Antivenom is the treatment of choice for snakebite, which annually kills an estimated 32,000 people in sub-Saharan Africa and leaves approximately 100,000 survivors with permanent physical disabilities that exert a considerable socioeconomic burden. Over the past two decades, the high costs of the most polyspecifically-effective antivenoms have sequentially reduced demand, commercial manufacturing incentives and production volumes that have combined to create a continent-wide vacuum of effective snakebite therapy. This was quickly filled with new, less expensive antivenoms, many of which are of untested efficacy. Some of these successfully marketed antivenoms for Africa are inappropriately manufactured with venoms from non-African snakes and are dangerously ineffective. The uncertain efficacy of available antivenoms exacerbates the complexity of designing intervention measures to reduce the burden of snakebite in sub-Saharan Africa. The objective of this study was to preclinically determine the ability of antivenoms available in Kenya to neutralise the lethal effects of venoms from the most medically important snakes in East Africa. We collected venom samples from the most medically important snakes in East Africa and determined their toxicity in a mouse model. Using a ‘gold standard’ comparison protocol, we preclinically tested the comparative venom-neutralising efficacy of four antivenoms available in Kenya with two antivenoms of clinically-proven efficacy. To explain the variant efficacies of these antivenoms we tested the IgG-venom binding characteristics of each antivenom using in vitro IgG titre, avidity and venom-protein specificity assays. We also measured the IgG concentration of each antivenom. None of the six antivenoms are preclinically effective, at the doses tested, against all of the most medically important snakes of the region. The very limited snake polyspecific efficacy of two locally available antivenoms is of concern. In vitro assays of the abilities of ‘test’ antivenom IgGs to bind venom proteins were not substantially different from that of the ‘gold standard’ antivenoms. The least effective antivenoms had the lowest IgG content/vial. Manufacture-stated preclinical efficacy statements guide decision making by physicians and antivenom purchasers in sub-Saharan Africa. This is because of the lack of both clinical data on the efficacy of most of the many antivenoms used to treat patients and independent preclinical assessment. Our preclinical efficacy assessment of antivenoms available in Kenya identifies important limitations for two of the most commonly-used antivenoms, and that no antivenom is preclinically effective against all the regionally important snakes. The potential implication to snakebite treatment is of serious concern in Kenya and elsewhere in sub-Saharan Africa, and underscores the dilemma physicians face, the need for clinical data on antivenom efficacy and the medical and societal value of establishing independent preclinical antivenom-efficacy testing facilities throughout the continent.
Snakebite annually kills over 95,000 people [1] residing in some of the most disadvantaged rural communities [2], and leaves about 300,000 surviving victims with permanent physical disabilities and stigmatising disfigurements. Since it is the most economically-productive and educationally-vulnerable 10–30 year olds that suffer most, snakebite also poses a significant additional socioeconomic burden on these remote, already impoverished communities. Available mortality data clearly indicate that snakebite deaths are greatest in Asia, and particularly in India [1,3] followed by sub-Saharan Africa (Table 1). The increasing concern over the plight of sub-Saharan African snakebite victims [4,5, 6] focuses upon the higher case fatality in sub-Saharan Africa than elsewhere (Table 1) and upon the declining availability of effective antivenom to treat snakebite victims. The crisis in supply of effective and affordable antivenom to treat snakebite victims in sub-Saharan Africa was first reported in 2000 [7], and has since deteriorated. Akin to the late 1990s market failure of the Behringwerke-manufactured antivenom, Sanofi Pasteur had also supplied Africa with one of the most polyspecifically-effective and widely-used antivenoms, FavAfrique, but ceased its manufacture in early 2016 after a more than a decade of commercial disincentives. This latest market failure of effective antivenom particularly affected snakebite-treatment capability in those state, private (mostly city-based) and charity hospitals that could afford this relatively expensive antivenom ($140/vial; [8]). The SAIMR polyvalent antivenom, manufactured by the South African Vaccine Producers Pty (SAVP), was also widely used and recognised to be highly effective–but outside of the Southern Africa Economic Community it has become more expensive ($315/vial, SAVP, personal communication) than FavAfrique was and, also because of low production volumes, become increasingly difficult to source. Cognisant presumably of potential commercial opportunities and the public health needs engendered by the snakebite-therapy vacuum in sub-Saharan Africa, several non-Africa based antivenom manufacturers have in the past two decades produced polyspecific antivenoms marketed at costs considerably lower ($18–75) than the FavAfrique or SAIMR antivenoms, and supplied in vastly greater quantities [8]. Superficially, this influx of new, affordable antivenoms into sub-Saharan Africa would seem highly desirable. However, in too many cases and African countries, this has not been the case—because some of these antivenoms have proved dangerously ineffective. Thus, reports from Ghana, Chad and the Central African Republic [9,10,11] document an increased case fatality rate (from under 2% to over 12%) following discontinuation of effective antivenoms and introduction of replacement products. In at least one case this was because the antivenom had been manufactured from IgG purified from horses immunised with venoms from Indian snakes–instead of venoms from African snakes [12]. Antivenom efficacy is predominantly restricted to snakes whose venoms were used in manufacture [13]–because the highly snake species-specific protein composition of venom dictates an equally specific IgG response in the immunised horses/sheep. Thus, the greater the biogeographic difference between the venom/s used in antivenom manufacture and the venom injected into the snakebite patient, the weaker the efficacy of the antivenom. For this reason, antivenom manufacturers are required to preclinically test and state the snake species for which their product is effective. Fig 1 evidences another antivenom marketed specifically for Central Africa but clearly inappropriately manufactured with venoms from Asian vipers. There are very few published reports on the clinical effectiveness of the several antivenoms in current use in sub-Saharan Africa. Preclinical efficacy data is therefore the only information available to physicians and government purchasers to decide which antivenom to use/purchase. However, many, perhaps the majority, of sub-Saharan African countries do not apparently subject newly-imported antivenoms to independent preclinical efficacy and safety testing, and clinicians and purchasers per-force base clinical use/purchase decision making upon manufacture-stated efficacy statements. The above reports of antivenom ineffectiveness and rising case fatalities, seemingly throughout much of sub-Saharan Africa, demonstrate this trust can be misplaced. There is therefore an urgent need to establish independent preclinical antivenom-efficacy testing facilities and expertise in sites throughout sub-Saharan Africa. With substantive new funding, the Liverpool School of Tropical Medicine has partnered with colleagues in Kenya, Nigeria and Cameroon to form the African Snakebite Research Group and established ‘Snakebite Research and Intervention Centres’ (SRIC) in each of these countries. Our remit includes improving the (i) availability of effective snakebite treatment in rural remote hospitals in greatest need and (ii) access to treatment for snakebite victims. To ensure this new programme is equipped with effective antivenom, and to provide the host government with independent antivenom-efficacy information, we purchased a vial of as many different antivenom brands as available from local pharmacies and preclinically tested their efficacy against venoms of the most medically important snakes in the region. This first report from the African Snakebite Research Group emanates from Kenya-SRIC activity and demonstrates, for the first time in East Africa, that there is substantial variation in the preclinical efficacy of the available antivenoms against the lethal effects of venoms from black mambas, spitting and non-spitting cobras, puff adders and saw-scaled vipers–and that no one antivenom is preclinically effective at the doses tested, against all these life-threatening snake venoms.
The antivenoms used in this study are described in detail in Table 2 and were acquired from a commercial pharmacy in Nairobi, except the SAIMR antivenoms that were donated to the first author from expired stocks held by Public Health England and had 2012 expiry dates. We were unsuccessful in purchasing one of the ASNA antivenoms manufactured by Bharat Serums and Vaccines Ltd that we had seen in a rural hospital in Kenya. All the antivenoms are manufactured as F (ab' ) 2 fragments of IgG, and for clarity to non-specialist readers we have used the term IgG to describe these antivenoms. The comparative preclinical efficacy of the ‘test’ antivenoms was conducted before their respective expiry dates. We were unable to purchase the SAIMR polyvalent and ECHIS CARINATUS monovalent ‘gold standard’ antivenom in Kenya and therefore used 2012-expired vials donated to us from Public Health England. SDS-PAGE profiling of these antivenoms (Supplementary S3 Fig) reveal that the IgG in these SAIMR antivenoms possess the same structural integrity as IgG from the ‘in date’ test antivenoms. Further validation of using these expired SAIMR antivenoms for this study is provided by the comprehensive binding of venom proteins by IgG in these antivenoms. East Africa is resident to multiple medically important vipers, elapids and colubrids. For this analysis, we selected venoms from the most relevant representative species of each genus. Venom was extracted from over four specimens of wild-caught puff adders (Bitis arietans, Kenya); saw-scaled vipers (Echis pyramidum leakeyi, Kenya); black mambas (Dendroaspis polylepis, Tanzania); Egyptian cobras (Naja haje, Uganda); black-necked spitting cobras (N. nigricollis, Tanzania) and red spitting cobras (N. pallida, Kenya) maintained in the Liverpool School of Tropical Medicine herpetarium (a UK Home Office accredited and inspected animal research facility). Freshly collected venom was snap frozen, lyophilised and stored as a powder at 4°C prior to reconstitution in phosphate-buffered saline (PBS). The same batches of these venoms were used for each of the analyses below to provide cross-experiment continuity. We employed routine protocols in our laboratory [14] to measure the IgG titre, avidity, venom protein-specificity and protein (IgG) concentration/ml antivenom to provide a detailed immunological profile of the ‘gold standard’ and ‘test’ antivenoms.
We used the WHO-recommended antivenom effective dose (ED50) assay, which measures the amount of antivenom required to prevent venom-induced lethality in 50% of mice (5/dose group) injected with venom/antivenom mixtures. To assess the efficacy of the SAIMR ‘gold standard’ polyvalent and monovalent antivenoms, we determined the ED50 dose of (i) the SAIMR ECHIS CARINATUS monovalent antivenom against only the saw-scaled viper venom and (ii) the SAIMR polyvalent antivenom against venoms of the black mamba, the Egyptian, red and black-necked spitting cobras and the puff adder (Table 4). We assigned the SAIMR antivenoms as the ‘gold standard’ comparators because of their sub-Saharan African clinical effectiveness [17] and because, unlike FavAfrique, these antivenoms are likely to be available for the foreseeable future. We elected a ‘gold standard comparison’ experimental design to achieve our objective instead of 24 conventional ED50 assays (4 ‘test’ antivenoms tested against 6 venoms), which would have required a minimum of 600 mice (25 mice/experiment). Instead, we tested the extent to which the ‘test’ antivenoms prevented the death of mice (5/dose group) injected with the venom/antivenom mixtures at volumes equivalent to half (0. 5x), equal (1x) or, where appropriate, 2. 5-fold more (2. 5x) the volume of the SAIMR antivenoms that impart 100% survival of the mice (calculated by doubling the ED50 dose volume). Thus, 100% survival of mice injected with 0. 5x volume of a ‘test’ antivenom indicates a higher venom-neutralising efficacy than the SAIMR ‘gold standard’ antivenoms, and a test antivenom providing less than 100% efficacy at 1x volumes would be deemed less dose-effective than the ‘gold standard’. Failure of an antivenom to neutralise the lethal effects of a venom at 2. 5x volumes in 100% of mice would raise serious concerns as to the potential clinical efficacy of that product. This protocol enabled us to comprehensively analyse the efficacy, compared to a gold standard, of several antivenoms against several venoms using only 40% of the number of mice had we used conventional ED50 testing (Fig 2). It is important to note that, for the sake of clarity for non-specialist readers, we have described the efficacy of the ‘test’ antivenoms relative to 0. 5x, 1x or 2. 5x volumes of the SAIMR antivenom that protect 100% of mice. This differs from the more conventional description as ED50 volumes (the volume of antivenom that protects 50% of the venom/antivenom injected mice), and antivenom preclinical testing practitioners are referred to Supplementary S1 Table that depicts the same data in Fig 2 in the context of antivenom volumes (μL) and amounts (mg). To ensure valid cross-comparison of antivenom-venom reactivity, we first standardised the IgG concentration of each antivenom to 5 mg IgG/ml. We next incubated serial dilutions of each antivenom with the same concentration of each venom (Fig 3). For space reasons we have excluded the graph showing the baseline reactivity of the naive control horse IgG to all the venoms. The OD readings of the antivenoms at the 1: 2,500 dilution, in the middle of the downward slope, provide the most immunologically meaningful comparison and, for clarity, are presented as tables in each panel of Fig 3. For example, at this dilution, the SAIMR ECHIS CARINATUS antivenom IgG shows (i) highest binding to the E. p. leakeyi venom, (ii) some cross-reactivity to other viper (puff adder) venom proteins, and (iii) near-zero binding to the four elapid venoms–results entirely consistent with an antivenom generated by immunisation with only Echis species venoms. While detectable venom-binding differences between the antivenoms exist, none of the antivenoms exhibited sufficiently poor IgG binding to venoms that account for the very poor polyspecific ED50 results of, for example, the VINS and INOSAN antivenoms (Fig 2). Thus, at the same IgG dilution (1: 2,500) the OD values (venom-binding) of VINS antivenom to all the venoms was greater or equivalent to that of the more preclinically effective Sanofi Pasteur antivenom. Furthermore, INOSAN’s inefficacy against D. polylepis venom contrasted with its higher OD to this venom than the considerably more efficacious Sanofi Pasteur antivenom. Finally, the OD values of the Premium Serums & Vaccines antivenom was consistently higher to all the venoms than the more efficacious SAIMR antivenoms. The ELISA IgG titration assay demonstrated that all the ‘test’ antivenoms contained venom-binding IgG titres not dissimilar to the ‘gold standard’ SAIMR antivenoms. Thus, while we identified many discreet IgG-venom binding differences between the antivenoms, we were unable to confidently attribute any of these as being responsible for the very different venom-neutralisation efficacies of these antivenoms. We therefore next performed an assay to identify whether the antivenoms possessed IgGs of variable avidity (binding strength) to the six different venoms that matched their distinct venom-neutralising efficacies. Ammonium thiocyanate (NH4SCN) is a potent disruptor of protein-protein binding (chaotrope) and, by measuring the ELISA OD readings of the same concentration of IgG and venom in the presence of increasing amounts of the chaotrope, is used to test antivenom IgG-venom protein binding strength. We incubated 1: 1,000 dilutions of each of the 5 mg/ml standardised antivenom solutions with the venoms (in the same concentration as for the IgG titre ELISA) and determined the OD readings after addition of 0,1, 2,4, 6 and 8 moles of NH4SCN (Fig 4; we have excluded the graph showing the baseline reactivity of the naive control horse IgG to all the venoms). The most immunologically-informative results were gained by comparing the percentage reduction in OD values of the antivenoms without NH4SCN to that in the middle of the downward slope at 4 M NH4SCN—as illustrated by the table inserted into each panel of Fig 4. This assay revealed that the Premium Serums & Vaccines (panel 4F) antivenom possesses the most consistent, and highest, cross-snake species IgG-venom binding avidity of all the antivenoms, including the ‘gold standard’ antivenoms. The INOSAN antivenom (panel 3E) exhibits the least consistent cross-species venom binding avidity. The chaotropic ELISA assay revealed closer links between antivenom IgG-venom binding avidity and antivenom efficacy than the IgG titre results. Thus, the notably higher IgG-binding avidity of the SAIMR polyvalent gold standard (panel 4A) to the B. arietans, N. haje and D. polylepis venoms, and lower avidity to the spitting cobra (N. nigricollis, N. pallida) venoms accurately reflect the venom-neutralising dose-efficacy of this antivenom (Table 4). Also, the substantially higher IgG avidity of the INOSAN antivenom to E. p. leakeyi venom also matched its venom-neutralising dose-efficacy profile. However, this assay did not provide data accounting for the snake-species distinct venom-neutralising efficacies of the Premium Serums & Vaccines, VINS and Sanofi Pasteur antivenoms, particularly in comparison with the superior venom-neutralising dose-efficacy of the SAIMR polyvalent antivenom against N. haje, D. polylepis and B. arietans venoms. Venoms consist of multiple distinct protein groups and the protein composition of venom is markedly snake genus/species specific–with obvious implications on the venom protein-specificity of IgGs from venom-immunised horses. Neither the IgG titre nor the IgG avidity ELISA assays are designed to examine IgG binding to specific venom proteins. We therefore next used an immunoblot assay to investigate whether the six antivenoms express differences in IgG venom protein specificities that match their distinct venom-neutralising efficacies. Fractionation of the six snake venoms by 15% SDS-PAGE revealed the numerical and molecular size diversity of the venom proteins (Fig 5A), with the cobra and mamba venoms (N. nigricollis, N. pallida, N. haje and D. polylepis) possessing a greater abundance of the low molecular mass neurotoxins/cytotoxins than the more evenly distributed molecular mass of the enzyme-rich, haemostasis-disruptive viper venoms (E. p. leakeyi and B. arietans). To determine the extent to which this wide spectrum of East African snake venom proteins are bound by IgG of the six antivenoms, we electrophoretically transferred these venom proteins to a membrane and incubated those (under identical conditions) with the antivenoms at 1: 5,000 dilutions (Fig 5). For this test, we did not adjust the antivenoms to a standard 5 mg/ml concentration. This analysis demonstrated that the intensity of the IgG-venom protein binding of the ‘gold standard’ SAIMR polyvalent and SAIMR ECHIS CARINATUS antivenoms was notably greater than all the ‘test’ antivenoms (Fig 5). It is important to note that, with minor brand-specific differences, that the difference between the ‘test’ and ‘gold standard’ antivenoms in this assay related to the intensity of venom protein binding and not the protein specificity. Thus, although the intensity of the IgG-venom protein binding was lower with the ‘test’ antivenoms, the immunoblots revealed that each antivenom possessed IgGs with similar venom protein specificities as the SAIMR ‘gold standard’ antivenoms. The lack of venom-reactivity of the control, naïve horse IgG (Fig 4B), evidences the venom-specificity of the antivenom IgGs. The three immune assays above identified detectable differences in the IgG titre, avidity and venom-protein specificities of the six antivenoms, but these differences were relatively minor and could not be consistently applied to the interaction of each antivenom with each venom. We were therefore unable to identify an IgG-venom binding deficiency with any of the ‘test’ antivenoms, and by inference a deficiency in their venom-immunisation protocols, that could account for the ineffectiveness/weak efficacy of some of the ‘test’ antivenoms in our preclinical assays. The substantially higher IgG-venom protein binding intensity of the SAIMR polyvalent antivenom in the immunoblot assay suggested to us that this antivenom may be formulated with a higher amount of IgG/vial than the others. Our final test was therefore to determine the protein (IgG) concentration (all antivenoms are formulated as F (ab’) 2 fragments of IgG) of each antivenom because this has an obvious bearing on dose-efficacy, and because it was not stated by any manufacturer, despite it being the active component of these therapies. We used a spectrophotometric instrument (NanoDrop) to measure protein content of each antivenom (in triplicate) and the results are presented in Table 5. We included control horse IgG of different known concentrations, to confirm the accuracy of the NanoDrop results. For the sake of completeness, we also used SDS-PAGE analysis of each antivenom to demonstrate their consistent IgG purity (Supplementary S3 Fig). This analysis demonstrated the substantial inconsistency in the total IgG content of the antivenoms, and, importantly, that the VINS and INOSAN antivenoms respectively contained 19% and 28% of the IgG concentration of the SAIMR polyvalent antivenom. The IgG content of antivenom therefore exhibited the closest association to their comparative efficacy in neutralising the venoms of N. nigricollis, N. pallida and E. p. leakeyi (Fig 2). It is not however a universally-applicable explanation, because, for example, IgG content alone does not explain the superior anti-B. arietans and anti-E. p. leakeyi efficacy of Premium Serums & Vaccines (63 mg/ml) antivenom over that of the Sanofi Pasteur (96. 7 mg/ml) antivenom. Assessing the antivenom efficacy data by mg antivenom did reveal that the anti-E. p. leakeyi venom efficacy of the INOSAN antivenom was equivalent to that of the SAIMR ECHIS CARINATUS ‘gold standard’ antivenom. Nevertheless, as stated above and depicted in Fig 2, it required 2. 5 times more the volume of the SAIMR antivenom for the INOSAN product to achieve this efficacy parity, which in the human-treatment context has cost and adverse effect implications. For completeness, we have presented the amount (mg) and volume (μl) of antivenom used for each of the ‘test’ antivenoms for each of the doses examined into Supplementary S1 Table. To facilitate comparison, we have also added the amount (mg) and volume (μl) of the calculated 2xED50 doses of the ‘gold standard’ antivenoms that provided 100% protection to the envenomed mice. This table therefore provides all the numerical data related to the preclinical assays, and interpretations of efficacy from this table is no different from that presented in Fig 2. No matter whether the data is examined by antivenom volume in μl or amount in mg, the least polyspecifically-effective antivenoms simply do not compare well to the ‘gold standard’ and to some of the other ‘test’ antivenoms. It was important that we tested the antivenoms by volume because that is the formulation in which the antivenom is provided by the manufacturer and used by the clinician. It is near impossible to envisage a clinician calculating the dose volume of antivenom he/she is going to administer based upon the mg/ml antibody content (and perhaps impossible because, as here, manufacturers rarely provide this information). Therefore, to ensure that our preclinical efficacy testing of the antivenoms is of value to clinicians and medicine-purchasing agencies in East Africa, we undertook this testing, and report the results by antivenom volume. The reality is that the efficacy of a monospecific antivenom to its homologous venom is dictated by multiple factors, including IgG concentration, titre, avidity and protein specificity, which are themselves affected by the quality of the immunising venoms, the quantitative ratio of the venoms used for immunisation, the selected adjuvant and other aspects of the immunisation and antivenom-manufacturing protocols. All these interlocking factors are made more complex, as here, by increasing the number of venoms used to manufacture polyspecific antivenom, making it very difficult/impossible to confidently assign any one factor as being primarily responsible for lack of efficacy.
The plight of snakebite victims, particularly those in sub-Saharan Africa has been the subject of considerable recent attention [see 6,8, 19,20,21] and the focus of recent Wellcome Trust and the Kofi Annan Foundation sponsored international meetings to identify remedial interventions. Reports from both meetings [4,5] identify the urgent need for the provision of effective antivenom and preclinical efficacy testing of existing and new antivenoms to ensure this. This recommendation aligns fully with the new WHO initiative to establish a prequalification programme for African antivenom, which includes establishing venom standards for sub-Saharan Africa and using those for preclinical antivenom-efficacy testing [22]. Over the past five years, we have generated a comprehensive inventory of sub-Saharan African snake venoms of defined gene and protein composition and murine toxicity [23,24,25]. This unique resource has enabled the Kenya Snakebite Research & Intervention Centre to conduct this first preclinical assessment of the efficacy of antivenoms available for clinical use in Kenya. This is important because the market failure of the Sanofi Pasteur antivenom and the very high costs of the SAIMR antivenoms leaves many East African countries with a choice of polyspecific antivenoms restricted to brands for which there is very little/no published data on their human effectiveness. Our preclinical results illustrate that the SAIMR polyvalent antivenom is considerably more effective in neutralising the murine lethality of the Egyptian cobra (N. haje), black mamba (D. polylepis) and puff adder (B. arietans) venoms than any of the ‘test’ antivenoms. The ‘test’ antivenoms exhibited a superior or equal dose-efficacy as the SAIMR polyvalent antivenom against the spitting cobra venoms (N. nigricollis and N. pallida). Preclinical neutralisation of the saw-scaled viper (E. p. leakeyi) venom was achieved by the Premium Serums & Vaccines and INOSAN antivenoms but required 2. 5-fold greater volumes than the SAIMR ECHIS monovalent antivenom. Perhaps the most important result of our study was that no single antivenom, at the doses tested (see below for a detailed consideration of this assay), was effective in neutralising the murine lethal effects of venoms of all six medically important snakes of East Africa—despite the pan-African efficacy claims inherent in the names of many of these products. The snake species-specific dose efficacy of each of the ‘test’ and ‘gold standard’ SAIMR polyvalent antivenoms suggests that the clinical management of envenoming by these snakes with any one of these antivenoms may require distinct, snake species-specific dose regimens. In the absence of a rapid snake species diagnostic test, this is clinically problematic and will likely result in the administration of too little or too much antivenom–both highly undesirable scenarios resulting in either inefficacy or increased risk of antivenom-induced adverse effects, respectively. On a more positive note, the preclinical efficacy of the Premium Serums & Vaccines product matched that of the highly regarded, but now unavailable Sanofi Pasteur product and approached that of the expensive SAIMR polyvalent antivenom. The Premium Serums & Vaccines product, at 26% of the cost of the SAIMR antivenoms (see Table 6), was the most affordable and effective antivenom of those tested here, although we note that preclinical efficacies against the two neurotoxic snake venoms (N. haje and D. polylepis) were weaker than against the other four species. Our results suggest that the preclinical efficacy of the VINS and INOSAN products could possibly be substantially improved by simply increasing the amount of IgG in each vial. The INOSAN product was the most expensive of the ‘test’ antivenoms, the most preclinically effective at neutralising the saw-scaled viper venom and one of the less effective antivenoms at neutralising the lethality of other snake venoms. It is notable that the IgG avidity of this antivenom to venoms of the six snakes varied more than the other antivenoms (Fig 4), and this avidity profile matched its snake-specific venom-neutralising efficacy. This may suggest that changes to the venom-immunisation regimen or analysis of the quality of the venoms could improve the venom-binding avidity and perhaps the efficacy of this antivenom. In consideration of the clinical efficacy of the SAIMR polyvalent and ECHIS monovalent antivenoms, it would be interesting to know whether SAVP has plans to combine the venom-immunising mixtures of these products to produce a truly pan-African polyspecific antivenom. We have carefully qualified our interpretation/extrapolation of the results of our preclinical ‘gold standard’ comparison assays to the efficacy of antivenom treatment of human snakebite patients, and urge readers to be similarly cautious. Our preclinical protocol enables a rapid efficacy comparison of a matrix of six venoms and four antivenoms using the minimum number of mice, but it differs from the recommended WHO antivenom-efficacy testing protocols in that it does not provide an ED50 value for each ‘test’ antivenom against each venom. Thus for example, we are unable to state whether, or not, the polyspecifically effectiveness of the VINS and INOSAN products could attain 100% efficacy against more of the venoms by using substantially greater volumes–because of the volume constraints inherent to this murine assay. From a human-treatment perspective, the necessity to administer multiple vials of an antivenoms carries important treatment costs (the INOSAN product was the most expensive ‘test’ antivenom) and adverse effects issues that risk poor uptake in rural tropical regions. On a more general note, these murine preclinical antivenom-efficacy testing assays are not infallible predictors of human efficacy. A recent study in Sri Lanka has questioned the value of predicting efficacy outcomes in human patients from ED50 results [26]. Conversely, the undoubted ability of the ED50 test to discriminate between effective and ineffective antivenoms in the murine model [13,27], and the effective use of ED50 data [15] to help design the dose regimen of a human antivenom clinical trial [28] suggests that while the ED50 preclinical test has inadequacies many practitioners share, it should be retained until more accurate assays are tested, validated and become available. Thus, while the results of this study identify a potentially serious therapeutic concern, one of the priorities of the Kenya, Nigerian and Cameroon Snakebite Research & Intervention Centres will be to undertake conventional ED50 tests so that the Ministries of Health can be provided with pharmacopoeia-compliant data that they can act upon to restrict human use of preclinically-ineffective antivenom. The dialogue above underscores the urgent need for more published data on the efficacy of the different antivenom brands in use in sub-Saharan Africa to treat human patients. The paucity of this information reflects the prohibitive expense, problems of recruiting sufficient patients envenomed by the myriad of venomous snake species, the lack of accurate diagnostic tools to distinguish such species-distinct envenoming, and time required to conduct full clinical trials. Until the funding and required clinical/diagnostic tools become available, we remain reliant upon clinical observation studies that have importantly identified the wide-spread use of some dangerously ineffective antivenoms [9–11]. However, only the first of these reports provided the clinically-vital information on the antivenom brand. Another objective of the recently-established African Snakebite Research Group will be to conduct surveys in many rural hospitals in Nigeria, Kenya and Cameroon experiencing high snakebite admissions. The outcomes of these surveys will include reporting antivenom availability, and assessments of the clinical outcomes of treating patients with the various brands of locally-available antivenom. In conclusion, this first report of the African Snakebite Research Group identifies a worrying differential in the preclinical efficacy of available antivenoms in Kenya and underscores the need for independent preclinical testing of antivenoms throughout sub-Saharan Africa, and the need for venom standards for all the most medically important snakes to resource this testing. There is also an urgent need for practitioners to publish data on the clinical outcomes of treating patients with brand-named antivenoms (despite the inherent problem of such transparency). The widespread availability of pan-African preclinical testing and clinical observation information will substantially help to improve the effectiveness of snakebite management and thereby reduce the high case fatality currently suffered by sub-Saharan African snakebite victims.
Snakebite is one of the most under-researched, under-resourced high morbidty/high mortality NTDs, as reflected by the fact that many of the antivenoms used to treat snakebite victims in sub-Saharan Africa are of uncertain and untested efficacy. This Kenya case study is the first examination of the preclinical efficacy of all available antivenoms to neutralize the venom toxic effects of the most medically important snakes in any region of sub-Saharan Africa. Our results identify serious preclinical efficacy limitations in two of the most commonly used antivenoms, that no single antivenom is effective against all regionally important snakes and that the least effective antivenoms had the lowest IgG concentrations. It is our aim that Ministry of Health medicine-supply regulators can use this data as evidence to demand more detailed efficacy evidence from manufacturers, and to justify the establishment of national/regional preclinical testing facilities. We hope this publication will also alert physicians treating African snakebite victims to check the efficacy of antivenom in their pharmacies. We have carefully qualified the extent and limitation of the results and of our interpretation of the clinical implications thereof.
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Preclinical antivenom-efficacy testing reveals potentially disturbing deficiencies of snakebite treatment capability in East Africa