several of the body’s vital systems simultaneously. The only specific treatment for snakebite envenoming is antivenom, consisting of antibodies from hyperimmunised animals. These antivenoms are typically only effective in treating envenomings caused by the snake venom(s) used during immunisation, as well as closely related venoms. The corollary is that it is necessary to determine which type of snake was involved in a bite, at least to a level corresponding to the neutralising capacities of the antivenom (which is often genus level). Currently, the common approach to diagnosis of snakebite patients is the syndromic approach, in which physicians assess the presence of characteristic venom syndromes. This approach can be very effective but requires highly skilled personnel. Unfortunately, many healthcare providers are never trained in clinical snakebite management, although they practise in areas where snakebites are common. Delays in diagnosis with subsequent delays in treatment or even administration of inappropriate treatment can worsen patient outcomes. Therefore, physicians, researchers, and key opinion leaders alike have advocated for improved training of healthcare personnel. The scientific community has also risen to this challenge, and several assays capable of detecting either venom components or biomarkers of envenoming have been reported in the literature. However, perhaps the only assay capable of detecting snake venom components that has found its way into clinical use is Seqirus’ Snake Venom Detection Kit, found only in Australia and Papua New Guinea.

This PhD project aimed to develop lateral flow assays capable of distinguishing venoms from the most medically relevant Brazilian snake genera, thereby having the potential to support snakebite diagnosis in Brazil. To this end, 121 monoclonal antibodies were tested for their binding to Brazilian snake venoms from the Bothrops, Lachesis, and Crotalus genera and 19 antibodies were selected for their ability to recognise several venoms from a single genus, without cross-reacting with venoms from closely related genera. These antibodies were screened in sandwich enzyme-linked immunosorbent assays (ELISAs), and 70 sandwich pairs were identified. These sandwich pairs were tested in further ELISAs to assess which pairs recognised venoms from most species from a single genus. The antibodies of the 29 most promising sandwich pairs were conjugated to biotin and gold-nanoparticles, respectively, and used to establish prototype lateral flow assays using commercially available lateral flow strips. These lateral flow assays were evaluated in terms of their sensitivity, and the most promising lateral flow assays capable of detecting Bothrops spp., Lachesis spp., and Crotalus spp. venoms, respectively, were chosen.

The Bothrops spp. lateral flow assay was characterised further, and a limit of detection of ca. 10 ng/mL was established, when using a commercial reader to detect B. atrox whole venom dissolved in buffer, pooled human urine, and pooled human serum. The visual limit of detection was < 25 ng/mL for venom dissolved in buffer. The ability of the Bothrops spp. lateral flow assay to detect 45 different venoms from snake species endemic to Brazil and nearby countries was assessed, and the assay was shown to detect several different Bothrops and Bothrocophias venoms, as well as a single Atropoides venom from a species not found in Brazil, while none of the other tested pit viper venoms were detected. This indicates that the Bothrops spp. lateral flow assay is selectively cross-reactive for bothropic venoms and could provide an indication that anti-bothropic antivenom is appropriate. Furthermore, 39 compounds were dissolved in serum and urine samples and were tested along with pure serum and urine samples adjusted to six different pHs, for their ability to cause false positives in the Bothrops spp. lateral flow assay. Two compounds and pHs ≤ 6 were found to cause false positives when measuring serum samples. None of the tested compounds or pHs were found to cause false positives when measuring urine samples. To make the lateral flow assay more thermally stable, and therefore potentially more viable in a tropical setting, it was decided to lyophilise the reagents. Six different lyophilisation solutions were evaluated, and it was concluded that the lyophilisation solutions containing casein with trehalose and casein with sucrose, respectively, provided the best protection. Towards the end of the project, a supply issue was encountered for the lateral flow assay strips. To circumvent this issue and continue the research, it was decided to develop a new version of the Bothrops spp. lateral flow assay using homemade strips. Additionally, it was decided to evaluate an alternative running buffer as well as labelling of the antibodies with carbon nanoparticles instead of gold nanoparticles. This resulted in a new Bothrops spp. lateral flow assay prototype, with a visual limit of detection of 1 ng/mL of B. atrox whole venom dissolved in running buffer. Neither the carbon-particle- based Bothrops prototype nor the gold-particle-based Bothrops prototype has yet been tested extensively on real snakebite patient samples, and thus their clinical utility remains to be seen. However, it is the hope that the development of the lateral flow assays described here can serve as a step in the direction of a future in which lateral flow assays will be more widely applied within clinical snakebite management to the benefit of the millions of people bitten every single year.