Standard Quality Characteristics and Efficacy of a New Third-Generation Antivenom Developed in Colombia Covering Micrurus spp. Venoms.

In Colombia, Micrurus snakebites are classified as severe according to the national clinical care guidelines and must be treated with specific antivenoms. Unfortunately, these types of antivenoms are scarce in certain areas of the country and are currently reported as an unavailable vital medicine. To address this issue, La Universidad de Antioquia, through its spin-off Tech Life Saving, is leading a project to develop third-generation polyvalent freeze-dried antivenom. The goal is to ensure access to this therapy, especially in rural and dispersed areas. This project aims to evaluate the physicochemical and preclinical parameters (standard quality characteristics) of a lab-scale anti-elapid antivenom batch. The antivenom is challenged against the venoms of several Micrurus species, including M. mipartitus, M. dumerilii, M. ancoralis, M. dissoleucus, M. lemniscatus, M. medemi, M. spixii, M. surinamensis, and M. isozonus, following the standard quality characteristics set by the World Health Organization (WHO). The antivenom demonstrates an appearance consistent with standards, 100% solubility within 4 min and 25 s, an extractable volume of 10.39 mL, a pH of 6.04, an albumin concentration of 0.377 mg/mL (equivalent to 1.22% of total protein), and a protein concentration of 30.97 mg/mL. Importantly, it maintains full integrity of its F(ab')2 fragments and exhibits purity over 98.5%. Furthermore, in mice toxicity evaluations, doses up to 15 mg/mouse show no toxic effects. The antivenom also demonstrates a significant recognition pattern against Micrurus venoms rich in phospholipase A2 (PLA2) content, as observed in M. dumerilii, M. dissoleucus, and M. isozonus. The effective dose 50 (ED50) indicates that a single vial (10 mL) can neutralize 2.33 mg of M. mipartitus venom and 3.99 mg of M. dumerilii venom. This new anti-elapid third-generation polyvalent and freeze-dried antivenom meets the physicochemical parameters set by the WHO and the regulators in Colombia. It demonstrates significant efficacy in neutralizing the venom of the most epidemiologically important Micrurus species in Colombia. Additionally, it recognizes seven other species of Micrurus venom with a higher affinity for venoms exhibiting PLA2 toxins. Fulfilling these parameters represents the first step toward proposing a new pharmacological alternative for treating snakebites in Colombia, particularly in dispersed rural areas, given that this antivenom is formulated as a freeze-dried product.

A generalized framework for estimating snakebite underreporting using statistical models: A study in Colombia.

BACKGROUND: Snakebite envenoming is a neglected tropical disease affecting deprived populations, and its burden is underestimated in some regions where patients prefer using traditional medicine, case reporting systems are deficient, or health systems are inaccessible to at-risk populations. Thus, the development of strategies to optimize disease management is a major challenge. We propose a framework that can be used to estimate total snakebite incidence at a fine political scale. METHODOLOGY/PRINCIPAL FINDINGS: First, we generated fine-scale snakebite risk maps based on the distribution of venomous snakes in Colombia. We then used a generalized mixed-effect model that estimates total snakebite incidence based on risk maps, poverty, and travel time to the nearest medical center. Finally, we calibrated our model with snakebite data in Colombia from 2010 to 2019 using the Markov-chain-Monte-Carlo algorithm. Our results suggest that 10.19% of total snakebite cases (532.26 yearly envenomings) are not reported and these snakebite victims do not seek medical attention, and that populations in the Orinoco and Amazonian regions are the most at-risk and show the highest percentage of underreporting. We also found that variables such as precipitation of the driest month and mean temperature of the warmest quarter influences the suitability of environments for venomous snakes rather than absolute temperature or rainfall. CONCLUSIONS/SIGNIFICANCE: Our framework permits snakebite underreporting to be estimated using data on snakebite incidence and surveillance, presence locations for the most medically significant venomous snake species, and openly available information on population size, poverty, climate, land cover, roads, and the locations of medical centers. Thus, our algorithm could be used in other countries to estimate total snakebite incidence and improve disease management strategies; however, this framework does not serve as a replacement for a surveillance system, which should be made a priority in countries facing similar public health challenges.

Estimating snakebite incidence from mathematical models: A test in Costa Rica.

BACKGROUND: Snakebite envenoming is a neglected public health challenge that affects mostly economically deprived communities who inhabit tropical regions. In these regions, snakebite incidence data is not always reliable, and access to health care is scare and heterogeneous. Thus, addressing the problem of snakebite effectively requires an understanding of how spatial heterogeneity in snakebite is associated with human demographics and snakes' distribution. Here, we use a mathematical model to address the determinants of spatial heterogeneity in snakebite and we estimate snakebite incidence in a tropical country such as Costa Rica. METHODS AND FINDINGS: We combined a mathematical model that follows the law of mass action, where the incidence is proportional to the exposed human population and the venomous snake population, with a spatiotemporal dataset of snakebite incidence (Data from year 1990 to 2007 for 193 districts) in Costa Rica. This country harbors one of the most dangerous venomous snakes, which is the Terciopelo (Bothrops asper, Garman, 1884). We estimated B. asper distribution using a maximum entropy algorithm, and its abundance was estimated based on field data. Then, the model was adjusted to the data using a lineal regression with the reported incidence. We found a significant positive correlation (R2 = 0.66, p-value < 0.01) between our estimation and the reported incidence, suggesting the model has a good performance in estimating snakebite incidence. CONCLUSIONS: Our model underscores the importance of the synergistic effect of exposed population size and snake abundance on snakebite incidence. By combining information from venomous snakes' natural history with census data from rural populations, we were able to estimate snakebite incidence in Costa Rica. The model was able to fit the incidence data at fine administrative scale (district level), which is fundamental for the implementation and planning of management strategies oriented to reduce snakebite burden.

Protocol to obtain targeted transcript sequence data from snake venom samples collected in the Colombian field.

Snake venoms are a mixture of different molecules that can be used in the design of drugs for various diseases. The study of these venoms has relied on strategies that use complete venom extracted from animals in captivity or from venom glands that require the sacrifice of the animals. Colombia, a country with political and geographical conflicts has difficult access to certain regions. A strategy that can prevent the sacrifice of animals and could allow the study of samples collected in the field is necessary. We report the use of lyophilized venom from Crotalus durissus cumanensis as a model to test, for the first time, a protocol for the amplification of complete toxins from Colombian venom samples collected in the field. In this protocol, primers were designed from conserved region from Crotalus sp. mRNA and EST regions to maximize the likelihood of coding sequence amplification. We obtained the sequences of Metalloproteinases II, Disintegrins, Disintegrin-Like, Phospholipases A2, C-type Lectins and Serine proteinases from Crotalus durissus cumanensis and compared them to different Crotalus sp sequences available on databases obtaining concordance between the toxins amplified and those reported. Our strategy allows the use of lyophilized venom to obtain complete toxin sequences from samples collected in the field and the study of poorly characterized venoms in challenging environments.

Neuromuscular effect of venoms from adults and juveniles of Crotalus durissus cumanensis (Humboldt, 1811) from Guajira, Colombia.

A toxinological study was performed to compare the neuromuscular effect of venom from adult and juvenile specimens from Crotalus durissus cumanensis from Guajira, Colombia. Both venoms exhibited neurotoxic activity in chick biventer cervicis nerve-muscle preparation. In addition, venom from juveniles was faster than adults to produce a neuromuscular blockade. In the contrary to the venom from juvenile's, at high doses, adult's venom affected the ACh and KCl contractures, indicating a myotoxic effect.