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. 2017 Oct 18;11(10):e0005969.
doi: 10.1371/journal.pntd.0005969. eCollection 2017 Oct.

Preclinical antivenom-efficacy testing reveals potentially disturbing deficiencies of snakebite treatment capability in East Africa

Affiliations

Preclinical antivenom-efficacy testing reveals potentially disturbing deficiencies of snakebite treatment capability in East Africa

Robert A Harrison et al. PLoS Negl Trop Dis. .

Erratum in

Abstract

Background: 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.

Methods: 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.

Findings: 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.

Conclusions: 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.

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Conflict of interest statement

None of the authors have competing interests.

Figures

Fig 1
Fig 1. Concerns over an antivenom widely marketed in sub-Saharan Africa.
Why are venoms from the Asian Russell’s viper (Daboia russelii—incorrectly labelled here as Vipera russelli) and saw-scaled viper (Echis carinatus) included in its efficacy statement?.
Fig 2
Fig 2. Variant efficacy of the ‘test’ antivenoms against the lethal effects of the six East African venoms in a mouse model.
The percent survival of mice injected with lethal doses of venom mixed with the ‘test’ antivenoms in volumes equivalent to half (0.5), the same (1x) or 2.5-fold more (2.5x) of the 100% effective volume of the SAIMR ‘gold standard’ antivenoms was used to identify whether the ‘test’ Premium Serums & Vaccines, VINS, INOSAN and Sanofi Pasteur antivenom were ineffective (red bar), partially effective (yellow bar), effective but requiring a higher dose of antivenom than the SAIMR products (light green bar), an equivalent dose efficacy as the SAIMR products (dark green bar) or a superior dose efficacy than the SAIMR antivenoms (blue bar). The test venoms were East African puff adders (B. arietans; 97.8 μg), saw-scaled vipers (E. p. leakeyi; 80.5 μg), black necked spitting cobras (N. nigricollis; 61.0 μg), red spitting cobras (N. pallida; 46.5 μg), Egyptian cobras (N. haje; 40.8 μg) and black mambas (D. polylepis; 30.8 μg). Please refer to Supplementary S1 Table for details on antivenom volumes, amounts of antivenom in mg, snake geographical origin and venom LD50 dose used for each experimental group. * Mice died from the high density of antivenom/venom complexes that precipitate out of solution, not from venom-induced effects. With experience, the symptoms of this cause of death can be readily distinguished from that caused by venom. This occurs occasionally in murine preclinical testing as a consequence of the 30 minute, 37°C incubation of the venom/antivenom mixture prior to injection. It likely has no clinical relevance, but can obfuscate preclinical results.
Fig 3
Fig 3. The IgG reactivity (titre) of six commercial antivenoms against six sub-Saharan Africa venoms determined by titration ELISA.
All antivenoms were adjusted to 5 mg/ml in PBS prior to being diluted 1 in 100 and then serially diluted 1 in 5 and applied to venoms. Panel (A) = SAIMR polyvalent, (B) = SAIMR ECHIS CARINATUS, (C) = Sanofi Pasteur, (D) = VINS (E) = INOSAN, (F) = Premium Serums & Vaccines (PS&V) antivenoms. Venoms: B. arietans (blue), E. p. leakeyi (red), N. nigricollis (green), N. pallida (purple), N. haje (orange) and D. polylepis (black). Results are the mean of three replicates with error bars representing standard deviation (SD). Error bars are not shown where SD is smaller than data point. The reactivity of control, naïve horse IgG to each venom was consistently low at each dilution for each venom (mean OD 405 nm = 0.07 ± 0.005). For interested readers, the same data as in Fig 3 is presented in Supplementary S1 Fig to facilely compare binding to each of the six venoms.
Fig 4
Fig 4. The relative IgG-venom binding avidity of six commercial antivenoms against six sub-Saharan Africa venoms determined by chaotropic ELISA.
1:1,000 dilutions of 5 mg/ml standardized antivenoms were allowed to bind to venom coated plates before being exposed to NH4SCN at increasing concentrations for 15 minutes. Panel (A) = SAIMR polyvalent, (B) = SAIMR ECHIS CARINATUS, (C) = Sanofi Pasteur, (D) = VINS (E) = INOSAN, (F) = Premium Serums & Vaccines (PS&V) antivenoms. Venoms: B. arietans (blue), E.p. leakeyi (red), N. nigricollis (green), N. pallida (purple), N. haje (orange) and D. polylepis (black). Results are the mean of three replicates with error bars representing standard deviation (SD). Error bars are not shown where SD is smaller than data point. The reactivity of control, naïve horse IgG to each venom was consistently low at each dilution for each venom (mean OD 405 nm = 0.05 ± 0.004). For interested readers, the same data as in Fig 4 is presented in Supplementary S2 Fig to facilely compare binding to each of the six venoms.
Fig 5
Fig 5. The IgG specificities of six commercial antivenoms to venom proteins of six sub-Saharan Africa venoms.
Venoms (10 μg) of B. arietans, E. p. leakeyi, N. nigricollis, N. pallida, N. haje and D. polylepis were separated by reduced SDS-PAGE and visualised by coomassie blue staining (Panel A). Venom proteins in identical gels were transferred to nitrocellulose blots and incubated with 1:5,000 dilutions of naïve Horse IgG (B), the ‘gold standard’ SAIMR polyvalent (C) and SAIMR ECHIS CARINATUS (D) antivenoms, and the ‘test’ Sanofi Pasteur (E), VINS (F), INOSAN (G) and Premium Serums & Vaccines (H) antivenoms. The antivenoms were not standardised to 5 mg/ml as in the ELISA assays.

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