Perspective Chapter: Diagnostic and Antivenom Immunotherapeutic Approaches in the Management of Snakebites

Abstract

Snakebite envenoming normally occurs as a result of the injection of venom following the bite of a venomous snake or the spraying of venom into a person’s eyes by snake species that are capable of spitting venom as a defense mechanism. According to World Health Organization, snakebite is considered to have high mortality among the neglected tropical diseases. The administration of toxin-specific therapy in snake envenoming is predicated on improving diagnostic techniques capable of detecting specific venom toxins. Various serological tests have been used in detecting snakebite envenoming. Comparatively, enzyme-linked immunosorbent assay has been shown to offer a wider practical application. On the other hand, the unavailability of effective antivenoms to treat snake envenoming has created a critical health need at global level. It has been reported that antivenom immunotherapy is the treatment of choice for snakebites. The generation of toxin-specific antibodies would lead to an increase in the dose efficacy of antivenoms and consequently reduce the risk of early anaphylactoid and late serum reactions that typify the administration of large volumes of horse and sheep-derived antivenoms. The aim of this chapter is to explore and discuss diagnostic and antivenom immunotherapeutic approaches to the management of snakebite envenoming.

1. Introduction

Snakebite envenoming is a potentially fatal disease that normally occurs as a result of the injection of venom following the bite of a venomous snake. Snakebite envenoming can also be caused by the spraying of venom into a person’s eyes by particular snake species that are capable of spitting venom as a defense tool. Envenoming results from an estimated 50–55% of all snakebites. Snake venoms are made up of complex mixtures of proteins and peptide toxins, which vary from one species to another and within species. Snakebite envenoming in humans and animals affects multiple organ systems based on the specific snake species and the groups of toxins present in the venom and can cause inter alia neuromuscular paralysis, hemorrhage and protracted disruption of hemostasis, cardiotoxicity, tissue necrosis, thrombosis, and myolysis (muscle degeneration) [1, 2].

Consistent with other neglected tropical diseases (NTDs), estimation of global morbidity, disability, and mortality occurring as a result of snakebite envenoming is problematic [2]. However, according to data [1], as many as 2.7 million people are affected by snakebite envenoming annually, majority of whom reside in some of the world’s most disadvantaged, poorly developed, and politically side-lined rural tropical communities. This results in an annual mortality of 81,000 to 138,000, and an estimated 400,000 surviving victims left with permanent physical and psychological disabilities, as well as stigmatizing disfigurements. Available data on mortality suggests that deaths from snakebites are highest in Asia, notably in India followed by sub-Saharan Africa [3]. In Africa, where data issues are even more problematic, an estimated 1 million snakebites occur annually, with about half requiring specific treatment [4]. Also, in Africa alone, about 8000 amputations are believed to occur each year as a result of snakebite envenoming [5, 6]. Snakebite envenoming is, thus, a disease that warrants urgent attention.

As the case with many poverty-related diseases, snakebite envenoming has been unsuccessful at attracting the needed public health policy inclusion and investment required to drive sustainable efforts aimed at reducing the medical and social burden. This is largely attributed to the demographics of the populations affected coupled with their lack of political voice [7]. Snakebites also pose significant additional socioeconomic burden on the remote and already penurious communities to the extent that the most economically productive (10–40-year-olds) are those who suffer the most [8].

Snakebite is a high morbidity/high mortality neglected tropical disease (NTD), and for that matter, one of the most under-researched and under-resourced NTDs. This is demonstrated by the fact that a majority of the antivenoms used to treat victims of snakebite in sub-Saharan Africa are of untested and indeterminate efficacy [3]. Furthermore, the plights of snakebite victims in this part of the continent have been exacerbated by a crisis in the supply of affordable and effective antivenoms, a phenomenon that was first reported in 2000 [3].

In the view of the World Health Organization, the unavailability of effective antivenoms to treat snake envenoming in various parts of the world has assumed a critical health status at the global level. This crisis is believed to have reached its highest intensity in sub-Saharan Africa [2]. It is, therefore, anticipated that the reintroduction of snakebite envenoming into the WHO NTD portfolio in 2017 following intense advocacy by stakeholders would give the needed attention to the disease and consequently lead to strategies to prevent, reduce, and control the snakebite burden [7].

The methods used in producing most antivenoms from the hyperimmune plasma of horses or sheep have not changed significantly in the last 50 to 60 years. As a result, the quality and safety of some of these antivenoms remain poor [9, 10]. The WHO recommends the development of polyspecific antivenoms, particularly for countries inhabited by several medically important snake species [2]. However, reports [3] suggest that the high costs of most polyspecific antivenoms over the last two decades resulted in decreased demand, and hence depressed commercial production volumes, resulting in the influx of less expensive antivenoms of untested efficacy. According to published data [11], some of these antivenoms have proven dangerously ineffective in many African countries. This is supported by reports from Ghana, Central African Republic, and Chad to the effect of an increased case fatality rate (from less than 2% to over 12%) due to the use of such antivenoms [11].

The generation of toxin-specific antibodies would lead to an increase in the dose efficacy of antivenoms and consequently reduce the risk of early anaphylactoid and late serum reactions that typify the administration of large volumes of horse and sheep-derived antivenoms. Toxin-specific antibodies have the potential to neutralize the venoms of medically important snake species [12]. The dose efficacy of monovalent antivenoms is usually higher than that of polyvalent antivenoms. For instance, the curative dose of EchiTAbG, a monovalent antivenom is reported to be one vial whiles that of EchiTAb-Plus-ICP, a polyvalent antivenom is three vials, implying that monovalent antivenoms present a better treatment alternative in terms of cost-effectiveness [13] but only if the specificity of the treatment can be assessed ahead of application. Also, in Nigeria, a monovalent Echis ocellatus antivenom was found to be preferred to a polyvalent product because the former was up to 4-fold more effective in neutralizing E. ocellatus venom relative to the latter [14]. Most parts of the world affected by snakebite envenoming depend on broad-spectrum polyspecific antivenoms that are known to contain a low content of case-specific efficacious immunoglobulins. Thus, advances in toxin-specific monoclonal antibodies hold much promise as far as future treatment strategies for snakebite envenoming are concerned. Monoclonal or other toxin-specific treatment options offer advantages such as fewer adverse reactions, case-specificity, increased efficacy, and cost-effectiveness as compared to past and present treatment approaches [15].

Furthermore, previous studies [16, 17] indicate that polyvalent antivenoms, although touted as effective and also credited with saving the lives of thousands of snakebite victims for nearly a century, are linked with serious allergic reactions, serum sickness, as well as other side effects. In addition, available data [16] show that monoclonal antibodies when included in antivenoms are able to neutralize all the medically important toxins present in a given venom and/or abrogate the synergistic effect of toxins in a venom sample. This, therefore, presupposes that the administration of toxin-specific or monoclonal-based monospecific antivenoms could mitigate the treatment-related challenges associated with polyspecific antivenoms, and hence make the former a preferred option for therapy. Thus, novel strategies, such as monoclonal antibodies, hold much promise with prognostic and diagnostic applications in snakebite envenoming (SBE). In this chapter, there will be a brief discussion of the modern concepts of snake envenoming and general discussion on the diagnostic and antivenom immunotherapeutic approaches to management of snake envenoming.

2. Snake venoms

Snake venoms are versatile and sophisticated weapons used for prey capture and defense by venomous snakes [17]. In all snakes considered to be venomous, the venom is made in a particular gland and usually released to the victim through modified teeth. Key among its functions includes facilitating the capture of preys via death or immobilization, aiding digestion in snakes, and acting as protective or defensive machinery against potential predators. Compositionally, nearly 90 to 95% of the total venom content in snakes is made up of proteins and peptides, described as biologically active, together with other elements that are nonprotein such as lipids, carbohydrates, inorganic salts, and amines. These proteins and peptides are classified as enzymes [such as snake venom metalloproteases (SVMP), snake venom serine proteases (SVSP), phospholipase A2 (PLA2), L-amino acid oxidases (LAAO)] or non-enzymes [such as three-finger toxins (3FTx), Kunitz peptides (KUN), and disintegrins (DIS)]. The constitution of snake venoms differs based on diverse factors, including among other things snake family, geographical location, genus, species, age, conventional prey preference, and snake size. For example, whereas SVMP, SVSP, and PLA2 are the most common in vipers, venoms of Elapids are generally predominated by 3FTx and PLA2, with the former basically absent in vipers [18].

3. Snakebite envenoming

Snake envenoming is a major health issue worldwide and often considered a rationalization for morbidity and mortality (morbi-mortality) and a variety of losses, both socially and economically. Conservative estimates of the global incidence of snakebite cases suggest that approximately 5.5 million incidents are reported each year [19]. Snakebite envenoming is a potentially fatal disease that normally occurs as a result of the injection of venom following the bite of a venomous snake. Snakebite envenoming can also be caused by the spraying of venom into a person’s eyes by particular snake species that are capable of spitting venom as a defense measure. Envenoming results from an estimated 50–55% of all snakebites. Snakebite envenoming in humans and animals affects multiple organ systems based on the specific snake species and the groups of toxins present in the venom and can cause inter alia neuromuscular paralysis, hemorrhage and protracted disruption of hemostasis, cardiotoxicity, tissue necrosis, thrombosis, and myolysis (muscle degeneration) [1, 2].

Taxonomically, snakes are carnivorous in nature, classified as reptiles and may be elapids, vipers, colubrids, boids, or pythons. Whereas the majority of bites occur when snakes are inadvertently stepped on by barefooted or unprotected victims, others may be instigated by alcohol intoxication and malevolence. In terms of classification, more than 3500 snake species are believed to exist, with venomous species accounting for approximately 600 (15–17%) [19].

3.1 Epidemiology of snakebite envenoming

Available epidemiological data on the control of snakebite envenoming has largely been fragmented, and for that matter inaccurate, a problem attributable to inadequate control efforts or measures. Rather than seeking help from health centers or hospitals, many snakebite victims resort to traditional remedies, a practice which further reduces the accuracy of existing data. Internationally, data available on snakebite reports is generally limited. Particularly, in developing countries, nearly all snakebites and the consequent deaths go unreported. In countries with poor infrastructure in rural areas, the extent of under-reporting is believed to be in excess of 70%. In spite of this, of the estimated 4.5–5.4 million snakebites yearly, close to half (1.8–2.7 million) result in clinical illness, with about 81,000–138,000 deaths due to complications. Globally, snakebites tend to affect rural populations of low socioeconomic status disproportionately, especially with groups considered as high risk including farmers or agricultural workers, hunters, fishermen, herders, occupants of poorly developed houses, working children, and people with little or no access to healthcare and education. High-case fatalities are more common among children, while young people are more frequently affected in terms of morbidities and mortalities. In addition, access to medical care is quite challenging for women from certain cultural backgrounds, with pregnant women being exceedingly vulnerable. Across the world, snakebite envenoming and its associated deaths are unevenly distributed, with the highest cases in Asia (South and South-East) and sub-Saharan Africa. A majority of the world’s population is thought to live in these areas, a phenomenon that brings about human-snake conflicts. On the other hand, Australia, Europe, and together with North America have the lowest cases of envenoming [2, 20].

3.2 Mechanism and pathogenesis of snakebite envenoming

Snake venom is produced and stored in a dedicated gland. During a bite, the venom is released through a compressor muscle-mediated action in which muscle surrounds the venom gland. Through the help of a duct, the venom is delivered to the fangs, which is then delivered into the tissues of a victim. Subsequent to snakebite envenoming, the venom toxins may act on different parts of the body systems. The venom of snakes is delivered or injected via a specialized delivery system. In elapids, viperids, and lamprophiids, the system is composed of a group of fangs located anterior to the maxillary bones. However, in colubrids with no front fangs, the fangs are positioned posteriorly. The injection of snake venom may either be subcutaneous or intramuscular based on the size of the fangs. Following envenoming, certain toxic components of the venom evoke pathological effects localized within the surrounding tissues, while some other toxins exert their effects on various organs as a result of their systemic distribution through the blood vessels and lymphatic system [21]. Following bites from vipers and certain cobras, local swelling is detected in a space of 2–4 hours with the potential of extending rapidly to reach its peak 2 or 3 days later. Within 2–12 hours of the bite, blistering develops and tissue necrosis becomes evident on the first day. In the subsequent weeks or month, the necrotic tissue undergoes sloughing while secondary infections such as osteomyelitis develops. Swelling of the bitten limb may be completely resolved, and normal function restored only after some weeks. Systemic envenoming may be indicated by syncope or vomiting occurring within minutes of the bite. A few hours after the bite, coagulopathy and bleeding set in and can persevere for two or more weeks without treatment. Signs of neurotoxicity can advance to widespread flaccid paralysis and respiratory arrest within half an hour to a few hours [22, 23]. Paralysis resulting from elapid bites including mambas, cobras, and kraits may be reversed if treated with specific antivenoms or acetylcholinesterase inhibitors, with recovery over a period of time in all instances, so long as respiration is supported sufficiently. Occasionally, drowsiness may also be observed with other local symptoms such as moderate or no pain, local swelling (mostly devoid of blisters or necrosis), and paraesthesia [22, 23], In addition, envenomings from non-front-fanged colubrids including boomslangs of African origin and vine snakes are usually typified by bruising (also called ecchymosis), which tends to evolve late or slowly, coagulopathy, systemic bleeding, and severe injury to the kidney, even when envenoming is minimal [24].

3.3 Diagnosis of snakebites

Snakebite envenoming is an emergency that presents clinical and diagnostic challenges due to the potentially rapid lethal effect. Decision-making can be complicated by the doubts surrounding the identity of a snake species, the amount of venom injected during a bite, and the composition therein, which may vary depending on the age of the snake and intra-species within its location. Nurses and health assistants are the personnel who manage most cases of snakebites in health posts, dispensaries, clinics, as well as district and rural-level hospitals. Sometimes, it is possible to refer cases to tertiary-level facilities with laboratories, specialists, and intensive care units [1].

3.3.1 Clinical diagnosis

The clinical diagnosis of snakebite envenoming relies on identifying specific signs in the patient. These include local signs such as swelling, blistering, and local necrosis. For the purposes of accurate diagnosis, systemic signs such as characteristic of viper bites, while signs of neurotoxicity and muscle damage (rhabdomyolysis) are primarily common with elapid bites and sea snake bites, respectively. There may, however, be some exceptions to these observations pertaining to snakebites. For instance, bites from some viper snakes, such as the tropical rattlesnake and berg adder, exhibit signs of neurotoxicity in patients with systemic envenomation. Following the bite of vipers particularly, localized effects including necrosis may occur; however, necrosis tends to manifest slightly later on and therefore may not necessarily be of diagnostic value. Also, in addition to neurotoxicity, certain Australian elapids can cause incoagulable blood and hemorrhage. In a similar way, certain cobras that have the capacity to spit venom into the eyes can cause serious localized painful conjunctivitis together with swelling. Whether or not fang marks are present cannot be used as a basis for diagnosis albeit the distance between the fang marks may offer a clue about the size of the snake implicated in the bite. Nonetheless, the identification of fang marks does not form sufficient basis to suggest that venom has indeed been introduced because in close to 50% of bites, no venom is injected [14].

3.3.2 Laboratory and other diagnostic methods

The diagnosis of snakebites in the laboratory operates on the basis of the changes, which take place following envenomation in victims. Such changes may include detecting irregular changes in blood parameters [such as incoagulable blood assessable using the 20-minute whole blood clotting test (WBCT20), dramatic drop in platelet count, and changes in white and red blood cell counts], changes in some enzyme levels (e.g. creatine phosphokinase), and presence or absence of myoglobinuria, as well as detecting specific venom antigens in the blood of envenomed individuals (biodetection techniques using immunologically based methods) [14].

Severe hemorrhage may be indicated by a low hematocrit value, while a high hematocrit value may be indicative of haemoconcentration, which occurs when plasma leaks into the tissues due to an increase in capillary permeability. Systemic envenoming by viperids, non-front-fanged colubrids, and oceanic elapids are mainly characterized by incoagulable blood. The 20-minute whole-blood clotting test (WBCT20) is a simple test that involves collecting a few milliliters (normally 2 mL) of venous blood into a new, clean, and dry glass tube, allowing it to stand undisturbed for 20 minutes at room temperature and then tilting the tube to see if it has clotted [25]. The absence of clotting is an indication of a venom that is anticoagulant or severe consumption coagulopathy [23]. Generalized fibrinolysis and intravascular coagulation may be detected by more sensitive laboratory tests including prothrombin and activated partial thromboplastin times, D-dimer, and products of fibrin degradation. Severe rhabdomyolysis may be indicated by a creatine kinase level greater than 10,000 units per liter. For patients who are in danger of acute kidney damage, serum creatinine and potassium concentrations or blood urea should be measured. Urine tests should be conducted for the presence of myoglobin, hemoglobin, as well as other proteins and blood. Abnormalities from electrocardiograms may include ST-T changes, sinus bradycardia, and signs of myocardial ischemia or different levels of atrioventricular block. The presence of myocardial dysfunction and pericardial effusion can be detected by electrocardiography. Magnetic resonance imaging (MRI) and computed tomography (CT) are increasingly utilized in the assessment of infarcts and intracranial hemorrhages [1]. Wound ultrasonography has also been advocated for use in detecting tissue damage [26].

3.4 Snakebite management

In treating snakebite envenoming, antivenom therapy remains the backbone of therapy. However, the choice of an antivenom is contingent on the species diagnosed. In the absence of the implicated snake being identified or brought in, the species diagnosis is normally based on the conformation of the bite, information on the species frequently recognized for a given geographical location, distinguishing the clinical manifestations, as well as information collected from the victim or witness. In various parts of the world, clinical manifestations form the basis of species differentiation. Nevertheless, this is difficult to the extent that the toxins that constitute venoms of different species are a lot of the time pharmacologically and physiochemically similar and as such may provoke similar clinical effects. For instance, venoms of Bungarus fasciatus, Naja naja siamensis, and Ophiophagus hannah all comprise postsynaptic neurotoxins that inhibit neuromuscular transmission resulting in the same major clinical features, notably paralysis, respiratory failure, and ultimately death. When the culprit snake is even brought, there is a likelihood of misidentification, and therefore antivenom misadministration resulting in complications as witnessed in the past. Additionally, it is observed that morphologically similar species may exhibit different clinical manifestations due to the difference in venom composition based on differences in geographical locations. Owing to the shortcomings for accurate snake species identification, clinicians may find it problematic to administer suitable antivenoms upon presentation [27]. According to Theakston & Laing [14], the difficulties in identifying the species implicated in a case of envenoming make it even more challenging in the choice of the appropriate antivenom for treatment, particularly in areas where only monospecific antivenoms exist. Accordingly, this dilemma became a key consideration that stimulated the development of sensitive assay methods employing immunodiagnostic, as well as other laboratory-based methods. At the initial stages of investigation, it was demonstrated that immunodiagnosis using enzyme-linked immunosorbent assay (ELISA) or enzyme immunoassay (EIA) was helpful in identifying the species responsible for snakebite envenoming and also in detecting specific venom antibody following the use of radioimmunoassay (RIA) to detect venom in Australia by the Sutherland’s group. Subsequently, EIA, a much cheaper technique relative to RIA, was developed in a manner that allowed the diagnostic patterns of envenomation by diverse, occasionally closely related snake species to be accurately detected. Rapid tests for snakebite envenoming are available in Australia, regrettably; however, they are believed to be extremely expensive, coupled with challenges with sensitivity. EIA is, however, valuable in studying both new and existing antivenoms since it offers a significant objective appraisal of antivenom efficacy and has the potential for application in various aspects of venom research [14, 28, 29].

3.4.1 Antivenom treatment and its related problems

Antivenom immunotherapy remains the only accepted and effective way of treating systemic snakebite envenoming that is yet to confront the regime of rigorous scientific testing. It is also credited as the only therapy that has rescued the lives of snakebite envenoming victims for nearly a century. In spite of this, antivenoms are replete with significant challenges including poor stability of antivenom in its liquid form, adverse side effects/reactions, efficacy issues, and huge production difficulties often resulting in antivenoms that are extremely expensive, especially for those most affected. Antivenoms are produced by immunizing animals, especially large mammals such as horses and in rare instances donkeys and sheep. The large size of these animals allows potentially the collection of large volumes of plasma, and consequently the generation of large volumes of antivenom. The immune system of the animal is, thus, exposed to either a single venom in which case a monospecific/monovalent antivenom is generated or multiple venoms resulting in a polyspecific/polyvalent antivenom. An immune response is elicited by the animal, with antibodies (notably IgGs) been raised to bind specifically to the antigens/immunogens available in the venom (s). Fractionation procedures, such as centrifugation or sedimentation, are then used to separate plasma from the blood and red blood cells reinfused or reinjected into the animal. Subsequently, additional purification activities may be carried out to reduce the non-IgG serum protein content in certain antivenoms. Nonspecific IgGs may sometimes be removed using affinity chromatography. Sometimes, fragment crystallizable regions (Fc) may be removed through enzymatic digestion using pepsin or papain, resulting in F(ab’)2 or Fab fragments, respectively, which are used to produce antivenom by most producers in Western countries, albeit intact IgGs are also utilized [1, 2].

3.4.1.1 Challenges in ensuring reproducibility in antivenom production

The production of conventional antivenoms is a challenging task. The risk involved is not just limited to milking venoms from the species responsible for the potentially deadly bites, but also the fact that an animal has to be immunized with a nonlethal/safe dose of the venom extracted, or in some instances, the venom is detoxified in a manner that ensures that its immunogenicity is not lost. These issues reflect the inherent problems associated with antivenom production. Aside the fact that the animal can be stressed up, its maintenance is quite expensive and the antivenom yields, which are predicated on the immune responses can be threatened by the stress [30]. Antivenom production processes are not only enormously laborious with low yields but are also confronted with vast batch-to-batch variability [31]. This is, however, expected because when animals are injected with venoms, which vary compositionally, they elicit varied levels of immune responses to the immunogen. Thus, a pool of at least 20–50 venom samples from the same area or region is recommended as a way of mitigating these difficulties [1].

3.4.1.2 Poor stability of antivenoms

The poor stability of liquid antivenom renders it difficult to make it available for use in remote areas that need it the most and freeze-dried preparations are difficult. Antivenom in its liquid form requires, in addition to preservatives, and more challengingly, the need for refrigeration at 4–6°C in order to ensure that its potency is maintained. Although freeze-dried or lyophilized antivenom products are also available, the liquid form is distributed in order to cut down cost and maximize its use [32]. Also, the incidence of adverse reactions is believed to be increased by the formation of protein aggregates, particularly in liquid antivenom, a phenomenon triggered by high temperatures [33]. Owing to the poor stability, antivenoms are sold with dates of expiration, while also warning users to avoid those that have undergone several cycles of freeze-thawing, even though a handful of the available evidence suggests that the efficacy of an antivenom is not significantly affected by either of these two [34].

3.4.1.3 Adverse effects/reactions associated with antivenom therapy

Antivenoms by their architecture contain antigenic proteins, which cause the immune system to be activated, thereby leading to adverse reactions. These reactions are classified as acute (anaphylactic or pyrogenic) and delayed “plasma” sickness (given that most antivenoms are made from plasma), with the latter still been referred to as late “serum” sickness [35]. The early type reactions are characterized by mild symptoms such as headaches, coughing, nausea, vomiting, and diarrhea, and others may be sparked immediately, although symptoms can start after about 1 hour. Severe anaphylactic reactions are also capable of developing and may be associated among other symptoms with angioedema, hypotension, and bronchospasm [23]. Endotoxins, occurring as a result of bacterial contamination, may give rise to pyrogenic reactions associated with symptoms such as fever, reduced blood pressure, chills, and vasodilation. On the other hand, late-type reactions mostly show up a number of days following administration of the initial dose and may be characterized by the same symptoms not only as happens with the early reactions but also other symptoms including joint pains, albuminuria, adenopathy, and occasionally encephalopathy. Prophylactic drugs, including hydrocortisone and adrenaline, are used to frequently treat anaphylactic reactions successfully [36]. How frequently adverse reactions occur and their nature depends partly on the level of purification of the antivenom. While in about 54% of patients, adverse reactions have been reported to be caused by crude first-generation antivenoms and second-generation antivenoms, which are obtained by affinity chromatography, and contain only intact IgGs without plasma proteins, have been reported to decrease adverse side effects to below 25% of victims. Third-generation antivenoms are generated by the enzymatic digestion of antibodies with either pepsin or papain leading to the removal of Fc regions, which are believed to cause fewer adverse side effects. In the midst of all these purification processes, however, the full neutralization effect of antivenoms is hardly achieved [15].

3.4.1.4 Cost of antivenoms

The cost of maintaining animals coupled with increasing immunoglobulin purification procedures are thought to account for the rise in treatment costs. In the developed world, more effective and expensive generations of antivenoms are available for use, where cost may not be an issue. However, in the developing world with rampant snakebites, such exorbitant antivenoms have the potential to cripple victims both financially and physically following a snakebite, with most of the time friends and family having to share in the financial burden. Treatment cost in India is estimated around $5000, a cost which is more than 2-fold the per capita GDP of India, and represents in excess of 10 years financial entitlements (in terms of salary) of a normal farm worker [37]. In the United States, there are reports of extremely high treatment costs (with antivenom inclusive) of about $153,000, with the possibility of even costlier treatments taking place [38]. In the face of this, however, inexpensive antivenoms are still possible to produce. At a treatment cost of just about $40, this is regarded as one of the most cost-effective antivenom treatments anywhere in the world, developed from a collaboration between Nigeria and the UK [30]. According to [38], treatment costs in some countries can go as high as 1000 times the cost involved in producing a vial of antivenom after considering expenses from clinical trials and hospital charges.

3.5 Emerging and future diagnosis and treatment options for snakebite envenoming

3.5.1 Enzyme-linked immunosorbent assay-based (ELISA) methods for detection of snake venoms

The application of ELISAs or enzyme immunoassays (EIAs) in detecting specific venoms as well as the indirect detection of specific venom antibodies (including antivenom) was first reported in 1977 using the double sandwich method carried out in a microliter plate with 96 wells [15]. According to [27], the ELISA was able to detect 1–5 ng of venom/mL in 3 hours and has since then been applied in detecting various venoms in some parts of the world. ELISAs have been widely used and have become handy for studying the kinetics of snake venoms in blood, magnitude of envenomation, and the adequacy or otherwise of antivenom serotherapy. ELISA continues to be the appropriate method for detecting snake venoms, toxins, and venom antibodies in body fluids and is considered to offer more practical value than any other immunoassay [28, 39, 40].

In principle, soluble antigens are linked to the wells contained in the ELISA plate, allowing the individual components to retain their reactivity. In a double sandwich method, a given venom antibody binds to the plate, which is then washed to remove any unbound material followed by adding a test substance, which contains a venom-specific antigen. The complex, thus, formed between the venom and antibody is detected after further washing by using an enzyme-conjugated specific antibody such as alkaline phosphatase or horseradish peroxidase. After a further washing step, an enzyme-specific substrate is added with the resulting hydrolysis or color change measured spectrophotometrically or visually considered proportionate to the quantity of venom or antigen available in the test sample [14]. In respect of sensitivity of ELISAs, significant improvements have been made to enable the detection of venom concentrations at picogram levels. In addition to using avidin-biotin amplification, further improvement in the sensitivity of ELISA can be achieved by an increase in the affinity of antibodies, attainable by increasing the length of immunization and the rate at which booster injections are given. Also, monoclonal antibodies and venom-specific antibodies purified via affinity chromatography are regularly used to accomplish species specificity of an ELISA, and the latter appears to be the ideal for detecting venoms [27].

In a study by Liu and colleagues [41], a sandwich ELISA test and lateral flow assay were developed as a way of improving the clinical management of snakebites in Taiwan. These tests were meant to detect both hemorrhagic and neurotoxic venom proteins of four main snake species responsible for over 90% of all clinical envenomation cases. In the study, species-specific antibodies were generated from antivenoms using a two-step affinity purification procedure. The neurotoxic and hemorrhagic species-specific antibodies were biotinylated and subsequently used to develop sandwich and lateral flow assays. The sandwich ELISA assay was developed based on an interaction between the biotinylated antibodies and alkaline phosphatase-conjugated streptavidin with 4-methyl umbelliferyl phosphate as substrate. A SpectraMax M5 microplate reader was then used to measure the fluorescence. Findings from the study showed that both diagnostic assays were able to successfully discriminate between hemorrhagic and neurotoxic venoms. For the ELISA, the limits of quantification for hemorrhagic and neurotoxic venoms were determined as 0.78 and 0.39 ng/mL, respectively, whereas the lateral flow assay detected hemorrhagic and neurotoxic venoms at concentrations lower than 50 and 5 ng/mL, respectively within 10 to 15 minutes.

In another study by Shaikh et al. [42], a rapid and sensitive assay and sandwich ELISA assay were developed to detect snake envenoming [notably the Indian Cobra (N. naja), Russell’s viper (Daboia russelii), common Krait (Bungarus caeruleus), and saw-scaled viper (Echis carinatus)] prior to the administration of antivenoms in India. Here, monovalent antisera were prepared by immunizing rabbits with specific venoms in order to obtain venom-specific antibodies. Immunoaffinity chromatography was then used to remove cross-reacting antibodies so as to obtain venom-specific antibodies. Thus, the venom detection ELISA test was developed using two different species of antibodies that provided increased sensitivity and also ensured that venoms of offending snakes were selectively identified. From the results obtained, the sensitivity of the sandwich ELISA was observed to detect venom up to 0.01 ng/mL. It also revealed that the venom detection ELISA test developed was rapid, specific, and sensitive, detecting venoms of culprit snakes at a venom concentration of up to 1 ng/mL. When experimentally envenomed samples were used, the venom detection ELISA test was able to quantitatively detect venom concentration in the range of <1 ng/mL within 20–25 minutes. At between 1.0–0.1 ng/mL, the device demonstrated the lowest venom detection limit with a 100% agreement recorded between the sandwich ELISA and the venom detection ELISA test device.

In another study by Dong et al. [40], an avidin-biotin ELISA (AB-ELISA) assay was developed for detection of venoms of four common snake species (Calloselasma rhodostoma, Trimeresurus popeorum, O. hannah, and N. naja) in the South of Vietnam. In the study, species-specific antivenom antibodies were generated using a three-step affinity purification protocol and antibodies subsequently used to develop an ELISA test kit for venom detection. From the results obtained, the ELISA kit was able to discriminate between the venoms from the four snakes in different sample types tested. The sensitivity of the AB-ELISA reported in the study was high, with a venom detection limit lower than 1.6 ng/mL in most of the samples tested. Notably, the detection limit was as low as 0.2 ng/mL of the serum or sample buffer in the case of N. naja venom. Thus, the sensitivity of the AB-ELISA assay was attributed to the selection of high titer serum samples containing high affinity, avidity antibodies, and antibody purification. In addition, the efficacy of the ELISA kit in detecting snakebite envenoming was successfully illustrated in experimentally envenomed rats. The findings also revealed that of the 140 human samples (blood, urine, wound exudates, and blister fluids) tested for venom detection by the kit, venom was detected in all the body fluids.

In a recent study [43], described an inhibition ELISA prototype for detecting cytotoxic three-finger toxins present in the venoms of African spitting cobra species. Such a diagnostic technique capable of detecting specific venom toxins is crucial for the administration of toxin-specific therapy for snakebite envenoming. Different ELISA parameters were optimized employing an indirect ELISA method. The specificity of the assay was assessed by the observed percent inhibition at various sample antigen concentrations (6–0.008 μg/mL) in three homologous (N. ashei, N. nigricollis, and N. haje) and two heterologous (Bitis arietans and D. polylepsis) venom samples. Similarly, to determine the sensitivity of the inhibition ELISA, crude N. ashei venom was analyzed from an initial concentration of 27 μg/mL to a final concentration of 0.04 μg/mL, following which the limit of detection (LOD) of the assay was determined. ELISA plates were coated with purified 3FTx antigen overnight, while sample antigen (crude N. ashei venom) was constituted in blocking buffer and diluted 3-fold serially. Following the incubation and washing steps, the plate was analyzed and percent inhibition calculated (Eq. (1)) as:

$$\text{Percent\hspace{0.17em}inhibition}=\frac{\mathrm{NAC}\mathrm{OD}-\text{Test\hspace{0.17em}sample}}{\mathrm{NAC}\mathrm{OD}}\times 100$$E1

The prototype was further tested for its ability to discriminate between venoms containing 3FTxs and those without using crude venoms from two other Naja sp. (N. nigricollis and N. haje) and two non-Naja sp. (B. arietans and D. polylepsis). These sample antigens were prepared as previously described, ELISA implemented, plate analyzed, and percent inhibition determined. At all the inhibitor concentrations tested, a percent inhibition of greater than 18% was observed across all homologous venoms. On the other hand, a% inhibition less than 16% was observed among the heterologous samples across all the inhibitor concentrations tested (Table 1).

Inhibitor Concentration (μg/mL)	N. ashei		N. nigricollis		N. haje		B. arietans		D. polylepsis
	OD	% Inhibition	OD	% Inhibition	OD	% Inhibition	OD	% Inhibition	OD	% Inhibition
6.000	0.094	76.90	0.085	77.68	0.088	79.75	0.474	7.06	0.458	8.77
2.000	0.118	71.13	0.087	77.01	0.091	79.17	0.464	9.02	0.439	12.46
0.667	0.174	57.25	0.098	74.24	0.098	77.45	0.454	11.08	0.458	8.67
0.222	0.259	36.49	0.125	66.97	0.113	73.99	0.467	8.53	0.465	7.28
0.074	0.295	27.64	0.177	53.37	0.131	69.85	0.471	7.65	0.445	11.27
0.025	0.319	21.62	0.252	33.42	0.182	58.11	0.447	12.45	0.434	13.46
0.008	0.331	18.80	0.299	21.00	0.260	40.28	0.447	12.35	0.424	15.55
NAC	0.407		0.379		0.435		0.510		0.502

Table 1.

Percent inhibition of 3FTxs induced by N. ashei, N. nigricollis, N. haje, B. arietans, and D. polylepsis venoms.

With a mean and standard deviation (of the negative control) of 0.835 and 0.207, respectively, the limit of detection (LOD) of the assay was determined to be approximately 0.01 μg/mL. From the six-point three-fold dilution of the sample antigen, the percent inhibition was observed to be highest (77.70%) at 27.00 μg/mL and lowest (36.56%) at 0.04 μg/mL of the antigen, respectively (Figure 1).

Figure 1.

Inhibition ELISA curve showing % inhibition of the coated antigen by the sample-containing antigen at various concentrations of crude N. ashei venom (inhibitor). Sample antigen was diluted 3-fold from an initial concentration of 27 μg/mL to a final concentration of 0.04 μg/mL and run in duplicates [43].

It was also observed that at high inhibitor concentrations, the ODs of all three Naja species were low. The ODs, however, increased with decreasing inhibitor concentrations. As low as 0.008 μg/mL inhibitor concentration, the sample antigens induced varying levels of inhibition (18.80, 21.00, and 40.28% for N. ashei, N. nigricollis, and N. haje, respectively). Conversely, increasing inhibitor concentrations resulted in an increase in the percent inhibition for the same species (Figure 2).

Figure 2.

Inhibition ELISA curves showing % inhibition of the coated antigen (pure 3FTx fraction) by sample antigens from crude N. ashei, N. nigricollis, N. haje, B. arietans, and D. polylepsis venoms at different concentrations. The different sample antigen-containing venoms were diluted 3-fold from an initial concentration of 6 μg/mL to a final concentration of 0.008 μg/mL. Samples were then analyzed in duplicates at each concentration [43].

The relatively high and comparable inhibition observed in N. ashei, N. nigricollis, and N. haje may confirm two things; first, the similarity in the venom proteomes of the cobras (both spitting and non-spitting) and second, the venom proteomes of Naja sp. contain more 3FTxs. ODs obtained for B. arietans and D. polylepsis suggest that there was no or minimal inhibition mainly because the venoms either contain little or none of the antigen of interest. Furthermore, the detection of envenoming by the inhibition ELISA model (LOD of 0.01 μg/mL) is consistent with previously reported ELISA methods, although different LODs were found.

3.5.2 Small molecule therapeutics

Venom components, particularly those of viperids, are known to contain distinctive active sites that usually depend on three amino acid residues. The ability therefore, of compounds to block such an active site makes it possible to functionally disable the venom component. Lately, various small molecule inhibitors have caused excitement, with the potential to expedite the utility of such compounds through molecular docking approaches given the already encouraging results available [15]. One of the most interesting small molecule therapeutics (SMTs) to have recently emerged is varespladib and its orally available prodrug format, methyl-varespladib. As a treatment drug initially meant for acute coronary syndrome, varespladib has been shown to successfully inhibit phospholipase A2 activity of various snake venoms obtained from different parts of the world. The effect of varespladib against venom-derived PLA2s is relevant given their poor immunogenicity, which may elicit a poor immune response during immunization of animals as part of the development of conventional antivenoms. Consequently, such antivenom products obtained from poorly immunogenic components may not be very effective against PLA2s. In an experimental set up, pretreating mice with 4 mg/kg varespladib and then injecting the same with the lethal dose of Micrulus fulvius venom was shown to prolong survival for nearly 24 hours following, which the protective effect wore off and reduce signs of hemorrhage in the mice. Also, the co-injection of an LD50 of Vipera belus venom and 4 mg/kg varespladib subcutaneously successfully ensured the survival of 3/7 mice, while all controls died. When varespladib was given following a little delay in the injection of V. belus venom, the same result was produced. A 100% survival was also observed in treated mice after the intravenous administration of 8 mg/kg varespladib followed by the subcutaneous injection of an LD50 of V. belus venom. In yet another experiment, rats injected with M. fulvius venom subcutaneously were entirely rescued following the intravenous administration of varespladib within 5 minutes of the challenge. In addition, varespladib was shown to suppress the increase in PLA2 activity induced by the venom, as well as haemolysis of the venom [44].

In another study conducted quite recently, varespladib was reported to inhibit the in vitro PLA2-induced toxic effects of Agkistrodon halys, Deinagkistrodon acutus, Naja atra, and Bungarus multicinctus. At a dose of 4 mg/kg, varespladib resulted in a reduction in the density of hemorrhagic plagues provoked by both D. acutus and A. halys venoms and also decreased edema and hemorrhage instigated by all four venom samples in vivo, varespladib-treated mice recorded a 31–81% decrease in edema relative to control animals. Again, signs of muscle damage induced by the venoms including desmin degradation were reduced by varespladib. At median effective doses (ED50s) of 0.45 and 1.14 μg/g, the inhibition of venom-induced lethality by varespladib was found to be more effective in viperid venoms of A. halys and D. acutus, respectively, as opposed to those of elapids; 22.09 and 15.23 μg/g for N. atra and B. multicinctus, respectively [45]. Venoms from most snakes contain toxic components, especially PLA2s that act synergistically with other toxins. To this end, it could be conjectured that the administration of varespladib could potentially obstruct the synergistic effect of key toxins, resulting in the complete inhibition of venom toxicity. Nonetheless, the usefulness of varespladib may be limited because the extensive reliance on PLA2s does not apply to all snake venoms. For instance, venom from the genus Dendroapsis is nearly exclusively devoid of phospholipases A2; thus, it is not probable that varespladib would be beneficial against snakebites from this genus [46, 47]. However, whereas varespladib on its own may possess exciting applications, methyl-varespladib, its corresponding prodrug format, is available orally for administration, rendering it a potential first-choice candidate drug for protection. Therefore, when methyl-varespladib is used alone or combined with other drugs, it may be capable of providing some respite to victims, whiles efforts are made to access further antivenom treatment at appropriate health facilities [48].

Another example of a SMT that has shown great promise is the matrix metalloproteinase batimastat and its orally available prodrug format marimastat. The incubation of these SMTs with a challenge dose of E. ocellatus venom (4 LD50) and subsequent co-injection into the tail vein of mice resulted in prolonged survival, although it was not fully protective. However, administering batimastat abrogated the hemorrhagic, in vitro coagulant, defibrinogenating, and proteinase activities induced by E. ocellatus venom sourced from Cameroon. When E. ocellatus venom samples from Ghana were used, an increased abrogation in hemorrhagic activity was observed when batimastat was quickly administered. Conversely, the delayed administration of batimastat resulted in better abrogation of defibrinogenating activity, with the possibility of completely abrogating the effect following a 60-min delay in administration of the molecule. While batimastat proved to be more efficacious in abrogating hemorrhagic activity, defibrinogenating activity was more effectively abrogated by marimastat [49]. Other SMTs including acetylcholinesterase inhibitors (e.g., atropine and neostigmine) are being investigated with some encouraging results in the reduction of mortality among venoms of certain elapids. In addition, nanoparticles and C60 fullerene are also being investigated, with the latter showing antivenom features in an insect model [27].

3.5.3 Protein, peptide, and oligomer-based technologies

Apart from antivenom, protein, peptide, and oligomer-based therapies are being explored. Human monoclonal-based single-chain variable fragments and fully humanized monoclonal IgGs have been developed. There are indications that these technologies may possibly be crucial in inhibiting various snake venom components in the future given that they are associated with relatively less adverse reactions, as well as the potential to be competitive in terms of cost [27]. According to El-Aziz et al. [50], oligonucleotides are without most of the problems that usually characterize the immunization approach of antibody production including the use of animals, protracted production time, low immunogenicity mostly with small-sized toxins, and production cost, as well as treatment-related challenges such as requirement for refrigeration, specificity issues, reduced shelf-life, and immunogenicity of animal-based antibodies. In exploring the neutralization action of αC-conotoxin PrXA found in Conus parius, a species of cone snail by oligonucleotides, a sample of oligonucleotide tested demonstrated capacity to neutralize the in vitro activity of αC-conotoxin PrXA. Although at the doses tested, the oligonucleotide was not fully protective, it, however, provided extended survival. Nonetheless, at much higher concentrations, the oligonucleotide provided full protection. Furthermore, [44] argue that an added benefit that comes with the use of oligonucleotides in the laboratory is that the cost of small-scale synthesis is low for the purposes of research and development (R&D), allowing researchers to easily and rapidly appraise a wide variety of molecules at reduced cost. There is the need, however, for more studies to evaluate the cost of manufacturing oligonucleotides on a large scale before its evaluation for clinical-based application. Lakkappa et al. [27] also report that aptamers (short DNA or RNA sequences) have been demonstrated to neutralize cardiotoxins and α-bungarotoxin, as well as toxins present in corn snails. In addition, a large number of alternative binding scaffolds (AbScaffs) are being investigated for their therapeutic prospects in snakebite envenoming because they cost low to produce, highly stable, and easy to engineer.

3.5.4 Monoclonal antibodies

Advances in basic research culminated in the development of the breakthrough hybridoma technology in 1975 by Kohler and Milstein in which hybrid cells producing rodent-derived monoclonal antibodies (mAbs) were generated in unlimited quantities [51]. These hybrid cells were achieved by fusing B cells obtained from an immunized animal with myeloma cells and the resulting selected cell secreting one specific antibody. Thus, the interest in the use of antibodies in therapeutics was again awoken by the hybridoma technology following its discovery. Subsequently, techniques were developed to overcome the safety, efficacy, and immunogenicity problems associated with the use of rodent-derived antibodies by transforming same into structures akin to human antibodies, ensuring that the binding properties to the target are retained. Consequently, the first humanization method resulted in the development of chimeric antibodies through combining sequences of human constant region domain and murine variable domain, with the resulting antibody preserving the specificity and reducing its immunogenicity [52].

In a previous study [53], the possibility of antivenom based on monoclonal antibodies was investigated. In this study, neutralizing mAbs were developed and tested against three key toxic constituents of Bothrops atrox, the main snake group causing accidents in the Northern region of Brazil. Like the venom of other Bothrops species, B. atrox venom contains a complex mixture of venom proteins including proteases whose substrates include blood clotting system components such as factors X, XII, and fibrinogen. The venom of B. atrox is also made up of proteins that are zinc-dependent metalloproteinases, majority of which are hemorrhagic and phospholipase A2, which has been demonstrated to play a role in myonecrosis and inflammation. Hybridoma cells secreting neutralizing mAbs PLA2 clone A85/9-4, Zn-metalloproteinase clone 59/2-E4, and serine proteinase clone 6 AD2-G5 of B. atrox were cultured, expanded and cells injected i.p. into BALB/c mice. Ascitic fluid was subsequently collected via abdominal puncture and mAbs purified via caprylic acid followed by ammonium sulfate precipitation. The results obtained showed that purified mAbs specific to these three venom proteins were successfully generated. When submitted to immunochemical analysis via SDS-PAGE, all three mAbs showed two main protein bands, one around 55 kDa and the other approximately 29 kDa, indicative of immunoglobulin heavy and light chains, together with several minor contaminant bands. On performing western blot analysis with anti-mouse IgG as the primary antibody, the two major bands were confirmed to correspond to mouse IgG heavy and light chains. Furthermore, estimation of the purity of the mAb preparations was performed by densitometry analysis of SDS-PAGE, which revealed purity grades of 46, 45, and 37% for A85/9-4, 59/2-E4, and 6 AD2-G5, respectively.

In another study by Laustsen et al. [54], an experimental recombinant antivenom based on a mixture of fully humanized IgG monoclonal antibodies, able to neutralize neurotoxicity facilitated by dendrotoxins of the notorious black mamba (Dendroaspis polylepis) is described. The approach to discovering the suite of human IgGs combined among other things toxicovenomics, antibody phage display technology and engineering, and mammalian cell expression. A human antibody phage display library of clones and single-chain variable fragments (scFvs) antibodies were constructed from B-lymphocytes obtained from nonimmunized human donors. ScFv antibodies that produced the highest binding signals from an expression-normalized capture (ENC) assay were selected and converted to IgG format. The results of the study demonstrate the possibility of exploiting oligoclonal cocktails of monoclonal human IgGs to treat black mamba envenoming. The findings further showed that individual monoclonal IgGs targeting neutralization of dendrotoxins cannot do that alone and for which reason it could be beneficial to employ antibody cocktails that neutralize multiple black mamba venom toxins in order to achieve full protection.

In a recent study by Manson et al. [55], anti-3FTxs monoclonal antibodies were developed against N. ashei venom in mice. Preliminary activity of the purified mAbs against 3FTx antigen was assessed by testing the ability of the mAbs to identify and bind to the target antigen in an ELISA titration. All three mAbs demonstrated capacity to recognize 3FTxs, and thus were able to bind to the antigen at the lowest concentration tested (0.0002 mg/mL). When compared with the control sample, all three clones were found to bind to the target antigen with comparatively higher efficacy as assessed by optical density. Further confirmation of the recognition, binding, and activity of the mAbs was evaluated using an inhibition ELISA assay previously described [43]. A cocktail of the purified mAbs was tested against two commercial antivenoms available in the Kenyan market (VINS™ and Inoserp™) and 3FTxs-challenged mice polyclonal antibodies. Tukey’s multiple comparison test showed that the 3FTxs-induced inhibition by the test mAbs was significantly different when compared with inhibition by the two antivenoms. Both the mAbs and polyclonal antibodies induced comparable inhibition. Similarly, there was a significant difference in the inhibition induced by the polyclonal antibodies relative to both antivenoms. Thus, the results demonstrate that the immunoaffinity-purified test mAbs have higher binding efficacy, and hence higher specificity for the target antigen relative to both the negative control and the two leading commercial brands of antivenoms on the Kenyan market. The results further demonstrate the prospects of developing toxin-specific monoclonal-based antivenoms for snakebite immunotherapy.

4. Conclusion

In conclusion, the advent of using diagnostic kits with the ability to detect specific venom toxins and support of their access could potentially permit species implicated in snakebites to be deduced and ensure that treatments are targeted at toxins present in the victim’s blood, as well as those that are known to be connected to the bites of particular species. Making available low-cost and heat-stable SMTs at the local level to high-risk areas could allow the administration of pre-hospital treatments after establishing envenomation that could lower the risks of tissue damage and paralysis. Subsequently, surveillance and treatment at a hospital may be procured using improved treatment approaches such as more specific monoclonal antibodies or AbScaffs [15, 56].

Acknowledgments

The authors acknowledge the support of Pan African University Institute of Basic Sciences, Technology and Innovation (PAUSTI) and the Japan International Cooperation Agency (Africa-ai-Japan Project).