Urinary proteomics reveals biological processes related to acute kidney injury in Bothrops atrox envenomings.

Acute kidney injury (AKI) is a critical systemic complication caused by Bothrops envenoming, a neglected health problem in the Brazilian Amazon. Understanding the underlying mechanisms leading to AKI is crucial for effectively mitigating the burden of this complication. This study aimed to characterize the urinary protein profile of Bothrops atrox snakebite victims who developed AKI. We analyzed three groups of samples collected on admission: healthy subjects (controls, n = 10), snakebite victims who developed AKI (AKI, n = 10), and those who did not evolve to AKI (No-AKI, n = 10). Using liquid-chromatography tandem mass spectrometry, we identified and quantified (label-free) 1190 proteins. A panel of 65 proteins was identified exclusively in the urine of snakebite victims, with 32 exclusives to the AKI condition. Proteins more abundant or exclusive in AKI's urine were associated with acute phase response, endopeptidase inhibition, complement cascade, and inflammation. Notable proteins include serotransferrin, SERPINA-1, alpha-1B-glycoprotein, and NHL repeat-containing protein 3. Furthermore, evaluating previously reported biomarkers candidates for AKI and renal injury, we found retinol-binding protein, beta-2-microglobulin, cystatin-C, and hepcidin to be significant in cases of AKI induced by Bothrops envenoming. This work sheds light on physiological disturbances caused by Bothrops envenoming, highlighting potential biological processes contributing to AKI. Such insights may aid in better understanding and managing this life-threatening complication.

Urinary proteomics reveals biological processes related to acute kidney injury in Bothrops atrox envenomings

Acute kidney injury (AKI) is a critical systemic complication caused by Bothrops envenoming, a neglected health problem in the Brazilian Amazon. Understanding the underlying mechanisms leading to AKI is crucial for effectively mitigating the burden of this complication. This study aimed to characterize the urinary protein profile of Bothrops atrox snakebite victims who developed AKI. We analyzed three groups of samples collected on admission: healthy subjects (controls, n = 10), snakebite victims who developed AKI (AKI, n = 10), and those who did not evolve to AKI (No-AKI, n = 10). Using liquid-chromatography tandem mass spectrometry, we identified and quantified (label-free) 1190 proteins. A panel of 65 proteins was identified exclusively in the urine of snakebite victims, with 32 exclusives to the AKI condition. Proteins more abundant or exclusive in AKI’s urine were associated with acute phase response, endopeptidase inhibition, complement cascade, and inflammation. Notable proteins include serotransferrin, SERPINA-1, alpha-1B-glycoprotein, and NHL repeat-containing protein 3. Furthermore, evaluating previously reported biomarkers candidates for AKI and renal injury, we found retinol-binding protein, beta-2-microglobulin, cystatin-C, and hepcidin to be significant in cases of AKI induced by Bothrops envenoming. This work sheds light on physiological disturbances caused by Bothrops envenoming, highlighting potential biological processes contributing to AKI. Such insights may aid in better understanding and managing this life-threatening complication.

Venom composition of neglected Bothropoid snakes from the Amazon Rainforest: ecological and toxinological implications

Snake venoms have evolved in several families of Caenophidae, and their toxins have been assumed to be biochemical weapons with a role as a trophic adaptation. However, it remains unclear how venom contributes to the success of venomous species for adaptation to different environments. Here we compared the venoms from Bothrocophias hyoprora, Bothrops taeniatus, Bothrops bilineatus smaragdinus, Bothrops brazili, and Bothrops atrox collected in the Amazon Rainforest, aiming to understand the ecological and toxinological consequences of venom composition. Transcriptomic and proteomic analyses indicated that the venoms presented the same toxin groups characteristic from bothropoids, but with distinct isoforms with variable qualitative and quantitative abundances, contributing to distinct enzymatic and toxic effects. Despite the particularities of each venom, commercial Bothrops antivenom recognized the venom components and neutralized the lethality of all species. No clear features could be observed between venoms from arboreal and terrestrial habitats, nor in the dispersion of the species throughout the Amazon habitats, supporting the notion that venom composition may not shape the ecological or toxinological characteristics of these snake species and that other factors influence their foraging or dispersal in different ecological niches.

Inflammatory Profile Associated with Secondary Infection from Bothrops atrox Snakebites in the Brazilian Amazon.

Bothrops snakebite envenomation (SBE) is consider an important health problem in Brazil, where Bothrops atrox is mainly responsible in the Brazilian Amazon. Local effects represent a relevant clinical issue, in which inflammatory signs and symptoms in the bite site represent a potential risk for short and long-term disabilities. Among local complications, secondary infections (SIs) are a common clinical finding during Bothrops atrox SBE and are described by the appearance of signs such as abscess, cellulitis or necrotizing fasciitis in the affected site. However, the influence of SI in the local events is still poorly understood. Therefore, the present study describes for the first time the impact of SBE wound infection on local manifestations and inflammatory response from patients of Bothrops atrox SBE in the Brazilian Amazon. This was an observational study carried out at the Fundação de Medicina Tropical Dr. Heitor Vieira Dourado, Manaus (Brazil), involving victims of Bothrops SBE. Clinical and laboratorial data were collected along with blood samples for the quantification of circulating cytokines and chemokines before antivenom administrations (T0) and 24 h (T1), 48 h (T2), 72 h (T3) and 7 days after (T4). From the 94 patients included in this study, 42 presented SI (44.7%) and 52 were without SI (NSI, 55.3%). Patients classified as moderate envenoming presented an increased risk of developing SI (OR = 2.69; CI 95% = 1.08-6.66, p = 0.033), while patients with bites in hands showed a lower risk (OR = 0.20; CI 95% = 0.04-0.96, p = 0.045). During follow-up, SI patients presented a worsening of local temperature along with a sustained profile of edema and pain, while NSI patients showed a tendency to restore and were highlighted in patients where SI was diagnosed at T2. As for laboratorial parameters, leukocytes, erythrocyte sedimentation ratio, fibrinogen and C-reactive protein were found increased in patients with SI and more frequently in patients diagnosed with SI at T3. Higher levels of circulating IL-2, IL-10, IL-6, TNF, INF-γ and CXCL-10 were observed in SI patients along with marked correlations between these mediators and IL-4 and IL-17, showing a plurality in the profile with a mix of Th1/Th2/Th17 response. The present study reports for the first time the synergistic effects of local infection and envenoming on the inflammatory response represented by local manifestations, which reflected on laboratorial parameters and inflammatory mediators and thus help improve the clinical management of SI associated to Bothrops SBE.

The Venom Composition of the Snake Tribe Philodryadini: 'Omic' Techniques Reveal Intergeneric Variability among South American Racers.

Snakes of the Philodryadini tribe are included in the Dipsadidae family, which is a diverse group of rear-fanged snakes widespread in different ecological conditions, including habitats and diet. However, little is known about the composition and effects of their venoms despite their relevance for understanding the evolution of these snakes or even their impact on the occasional cases of human envenoming. In this study, we integrated venom gland transcriptomics, venom proteomics and functional assays to characterize the venoms from eight species of the Philodryadini tribe, which includes the genus Philodryas, Chlorosoma and Xenoxybelis. The most abundant components identified in the venoms were snake venom metalloproteinases (SVMPs), cysteine-rich secretory proteins (CRISPs), C-type lectins (CTLs), snake endogenous matrix metalloproteinases type 9 (seMMP-9) and snake venom serinoproteinases (SVSPs). These protein families showed a variable expression profile in each genus. SVMPs were the most abundant components in Philodryas, while seMMP-9 and CRISPs were the most expressed in Chlorosoma and Xenoxybelis, respectively. Lineage-specific differences in venom composition were also observed among Philodryas species, whereas P. olfersii presented the highest amount of SVSPs and P. agassizii was the only species to express significant amounts of 3FTx. The variability observed in venom composition was confirmed by the venom functional assays. Philodryas species presented the highest SVMP activity, whereas Chlorosoma species showed higher levels of gelatin activity, which may correlate to the seMMP-9 enzymes. The variability observed in the composition of these venoms may be related to the tribe phylogeny and influenced by their diets. In the presented study, we expanded the set of venomics studies of the Philodryadini tribe, which paves new roads for further studies on the evolution and ecology of Dipsadidae snakes.

Rhomb-I, a P-I metalloproteinase from Lachesis muta rhombeata venom degrades vessel extra cellular matrix components and impairs platelet aggregation.

Rhomb-I, a 23-kDa metalloproteinase was isolated from L. m. rhombeata venom. Its dimethylcasein proteolysis was abolished by metal chelators, and slightly enhanced by Ca2+ and Mg2+ ions, but inhibited by Co2+, Zn2+ and α2-macroglobulin. In aqueous solution, rhomb-I autoproteolyzed to a 20- and 11-kDa fragments at 37 °C. The amino acid sequence showed high homology with other snake venom metalloproteinases. Rhomb-I causes hemorrhage that may be ascribed to hydrolysis of essential basement membrane, extracellular matrix and plasma proteins. It preferentially cleaves the α-chains of fibrin (ogen). Rhomb-I inhibited convulxin- and von Willebrand factor (vWF)-induced aggregation on human platelets without significant effect on collagen-stimulated aggregation or other effectors. It digests vWF into a low-molecular-mass multimers of vWF and a rvWF-A1 domain to a 27-kDa fragment as revealed by western blotting with mouse anti-rvWF A1-domain IgG. Incubation of platelets with rhomb-I resulted in adhesion to and cleavage of platelet receptors glycoprotein (GP)Ibα and GPVI to release a 55-kDa soluble form. Both membrane glycoproteins GPIbα that binds vWF, together with GPVI which binds collagen, play a key role in mediating platelet adhesion/activation and can initiate (patho)physiological thrombus formation. Conclusions: rhomb-I is implicated in the pathophysiology of Lachesis envenoming by disrupting vasculature, hemostasis and platelet aggregation through impairing vWF-GPIb axis and blocking GPVI-collagen binding.

"Two Cultures in Favor of a Dying Patient": Experiences of Health Care Professionals Providing Snakebite Care to Indigenous Peoples in the Brazilian Amazon.

In the Brazilian Amazon, deaths and disabilities from snakebite envenomations (SBEs) are a major and neglected problem for the indigenous population. However, minimal research has been conducted on how indigenous peoples access and utilize the health system for snakebite treatment. A qualitative study was conducted to understand the experiences of health care professionals (HCPs) who provide biomedical care to indigenous peoples with SBEs in the Brazilian Amazon. Focus group discussions (FGDs) were carried out in the context of a three-day training session for HCPs who work for the Indigenous Health Care Subsystem. A total of 56 HCPs participated, 27 in Boa Vista and 29 in Manaus. Thematic analysis resulted in three key findings: Indigenous peoples are amenable to receiving antivenom but not to leaving their villages for hospitals; HCPs require antivenom and additional resources to improve patient care; and HCPs strongly recommend a joint, bicultural approach to SBE treatment. Decentralizing antivenom to local health units addresses the central barriers identified in this study (e.g., resistance to hospitals, transportation). The vast diversity of ethnicities in the Brazilian Amazon will be a challenge, and additional studies should be conducted regarding preparing HCPs to work in intercultural contexts.

The venom composition of the snake tribe Philodryadini: ‘Omic’ techniques reveal intergeneric variability among South American racers

Snakes of the Philodryadini tribe are included in the Dipsadidae family, which is a diverse group of rear-fanged snakes widespread in different ecological conditions, including habitats and diet. However, little is known about the composition and effects of their venoms despite their relevance for understanding the evolution of these snakes or even their impact on the occasional cases of human envenoming. In this study, we integrated venom gland transcriptomics, venom proteomics and functional assays to characterize the venoms from eight species of the Philodryadini tribe, which includes the genus Philodryas, Chlorosoma and Xenoxybelis. The most abundant components identified in the venoms were snake venom metalloproteinases (SVMPs), cysteine-rich secretory proteins (CRISPs), C-type lectins (CTLs), snake endogenous matrix metalloproteinases type 9 (seMMP-9) and snake venom serinoproteinases (SVSPs). These protein families showed a variable expression profile in each genus. SVMPs were the most abundant components in Philodryas, while seMMP-9 and CRISPs were the most expressed in Chlorosoma and Xenoxybelis, respectively. Lineage-specific differences in venom composition were also observed among Philodryas species, whereas P. olfersii presented the highest amount of SVSPs and P. agassizii was the only species to express significant amounts of 3FTx. The variability observed in venom composition was confirmed by the venom functional assays. Philodryas species presented the highest SVMP activity, whereas Chlorosoma species showed higher levels of gelatin activity, which may correlate to the seMMP-9 enzymes. The variability observed in the composition of these venoms may be related to the tribe phylogeny and influenced by their diets. In the presented study, we expanded the set of venomics studies of the Philodryadini tribe, which paves new roads for further studies on the evolution and ecology of Dipsadidae snakes.

Independent Recruitment of Different Types of Phospholipases A2 to the Venoms of Caenophidian Snakes: The Rise of PLA2-IIE within Pseudoboini (Dipsadidae)

Snake venoms harbor a wide and diverse array of enzymatic and nonenzymatic toxic components, allowing them to exert myriad effects on their prey. However, they appear to trend toward a few optimal compositional scaffolds, dominated by four major toxin classes: SVMPs, SVSPs, 3FTxs, and PLA2s. Nevertheless, the latter appears to be restricted to vipers and elapids, as it has never been reported as a major venom component in rear-fanged species. Here, by investigating the original transcriptomes from 19 species distributed in eight genera from the Pseudoboini tribe (Dipsadidae: Xenodontinae) and screening among seven additional tribes of Dipsadidae and three additional families of advanced snakes, we discovered that a novel type of venom PLA2, resembling a PLA2-IIE, has been recruited to the venom of some species of the Pseudoboini tribe, where it is a major component. Proteomic and functional analyses of these venoms further indicate that these PLA2s play a relevant role in the venoms from this tribe. Moreover, we reconstructed the phylogeny of PLA2s across different snake groups and show that different types of these toxins have been recruited in at least five independent events in caenophidian snakes. Additionally, we present the first compositional profiling of Pseudoboini venoms. Our results demonstrate how relevant phenotypic traits are convergently recruited by different means and from homologous and nonhomologous genes in phylogenetically and ecologically divergent snake groups, possibly optimizing venom composition to overcome diverse adaptative landscapes.

Independent recruitment of different types of Phospholipases A2 to the venoms of Caenophidian snakes: the rise of PLA2-IIE within Pseudoboini (Dipsadidae)

Snake venoms harbor a wide and diverse array of enzymatic and nonenzymatic toxic components, allowing them to exert myriad effects on their prey. However, they appear to trend toward a few optimal compositional scaffolds, dominated by four major toxin classes: SVMPs, SVSPs, 3FTxs, and PLA2s. Nevertheless, the latter appears to be restricted to vipers and elapids, as it has never been reported as a major venom component in rear-fanged species. Here, by investigating the original transcriptomes from 19 species distributed in eight genera from the Pseudoboini tribe (Dipsadidae: Xenodontinae) and screening among seven additional tribes of Dipsadidae and three additional families of advanced snakes, we discovered that a novel type of venom PLA2, resembling a PLA2-IIE, has been recruited to the venom of some species of the Pseudoboini tribe, where it is a major component. Proteomic and functional analyses of these venoms further indicate that these PLA2s play a relevant role in the venoms from this tribe. Moreover, we reconstructed the phylogeny of PLA2s across different snake groups and show that different types of these toxins have been recruited in at least five independent events in caenophidian snakes. Additionally, we present the first compositional profiling of Pseudoboini venoms. Our results demonstrate how relevant phenotypic traits are convergently recruited by different means and from homologous and nonhomologous genes in phylogenetically and ecologically divergent snake groups, possibly optimizing venom composition to overcome diverse adaptative landscapes.

Rhomb-I, a P–I metalloproteinase from Lachesis muta rhombeata venom degrades vessel extra cellular matrix components and impairs platelet aggregation

Rhomb-I, a 23-kDa metalloproteinase was isolated from L. m. rhombeata venom. Its dimethylcasein proteolysis was abolished by metal chelators, and slightly enhanced by Ca2+ and Mg2+ ions, but inhibited by Co2+, Zn2+ and α2-macroglobulin. In aqueous solution, rhomb-I autoproteolyzed to a 20- and 11-kDa fragments at 37 °C. The amino acid sequence showed high homology with other snake venom metalloproteinases. Rhomb-I causes hemorrhage that may be ascribed to hydrolysis of essential basement membrane, extracellular matrix and plasma proteins. It preferentially cleaves the α-chains of fibrin (ogen). Rhomb-I inhibited convulxin- and von Willebrand factor (vWF)-induced aggregation on human platelets without significant effect on collagen-stimulated aggregation or other effectors. It digests vWF into a low-molecular-mass multimers of vWF and a rvWF-A1 domain to a 27-kDa fragment as revealed by western blotting with mouse anti-rvWF A1-domain IgG. Incubation of platelets with rhomb-I resulted in adhesion to and cleavage of platelet receptors glycoprotein (GP)Ibα and GPVI to release a 55-kDa soluble form. Both membrane glycoproteins GPIbα that binds vWF, together with GPVI which binds collagen, play a key role in mediating platelet adhesion/activation and can initiate (patho)physiological thrombus formation. Conclusions: rhomb-I is implicated in the pathophysiology of Lachesis envenoming by disrupting vasculature, hemostasis and platelet aggregation through impairing vWF-GPIb axis and blocking GPVI-collagen binding.

Two cultures in favor of a dying patient: experiences of health care professionals providing snakebite care to indigenous peoples in the Brazilian amazon

In the Brazilian Amazon, deaths and disabilities from snakebite envenomations (SBEs) are a major and neglected problem for the indigenous population. However, minimal research has been conducted on how indigenous peoples access and utilize the health system for snakebite treatment. A qualitative study was conducted to understand the experiences of health care professionals (HCPs) who provide biomedical care to indigenous peoples with SBEs in the Brazilian Amazon. Focus group discussions (FGDs) were carried out in the context of a three-day training session for HCPs who work for the Indigenous Health Care Subsystem. A total of 56 HCPs participated, 27 in Boa Vista and 29 in Manaus. Thematic analysis resulted in three key findings: Indigenous peoples are amenable to receiving antivenom but not to leaving their villages for hospitals; HCPs require antivenom and additional resources to improve patient care; and HCPs strongly recommend a joint, bicultural approach to SBE treatment. Decentralizing antivenom to local health units addresses the central barriers identified in this study (e.g., resistance to hospitals, transportation). The vast diversity of ethnicities in the Brazilian Amazon will be a challenge, and additional studies should be conducted regarding preparing HCPs to work in intercultural contexts.

Snakebite clinics and pathogenesis: from preclinical to resource mapping studies

Amidst the global healthcare landscape, the menace of snakebite envenoming (SBE) has persisted, silently afflicting millions and annually claiming tens of thousands of lives. Indeed, in 2017, the World Health Organization (WHO) reclassified snakebite envenoming as a Category A Neglected Tropical Disease (NTD), finally prompting worldwide recognition of the profound health and economic devastation caused by these venomous encounters. Then, in 2019, WHO unveiled an ambitious strategy: to slash snakebite envenoming-related mortality and disability by 50% before 2030 [1,2]. This editorial marks the inception of our Special Issue, “Snakebite Clinics and Pathogenesis: From Preclinical to Resource Mapping Studies”, which stands as a guiding light in our collective effort to confront SBEs. Gathering insights from research on snakebite envenoming outcomes, diagnostic advancements, uncommon case reports, therapeutic strategies, and healthcare professional training, this Special Issue is dedicated to disseminating knowledge and charting a course towards a future where snakebite envenomings cease to be a neglected tragedy and evolve into a preventable and manageable challenge. In their pioneering study, Murta et al. [3] explored the experiences of healthcare professionals (HCPs) providing medical care to indigenous people with SBEs in the Brazilian Amazon. They conducted group discussions during a three-day training session for HCPs from the Indigenous Health Care Subsystem, involving 56 participants split between Boa Vista (Roraima) and Manaus (Amazonas), which are state capitals located in the Brazilian Amazon Forest. The study revealed three key findings: indigenous people are open to receiving antivenom but prefer not to leave their villages for hospitals; HCPs require antivenom and additional resources to improve patient care; and HCPs advocate for a collaborative, culturally sensitive approach to SBE treatment. To address these challenges, the study suggests decentralizing antivenom distribution to local health units. However, the diverse ethnicities in the Brazilian Amazon pose a challenge, necessitating further research on preparing HCPs for intercultural contexts. Even when antivenom is available in low-resource areas, health workers do not receive adequate training to manage SBEs. The study of Rocha et al. (2022) [4] aimed to develop and validate a clinical practice guideline (CPG) for SBE management across Brazil. Content validation was performed by a panel of expert judges with academic and/or technical expertise in SBE management, and semantic validation was performed by analyzing focus group discussions with doctors and nurses from three municipalities of the Brazilian Amazon. This study presents the successful development and validation process of a CPG for SBE management, which is targeted to a specific low-resource, high-burden setting. This development and validation process can be adapted to other settings and/or other neglected tropical diseases. In the health system domain, this strategy involves ensuring the production and distribution of safe and effective antivenom treatment and strengthening local health systems. Bhatia et al. (2022) [5] highlight that there is an urgent need to replace the excessive use of animals in snake antivenom production. We tested the efficacy of a single batch of polyvalent antivenom from bioproducts limited to Echis carinatus venom collected from Tamil Nadu, Goa, and Rajasthan, using different in vitro assays. The use of both binding and functional assays allowed us to measure the efficacy of the antivenom. By normalizing the scale of measurements of the neutralization capacity of the Indian polyvalent antivenom using different in vitro assays, we were able to arrive at an efficacy score for Echis carinatus venoms that could be used to predict the ED50. This approach captures the variation in venom toxins shown by snake species and paves the way to replace the use of mice for evaluating antivenom potency. Protobothrops mucrosquamatus snakebites are frequent in Taiwan, and the species’ widespread distribution and diverse habitats drove Chiang et al. (2022) [6] to investigate envenoming effects and relevant venom variations. The results showed minor differences in the protein family, with variations in acidic phospholipases A2s, serine proteinases, metalloproteinases, C-type lectin-like proteins, and other less abundant components. Moreover, clinical manifestations of envenomed patients hospitalized in northern Taiwan revealed differences in local symptoms, such as ecchymosis and blistering. The mechanism of these local effects is probably related to the venom components’ geographical variability. These findings will help to improve the management of P. mucrosquamatus bites in Taiwan. Vera-Palacios et al. (2022) [7] investigated in vivo the ability of Urospatha sagittifolia (Araceae) to modulate the catalytic activity of Bothrops atrox venom, and their toxic consequences, such as edema, skin hemorrhage, and lethality. Ethanolic extract, which is rich in phenolics, alkaloids, coumarins, and flavonoids, reduced these three parameters. The authors concluded that these findings will support future studies with purified metabolites as new agents for the treatment of B. atrox snakebites, an important public health problem in the Amazon region. The study by Manson et al. [8] marks a groundbreaking leap forward in the realm of SBE treatment, with a particular focus on combating the toxicity of Three-Finger Toxins (3FTxs) of Naja ashei snake venom. These potent venom-derived toxins are prevalent in N. ashei venom and have posed a formidable challenge to effective antivenom therapy. What sets this research apart is the development of monoclonal antibodies (i.e., P4G6a, P6D9a, and P6D9b) meticulously designed to target these troublesome 3FTxs. Remarkably, the monoclonal antibodies demonstrated exceptional binding capabilities to the target 3FTxs, outperforming even the leading commercial antivenoms available in the Kenyan market. The true breakthrough lies in the combined use of these monoclonal antibodies, where their cocktail exhibited superior toxin inhibition compared to traditional antivenoms. Alsolaiss et al. [9] sheds essential light on the complex and diverse acute responses triggered by African snake venoms, a critical aspect of understanding the pathophysiology of SBEs. Using a well-designed murine model, the research systematically evaluated the acute-phase and inflammatory reactions induced by ten different African snake venoms, with a particular focus on sub-Saharan African species, including the spitting cobra (Naja nigricollis) and forest cobra (N. melanoleuca), as potent inducers of acute-phase and inflammatory responses, with N. nigricollis venom stimulating a remarkable 100-fold increase in systemic interleukin 6 (IL-6). Moreover, the study revealed species-specific changes in red blood cell morphology, lymphopenia, neutrophil leukocytosis, and marked hemolysis and platelet aggregation levels in response to these venoms. These findings underscore the intricate and diverse nature of acute responses to envenoming, paving the way for potential diagnostic and therapeutic advancements that could greatly benefit snakebite victims. A very interesting review was also presented in the Special Issue. Huang et al. [10] analyzes 35 cases of snakebites, primarily from front-fanged snakes, like vipers and cobras, as well as a few rare instances from rear-fanged snakes. Viper bites often result in severe complications, such as ischemic strokes and intracranial hemorrhages, leading to fatalities in some cases. In contrast, elapid bites are primarily manifested as neural, cardiac, and ophthalmic disorders. Remarkably, rear-fanged snakebites, characterized by shallow bites and minimal venom injection, rarely cause severe complications. An essential takeaway from the review is the pivotal role of antivenom (AV) treatment, although it also discusses various therapeutic agents that could potentially complement AV treatment for snakebite-induced complications. Furthermore, the Special Issue delved into two unconventional snakebite case reports, one conducted in Romania and the other in Brazil, subjecting them to comprehensive examination and discussion. Nitescu et al. [11] offers a unique perspective on snake envenomation, focusing on a specific exception within the European viper (Vipera berus) species. While most V. berus bites typically lack neurotoxic effects, their study highlights rare cases involving subspecies found in the Carpathian Basin of southeastern Europe that do induce such symptoms. The study presents a compelling case of a 5-year-old girl from southern Romania who experienced neurotoxicity, alongside systemic and local symptoms, following a bite from one of these Carpathian Basin V. berus subspecies. This case provides pivotal insights, affirming that venom from V. berus subspecies in the Carpathian Basin region can indeed induce neurotoxic effects. Additionally, it underscores the effectiveness of monospecific antivenom treatment in rapidly and completely mitigating the envenomation’s effects, offering valuable clinical guidance for the management of such rare cases. In contrast, Oliveira et al.’s [12] case report delves into the often-overlooked long-term musculoskeletal disabilities resulting from snakebites in indigenous communities in Brazil. The report focuses on a 32-year-old male indigenous patient envenomed by a Bothrops species (lancehead snake), highlighting the significant and enduring health challenges posed by snakebites. Over approximately 2 years and 6 months, the patient underwent various medical interventions, including debridement, tissue reconstruction, and physical therapy, resulting in improved mobility but a lasting impact on his gait. This case report emphasizes the need for a comprehensive healthcareapproach, including physiotherapy, plastic surgery, orthopedics, and social support, to aid in the reintegration of snakebite survivors into their communities. Antivenom treatments for SBE patients have existed for more than 130 years, remaining the only therapeutics available for this neglected problem. Remarkably, despite advances in the health system, access to antivenom treatment is poor in most areas of low-income countries. Better logistics for the transportation of antivenoms and other commodities is an issue to be addressed, as well as realistic and comprehensive health programs. In parallel, many investments are still needed for the research and development of more effective antivenoms for some species of snakes, as well as for the advance of small-molecule inhibitor-based drug therapies.

The severity of acute kidney injury correlates with plasma venom levels in Bothrops atrox envenomings.

The Brazilian Amazon has high rates of snakebite envenomings (SBEs), with ∼90% caused by Bothrops atrox. Envenomings by this species can trigger local and systemic effects, such as acute kidney injury (AKI). Our aim was to identify predictors of AKI in Bothrops SBEs in patients from Manaus, Western Brazilian Amazon. A total of 127 patients were enrolled, with a predominance of men between 16 and 45 years old from rural areas. Of the 127 patients, 38.6% developed AKI, with 61.2% presenting stage I, 34.7% presenting stage II and 4.1% presenting stage III severity. The age groups 0-10 years and ≥60 years presented a significantly higher frequency of AKI compared to the 11-40 years group. Moderate/severe edema in the affeccted limb was significantly associated with lower risk of AKI [p = 0.01; OR = 0.11 (95%CI 0.02-0.53)]. Nausea [p = 0.01; OR = 54.44 (95%CI = 3.26-909.27)] and high blood urea levels [p = 0.01; OR = 5.38 (95%CI = 2.12-13.66)] were risk factors for AKI. There was a significant positive correlation between circulating venom levels and the highest creatinine serum values during the hospital stay (p = 0.03) and with the difference between the maximum creatinine levels and the creatinine levels on admission (p = 0.02). A positive correlation between serum venom concentrations and creatinine levels suggests a direct or indirect dose-dependent participation of the venom toxins in the pathogenesis of AKI.

The severity of acute kidney injury correlates with plasma venom levels in Bothrops atrox envenomings

The Brazilian Amazon has high rates of snakebite envenomings (SBEs), with ∼90% caused by Bothrops atrox. Envenomings by this species can trigger local and systemic effects, such as acute kidney injury (AKI). Our aim was to identify predictors of AKI in Bothrops SBEs in patients from Manaus, Western Brazilian Amazon. A total of 127 patients were enrolled, with a predominance of men between 16 and 45 years old from rural areas. Of the 127 patients, 38.6% developed AKI, with 61.2% presenting stage I, 34.7% presenting stage II and 4.1% presenting stage III severity. The age groups 0–10 years and ≥60 years presented a significantly higher frequency of AKI compared to the 11–40 years group. Moderate/severe edema in the affeccted limb was significantly associated with lower risk of AKI [p = 0.01; OR = 0.11 (95%CI 0.02–0.53)]. Nausea [p = 0.01; OR = 54.44 (95%CI = 3.26–909.27)] and high blood urea levels [p = 0.01; OR = 5.38 (95%CI = 2.12–13.66)] were risk factors for AKI. There was a significant positive correlation between circulating venom levels and the highest creatinine serum values during the hospital stay (p = 0.03) and with the difference between the maximum creatinine levels and the creatinine levels on admission (p = 0.02). A positive correlation between serum venom concentrations and creatinine levels suggests a direct or indirect dose-dependent participation of the venom toxins in the pathogenesis of AKI.

Diversity of phospholipases A2 from Bothrops atrox snake venom: adaptive advantages for snakes compromising treatments for snakebite patients

The evolution of snake venoms resulted in multigene toxin families that code for structurally similar isoforms eventually harboring distinct functions. PLA2s are dominant toxins in viper venoms, and little is known about the impact of their diversity on human envenomings and neutralization by antivenoms. Here, we show the isolation of three distinct PLA2s from B. atrox venom. FA1 is a Lys-49 homologue, and FA3 and FA4 are catalytic Asp-49 PLA2s. FA1 and FA3 are basic myotoxic proteins, while FA4 is an acid non-myotoxic PLA2. FA3 was the most potent toxin, inducing higher levels of edema, inflammatory nociception, indirect hemolysis, and anticoagulant activity on human, rat, and chicken plasmas. FA4 presented lower anticoagulant activity, and FA1 had only a slight effect on human and rat plasmas. PLA2s presented differential reactivities with antivenoms, with an emphasis on FA3, which was not recognized or neutralized by the antivenoms used in this study. Our findings reveal the functional and antigenic diversity among PLA2s from B. atrox venom, highlighting the importance of assessing venom variability for understanding human envenomations and treatment with antivenoms, particularly evident here as the antivenom fails to recognize FA3, the most active multifunctional toxin described.

Differences in PLA2 constitution distinguish the venom of two endemic brazilian mountain lanceheads, Bothrops cotiara and Bothrops fonsecai

Interspecific differences in snake venom compositions can result from distinct regulatory mechanisms acting in each species. However, comparative analyses focusing on identifying regulatory elements and patterns that led to distinct venom composition are still scarce. Among venomous snakes, Bothrops cotiara and Bothrops fonsecai represent ideal models to complement our understanding of the regulatory mechanisms of venom production. These recently diverged species share a similar specialized diet, habitat, and natural history, but each presents a distinct venom phenotype. Here, we integrated data from the venom gland transcriptome and miRNome and the venom proteome of B. fonsecai and B. cotiara to better understand the regulatory mechanisms that may be acting to produce differing venom compositions. We detected not only the presence of similar toxin isoforms in both species but also distinct expression profiles of phospholipases A2 (PLA2) and some snake venom metalloproteinases (SVMPs) and snake venom serine proteinases (SVSPs) isoforms. We found evidence of modular expression regulation of several toxin isoforms implicated in venom divergence and observed correlated expression of several transcription factors. We did not find strong evidence for miRNAs shaping interspecific divergence of the venom phenotypes, but we identified a subset of toxin isoforms whose final expression may be fine-tuned by specific miRNAs. Sequence analysis on orthologous toxins showed a high rate of substitutions between PLA2s, which indicates that these toxins may be under strong positive selection or represent paralogous toxins in these species. Our results support other recent studies in suggesting that gene regulation is a principal mode of venom evolution across recent timescales, especially among species with conserved ecotypes.

Lower levels of CXCL-8 and IL-2 on admission as predictors of early adverse reactions to Bothrops antivenom in the Brazilian Amazon

Snakebite envenomings are considered a global health problem. The specific therapy for these envenomings consists of administering animal-derived antivenoms aiming to neutralize the venom toxins. Antivenoms have been used effectively to treat snakebites for more than a century; however, their administration may result in early and/or late adverse reactions. The present study presents the prevalence of early adverse reactions (EARs) towards Bothrops antivenom therapy in a health tertiary unit in the Brazilian Amazon and explores if specific plasma cytokines and chemokines from envenomed patients could be used as predictors of EARs. A cohort of patients bitten by Bothrops atrox was followed-up at the Fundação de Medicina Tropical Dr. Heitor Vieira Dourado (FMT-HVD), from 2014 to 2016. Patients were treated with the Brazilian Bothrops antivenom and CXCL-8, CCL-5, CXCL-9, CCL-2, CXCL-10, IL-6, TNF, IL-2, IL-10, IFN-y, IL-4, and IL-17A were evaluated in patients’ plasma samples before and after antivenom administration. From the total of patients (n = 186), mostly were male (82.3%), inhabiting rural areas (87.1%), with an average age of 35 years. Most of the patients (83.8%) were admitted to the hospital within 6 h after the accident, 26 (14%) reported having suffered a previous snakebite, and 97 (52.1%) received between 7 and 9 antivenom vials. The frequency of antivenom-induced EARs was 11.8% (22), resulting mostly of mild reactions. Urticaria was the major EAR manifestation (46.4%). Interestingly, CXCL-8 and IL-2 showed significantly lower levels in patients who progressed to EARs, although IL-2 levels might not represent biological relevance due the small magnitude difference between groups. This study reveals that CXCL-8 and IL-2 could play a role in the onset of EARs in pit viper envenomings.

Clinical implications of ontogenetic differences in the coagulotoxic activity of Bothrops jararacussu venoms.

Is snake venom activity influenced by size? This is a long-standing question that can have important consequences for the treatment of snake envenomation. Ontogenetic shifts in venom composition are a well-documented characteristic of numerous snake species. Although snake venoms can cause a range of pathophysiological disturbances, establishing the coagulotoxic profiles related to such shifts is a justified approach because coagulotoxicity can be deadly, and its neutralisation is a challenge for current antivenom therapy. Thus, we aimed to assess the coagulotoxicity patterns on plasma and fibrinogen produced by B othrops jararacussu venoms from individuals of different sizes and sex, and the neutralisation potential of SAB (anti bothropic serum produced by Butantan Institute). The use of a metalloproteinase inhibitor (Prinomastat) and a serine proteinase inhibitor (AEBSF) enabled us to determine the toxin class responsible for the observed coagulopathy: activity on plasma was found to be metalloprotease driven, while the activity on fibrinogen is serine protease driven. To further explore differences in venom activity, the activation of Factor X and prothrombin as a function of snake size was also evaluated. All the venoms exhibited a potent procoagulant effect upon plasma and were less potent in their pseudo-procoagulant clotting effect upon fibrinogen. On human plasma, the venoms from smaller snakes produced more rapid clotting than the larger ones. In contrast, the venom activity on fibrinogen had no relation with size or sex. The difference in procoagulant potency was correlated with the bigger snakes being proportionally better neutralized by antivenom due to the lower levels of procoagulant toxins, than the smaller. Thus, while the antivenom ultimately neutralized the venoms, proportionally more would be needed for an equal mass of venom from a small snake than a large one. Similarly, the neutralisation by SAB of the pseudo-procoagulant clotting effects was also correlated with relative potency, with the smaller and bigger snakes being neutralized proportional to potency, but with no correlation to size. Thromboelastography (TEG) tests on human and toad plasma revealed that small snakes' venoms acted quicker than large snakes' venom on both plasmas, with the action upon amphibian plasma consistent with smaller snakes taking a larger proportion of anuran prey than adults. Altogether, the ontogenetic differences regarding coagulotoxic potency and corresponding impact upon relative antivenom neutralisation of snakes with different sizes were shown, underscoring the medical importance of investigating ontogenetic changes in order to provide data crucial for evidence-based design of clinical management strategies.