A Cytotoxicity Assay as an Alternative to the Murine Model for the Potency Testing of Bothrops jararaca Venom and Antivenom: An Intralaboratory Pre-validation Study.

Antivenom therapy is the only specific treatment for snakebite envenomation, and antivenom potency determination is key in the efficacy assurance quality control process. Nowadays, this process relies on the in vivo murine model - thus, the development of alternative in vitro methods is imperative. In the current study, the principle of the proposed method is the ability of Bothrops venom to induce cytotoxic effects in Vero cells, and the capacity to evaluate the inhibition of this cytotoxicity by the respective antivenom. After exposure to the venom/antivenom, the relative proportions of adherent (viable) cells were evaluated by direct staining with Coomassie Blue. The optical density (OD) of the lysed cell eluate was directly proportional to the number of adherent cells. This cytotoxicity-based alternative method could represent a potential candidate for validation as a replacement for the current in vivo test. The in vitro-determined cytotoxicity of the Brazilian Bothrops reference venom (expressed as the 50% effective concentration; EC50) was 3.61 µg/ml; the in vitro-determined 50% inhibitory concentration (IC50) of the Brazilian Bothrops reference antivenom was 0.133 µl/ml. From these two values, it was possible to calculate the potency of the reference antivenom. The results from the assays exhibited a good linear response, indicating that the method could be a potential candidate replacement method for use in antivenom quality control prior to lot release, subject to further validation.

Analysis of commercially available snake antivenoms reveals high contents of endotoxins in some products.

As injectable therapeutics, snake antivenoms must meet specifications for endotoxin content. The Limulus amebocyte lysate (LAL) test was used to evaluate the endotoxin content in several commercially available antivenoms released for clinical use. It was found that some products have endotoxin concentrations higher than the accepted limit for these contaminants. These results emphasize the need to include endotoxin determination as part of the routine evaluation of antivenoms by manufacturers and regulatory agencies.

Intrageneric cross-reactivity of monospecific rabbit antisera against venoms of mamba (Elapidae: Dendroaspis spp.) snakes.

Snakebite envenomation is a neglected tropical disease posing a high toll of mortality and morbidity in sub-Saharan Africa. Polyspecific antivenoms of broad effectiveness and specially designed for this region require a detailed understanding of the immunological features of the mamba snake (Dendroaspis spp.) venoms for the selection of the most appropriate antigen combination to produce antivenoms of wide neutralizing scope. Monospecific antisera were generated in rabbits against the venoms of the four species of mambas. The toxic effects of the immunization scheme in the animals were evaluated, antibody titers were estimated using immunochemical assays, and neutralization of lethal activity was assessed. By the end of the immunization schedule, rabbits showed normal values of the majority of hematological parameters tested. No muscle tissue damage was noticed, and no alterations in most serum chemical parameters were observed. Immunological analyses revealed a variable extent of cross-reactivity of the monospecific antisera against the heterologous venoms. The venoms of D. jamesoni and D. viridis generated the antisera with broader cross-reactivity by immunochemical parameters. The venoms of D. polylepis and D. viridis generated the antisera with better cross-neutralization of lethality, although the neutralizing ability of all antisera was lower than 0.16 mg venom/mL antiserum against either homologous or heterologous venoms. These experimental results must be scaled to large animal models used in antivenom manufacture at industrial level to assess whether these predictions are reproducible.

Kounis syndrome after anti-snake venom intradermal skin test: A case report.

Snake bites are a concerning health problem in Egypt and other tropical countries that are effectively managed with anti-snake venom (ASV). ASV has common reactive complications that are usually mild. Rarely, anaphylaxis and severe systemic reactions may occur following ASV. In this case report, we present a rare condition of Kounis syndrome, acute allergic myocardial infarction, in a young man during anaphylaxis following an allergic intradermal test of VACSERA snake antivenom.

Advanced approaches for endotoxin detection and removal from snake antivenoms.

A comprehensive assessment of the literature on strategies for the detection and removing endotoxin from biotechnological preparations was conducted. This study highlighted the brief history of endotoxin. After that, a review of endotoxin's chemical and physical features, as well as its pathophysiological consequences when the body is exposed to LPS excessively or systemically, is presented. The procedures for determining endotoxin and the interaction of endotoxin with proteins are also discussed, considering both known approaches and cutting-edge technology in this sector. This review presented the endotoxin detection and removal approaches from antisera with an economical approach using several processes documented in the literature (e.g., adsorption, ultrafiltration, and chromatography). Different methods with relatively high protein recoveries are mentioned. This review concludes that heat activation at 70 °C-80 °C for 10 min and rehydration of the LAL reagent with endotoxin-specific buffer solution is the best technique to control the enhancement problem when testing polyvalent snake venom antiserum samples by the LAL method. The most efficient method for eliminating endotoxins has proven to be affinity resin-based chromatography.

Snakebites in Northeastern Brazil: accessing clinical-epidemiological profile as a strategy to deal with Neglected Tropical Diseases

ABSTRACT Background: Brazil ranks first in the number of snakebites in South America. A detailed analysis of these cases is required to improve the public health planning. In this study, we retrospectively examined the clinical and epidemiological profiles of snakebites in Maranhão between January 2009 and December 2019. Methods: Data were obtained from the compulsory notification forms provided by the Health Department of Maranhão. Results: A total of 17,658 cases were recorded during the study period. Most of the bites were from snakes belonging to the genus Bothrops. Medical care was mostly within three hours after the bite. Most cases were classified as mild and most victims recovered; however, 139 deaths were recorded. Most bites occurred among people aged 20-39 years, mainly among rural workers. The most frequent local clinical manifestations were pain, edema, and ecchymosis. The most common systemic clinical manifestations include neuroparalysis, vagal syndrome, and myolysis. Most snakebites occurred between January and March. The municipalities with the highest number of notifications were Buriticupu (936 cases), Arame (705 cases), and Grajaú (627 cases). Conclusions: The clinical profile of snakebites in Maranhão is similar to that observed in other states of Northeast Brazil. However, we found that some systemic manifestations are not compatible with the etiology of snakebites, which leads us to believe that the problem could be the lack of knowledge of the health professionals at the site of envenomation, who may not be ready for attendance, and an important lack of health centers with snake antivenom to treat snakebites.

The Clinical Usefulness of Taiwan Bivalent Freeze-Dried Hemorrhagic Antivenom in Protobothrops mucrosquamatus- and Viridovipera stejnegeri-Envenomed Patients.

Snakebites from Protobothrops mucrosquamatus (Taiwan habus) and Viridovipera stejnegeri (green bamboo vipers) account for the most venomous snakebites in Taiwan. The bivalent freeze-dried hemorrhagic (FH) antivenom is employed to treat these two snakebite patients without a strict clinical trial. We evaluated the clinical usefulness of Taiwan bivalent freeze-dried hemorrhagic (FH) antivenom in Taiwan habu- and green bamboo viper-envenomed patients. We checked ELISA- based serum venom antigen levels before and after FH antivenom to evaluate FH's ability to neutralize patients' serum snake venom and its usefulness in reducing limb swelling after snakebites. Patients who had higher serum venom antigen levels had more severe limb swelling. Of the 33 enrolled patients, most of their snake venom antigen levels were undetected after the appliance of antivenom. Most enrolled patients (25/33) had their limb swelling subside within 12 h after antivenom treatment. The failure to reduce limb swelling was probably due to an inadequate antivenom dose applied in more severely envenomated patients. Our data indicate the feasibility of the FH antivenom in effectively eliminating venom and resolving the affected limb swelling caused by Taiwan habu and green bamboo viper bites.

Antivenoms, hepatitis B vaccine and oral polio vaccine can be considered for storage and handling outside the cold chain following the innovative 'controlled temperature chain' approach.

Possible applicability of controlled temperature chain (CTC) for selected antisera and vaccines was evaluated. Bivalent oral polio vaccine (OPV), hepatitis B vaccine (HepB vaccine; monovalent and combined) and antisera (lyophilized and liquid scorpion-antivenom and liquid snake-antivenom) were tested. Samples were stored at accelerated (35 ± 5 °C) and freezing (-25 ± 5 °C) conditions for 24 h, one week and one month in addition to recommended storage condition (2-8 °C), except OPV samples that were tested at accelerated and refrigerated (2-8 °C) conditions compared to recommended storage conditions (-25 ± 5 °C). All samples were tested for potency. Protein content and composition were determined for antisera samples. All vaccine vial-monitors were evaluated. HepB vaccine was subjected to aluminum-content assay, shake test and microscopical examination. No significant change in antisera potency was detectable under accelerated condition for a week. OPV stored in refrigerator for a month and at accelerated condition for 48 h maintained acceptable potency. Monovalent and combined HepB vaccine maintained acceptable potency under accelerated condition for a month and a week, respectively. Freezing adversely affected HepB vaccine. In conclusion, reevaluation of storage conditions of tested products is urgently required; this can reduce storage costs and improves their availability. Other products should be tested for possible CTC applicability.

Venom-induced consumptive coagulopathy leading to thrombotic microangiopathy following Echis carinatus sochureki bite: is snake antivenom effective?

The incidence of thrombotic microangiopathy (TMA) following snake bite is reported to be ranging from 3.6 to 15%. We report a 10-year-old boy who developed TMA and due to venom-induced consumptive coagulopathy (VICC) despite receiving adequate and timely doses of snake antivenom following a bite of saw-scaled viper (Echis carinatus sochureki). VICC was managed by plasmapheresis. Though snake bite envenomation-associated renal complications are not uncommon, possibility of TMA should be considered early during management. Our patient developed TMA with subsequent acute cortical necrosis after saw-scaled viper bite despite an adequate and timely dose of snake antivenom which emphasizes the ineffectiveness of antivenom against the venom of given snake species leading to long-term complications.

Assessing a 6-h endpoint observation time in the lethality neutralization assay used to evaluate the preclinical efficacy of snake antivenoms.

The lethality neutralization assay in mice is the gold standard for the evaluation of the preclinical efficacy and specification fulfillment of snake antivenoms. However, owing to the animal suffering involved, this assay is a candidate to be replaced by in vitro alternatives or, at least, improved by the reduction of the number of animals used per experiment, the introduction of analgesia, and the refinement of the test. Since these tests are usually run for 24 or 48 h, one possibility to refine it is to shorten the endpoint observation time of the assay and so limiting the duration of suffering. To assess the effect of this modification of the standard procedure on the analytical properties of the assay, we compared the median lethal dose (LD50) and median effective dose (ED50) values, estimated through observation times of 6, 24 and 48 h. We used African and Latin American snake venoms and several batches of two polyspecific antivenoms. A significant correlation was found between LD50 and ED50 values estimated at the three observation times. Although some LD50 and ED50 values were significantly different at these time points, results of 6 h were robust enough to be used in the characterization of new antivenoms, the verification of specification compliance, and the parallel comparison of formulations. Our observations support the modification of the standard procedures used for assessing neutralizing ability of antivenoms by carrying out the observations at 6 h instead of 24 or 48 h, with the consequent reduction in the suffering inflicted upon mice during these assays. However, the shortening of the observation time in the lethality tests must be validated for each venom and antivenom before its introduction in the routine procedures.

Influence of sexual dimorphism on venom composition in Bothrops asper and Crotalus simus (Serpentes: Viperidae) and its potential implications on the snake antivenom production.

This work is aimed to bring insights on the potential sexual dimorphism differences on the venom composition of Bothrops asper and Crotalus simus to expand the knowledge of the venom variability that might improve the antivenom design. Biological characterization of venoms of each sex in both species did not show significant qualitative differences. Considerations on the sexual venom variations in these species are not relevant for choosing the snake donors for venom production.

Snake antivenom production in Ecuador: Poor implementation, and an unplanned cessation leads to a call for a renaissance.

Snakebite envenomation is a global health problem. This health problem asymmetrically affects rural populations in developing countries to such an extent that it recently has been listed as a priority neglected tropical disease (NTD). It is estimated that 5.4 million individuals are bitten by snakes each year, causing at least 2.7 million envenomations and more than 100,000 deaths each year. Ecuador has one of the highest snakebite envenomation incidence rates in Latin America, mostly in the coastal and Amazonian provinces. Envenomations in these regions are the result of bites primarily by species of snakes belonging to the Viperidae family. Ecuador was able to locally produce antivenoms, however serious flaws were revealed in the antivenom production process, leading to the decommissioning of the existing facility. In the interest of public health, we have summarized the political and social setbacks experienced by the antivenom serum production plant in Ecuador, while encouraging resuming local production of snake antivenom to improve the responsiveness of the already overburdened health system.

Reports of Doses Administered and Adverse Reactions to Snake Antivenom Used in Uruguay in 2018.

In Uruguay, around 60 cases of snakebite accidents occur every year that need to be treated with specific antivenom. They are caused by two snakes of Bothrops genus: Bothrops alternatus and Bothrops pubescens. Snakebite accidents are mandatory notification events, allowing the acquisition of an accurate registry and a fluent communication with the health care services. The aim of this study is to analyze and report the doses administered to achieve the neutralization of the venom and the adverse reactions caused by snake antivenoms used in Uruguay in 2018, when a change was made in the type of antivenom available. In this year, Uruguay started to use the BIOL® antivenom (lyophilized) and this use coexists with traditional antivenom liquid forms (Vital Brazil and Malbran). The number of patients treated with heterologous BIOL® antivenom were 28 and the ones treated with heterologous solutions Malbran and Vital Brazil antivenoms were 21. The initial dose of BIOL antivenom was 8 vials instead of 4 vials regularly used with the others antivenoms and it achieved the neutralization of most cases (27/28 cases). Early adverse reactions were detected in 4 patients (3 in children) treated with BIOL antivenom and there were no adverse reactions in those treated with Malbran or Vital Brazil antivenoms. Lyophilized antivenom BIOL is being used widely in Uruguay without major complications.

In vitro anti-venom potential of various solvent based leaf extracts of Andrographis serpyllifolia (Rottler ex Vahl) Wight against Naja naja and Daboia russelli.

ETHNOPHARMACOLOGICAL RELEVANCE: Snake bite is a major occupational hazard in tropical and subtropical countries including India as per the World Health Organization. Naja naja (Indian cobra) and Daboia russelli (Russell's viper) are the two poisonous snakes commonly associated with human mortality in India. Andrographis serpyllifolia (Rottler ex Vahl) Wight has been documented in ethnobotanical records as a plant possessing potent anti-snake venom activity. AIM OF THE STUDY: The present study is aimed for systematic evaluation of in vitro anti-venom potential of various solvent based leaf extracts of A. serpyllifolia against toxic venom enzymes of Naja naja and Daboia russelli. MATERIALS AND METHODS: Different solvent based leaf extracts of A. serpyllifolia were tested against the snake venoms of Naja naja and Daboia russelli obtained from Irula Snake Catchers Industrial Co-operative Society Limited, Kancheepuram, Tamil nadu, India. Three different in vitro neutralization assays such as indirect hemolysis, procoagulent and lytic activities and seven in vitro enzyme inhibition assays such as protease, acetylcholinesterase, phosphomonoesterase, phosphodiesterase, 5'nucleotidase, phospholipase A2, hyaluronidase and post synaptic acetylcholine receptor binding activity were carried out according to standard protocols. The results were analyzed using the standard ANOVA procedures. RESULTS: Among various solvent based leaf extracts of A. serpyllifolia tested, aqueous extract showed maximum neutralizing and inhibitory activities against Naja naja and Daboia russelli venoms. CONCLUSIONS: The various in vitro enzymatic studies reveal that the aqueous leaf extract of A. serpyllifolia plant could inhibit most of the toxic enzymes of the Naja naja and Daboia russelli venoms which could be further confirmed by in vivo studies.

Standardization of the first Korean national reference standard for snake (Gloydius brevicaudus) antivenom.

In 2017, the second national reference standard (NRS) for Gloydius snake venom was established to replace the first NRS for Gloydius snake venom. In connection with the second venom NRS, a candidate for the first NRS for Gloydius snake antivenom was produced in 2017. In this study, the qualification of the candidate was estimated and the potency was determined by a collaborative study. The potency (anti-lethal titer and anti-hemorrhagic titer) of the candidate was determined by measuring the capability of the antivenom to neutralize the lethal and hemorrhagic effects of the second NRS for Gloydius snake venom, which was calibrated against the regional reference standard for Gloydius snake antivenom established in 2006. Two Korean facilities contributed data from 20 independent assays. Subsequently, one foreign national control research laboratories participated in this collaborative study. The general common potency of the anti-lethal and anti-hemorrhagic titers was obtained from the results of a total of 25 tests performed at three facilities. According to the results of the present study, the candidate preparation showed good quality and is judged to be suitable to serve as the first NRS for Gloydius snake antivenom with the following potency: an anti-lethal titer of 3100 unit (U) (95% confidence interval 2991-3276 U) and anti-hemorrhagic titer of 3000 U (95% confidence interval 2849-3159 U). In conclusion, the first NRS for Gloydius snake antivenom was established in this study. This reference standard will be used routinely for quality control of a snake antivenom product by manufacturer in Korea, which also can be used for national quality control, including a national lot-release test of the snake antivenom product.

Envenomation by Trimeresurus stejnegeri stejnegeri: clinical manifestations, treatment and associated factors for wound necrosis.

BACKGROUND: Trimeresurus stejnegeri stejnegeri bite induces tissue swelling, pain, thrombocytopenia, rhabdomyolysis, and acute renal failure. However, the incidence of coagulopathy, factors associated with wound necrosis, and the appropriate management of this condition have not been well characterized yet. MATERIALS: This study included patients bitten by T. s. stejnegeri that were admitted to the study hospitals from 2001 to 2016. Patient characteristics, laboratory data, and management approaches were compared in victims with and without wound necrosis. RESULTS: A total of 185 patients were evaluated: three patients (1.6%) were asymptomatic; whereas tissue swelling and pain, local ecchymosis, wound necrosis, coagulopathy, thrombocytopenia, rhabdomyolysis, and renal impairment were present in 182, 53, 13, 15, 10, 1, and 3 patients, respectively. One patient died from coagulopathy and hemorrhagic shock. Antivenom was administered to all envenomed patients at a median time of 1.8 h after the bite. The median total dose of antivenom was five vials. Chi-square analysis showed that bitten fingers, using cold packs during first aid, presence of bullae or blisters, lymphangitis or lymphadenitis, local numbness and suspected infection to be significantly associated with wound necrosis. After adjustment using a multivariate logistic regression model, only cold packs as first aid, bulla or blister formation, and wound infection remained significant. CONCLUSIONS: The main effects of T. s. stejnegeri envenomation are tissue swelling, pain, and local ecchymosis. We do not recommend the use of cold packs during first aid to reduce wound pain, as this may be a risk factor for wound necrosis. In addition, patients with bulla or blister formation should be carefully examined for subsequent wound necrosis. Antiplatelet use may worsen systemic bleeding. No severe rhabdomyolysis or renal failure was observed in this large case series, we therefore considered that they were not prominent effects of T. s. stejnegeri bite.

Serum IgE against galactose-alpha-1,3-galactose is common in Laotian patients with snakebite envenoming but not the major trigger for early anaphylactic reactions to antivenom.

Snake antivenom is the only specific treatment for snakebite envenoming, but life-threatening anaphylaxis is a severe side effect and drawback for the use of these typically mammalian serum products. The present study investigates the hypotheses whether serum IgE antibodies against the epitope galactose-alpha-1,3-galactose (α-gal) located on the heavy chain of non-primate mammalian antibodies are a possible cause for hypersensitivity reactions to snake antivenom. Serum samples from 55 patients with snakebite envenoming were obtained before administration of snake antivenom and tested for serum IgE (sIgE) against α-gal and total IgE. Early anaphylactic reactions (EARs) during the first 3 h after antivenom administration were classified into mild, moderate or severe and correlated with the presence of sIgE against α-gal. Fifteen (27%) out of 55 patients (37 male, 18 female, median 34 years, range 9-90 years) developed EARs after antivenom administration. Eleven, three and one patients had mild, moderate and severe EARs, respectively. Serum IgE against α-gal was detected in 17 patients (31%); in five (33%) out of 15 patients with EARs and in 12 (30%) out of 40 patients without EAR (Odds Ratio = 1.2; 95%-confidence interval: 0.3-4.2) with no correlation to severity. Although the prevalence of serum IgE against α-gal was high in the study population, very high levels of total IgE in the majority of patients question their clinical relevance and rather indicate unspecific sIgE binding instead of allergy. Lack of correlation between α-gal sIgE and EARs together with significantly increased total IgE levels suggest that sIgE against α-gal is not the major trigger for hypersensitivity reactions against snake antivenom.

Cobra snakebite mimicking brain death treated with a novel combination of polyvalent snake antivenom and anticholinesterase.

In toxicology literature, snake bites were the second toxicology-relevant cause mimicking brain death. A 57-year-old woman with history of cobra snake bite. On examination, the brain stem reflexes were absent with Glasgow coma score of 3. The patient accomplished full neurological recovery after using a novel combination of Polyvalent Snake Antivenom (PSA) and anticholinesterases. This case highlights a unique presentation of cobra bite induced brain death mimicking. Thus, intensivist should exclude neuroparalytic effect of snakebite before considering withdrawal of ventilatory support or organ donation. Also, the life-threatening presentation of cobra envenomation mandates the use of higher doses of PSA to reverse the neuroparalytic toxicity. We should consider the rule of anticholinesterase as an adjunctive therapy to PSA in severe cobra envenomation.

Red-bellied black snake (Pseudechis porphyriacus) envenomation in 17 dogs: clinical signs, coagulation changes, haematological abnormalities, venom antigen levels and outcomes following treatment with a tiger-brown snake antivenom.

BACKGROUND: This report describes 17 cases of red-bellied black snake envenomation (RBBS; Pseudechis porphyriacus) in dogs in south-eastern Queensland. Patients were prospectively enrolled for the treatment with a new tiger-brown snake antivenom 8000 units, (TBAV; Padula Serums Pty Ltd, VIC, Australia). CASE REPORT: Clinical diagnosis of RBBS envenomation was made by either snake venom detection kit, snake identification using scale counting, or owner observed dog-snake interaction in patients with clinical signs of envenomation. An RBBS venom antigen sandwich ELISA was used to retrospectively quantify venom levels in frozen serum and urine. Mechanical ventilation was required in 11% (2/17) patients, whole blood transfusion in 12% (2/17), tissue swelling at the bite site occurred in 53% (9/17) and facial palsy in 12% (2/17). One dog was euthanised, and overall, 94% (16/17) survived to hospital discharge. Clinicopathological changes pre-TBAV included variable haemolysis, increased CK, pigmenturia and mildly prolonged active clotting time with a median of 134 s (n = 13, range 91-206 s). Haematological profiles post envenomation revealed anaemia (6/6) and spherocytosis (5/5), which resolved without the use of corticosteroids. Pre-TBAV, median RBBS venom antigen concentration was 22.6 ng/mL (n = 15, range 2-128) in serum and 58 ng/mL (range 1-452) in urine; RBBS venom antigen was undetectable in serum post-TBAV in all patients. CONCLUSION: Some RBBS envenomed dogs required, critical care including mechanical ventilation, blood transfusion, additional antivenom and prolonged hospitalisation. TBAV was effective with excellent prognosis despite stated specificity for tiger and brown snake.

Envenomation by Trimeresurus stejnegeri stejnegeri: clinical manifestations, treatment and associated factors for wound necrosis

Trimeresurus stejnegeri stejnegeri bite induces tissue swelling, pain, thrombocytopenia, rhabdomyolysis, and acute renal failure. However, the incidence of coagulopathy, factors associated with wound necrosis, and the appropriate management of this condition have not been well characterized yet. Materials: This study included patients bitten by T. s. stejnegeri that were admitted to the study hospitals from 2001 to 2016. Patient characteristics, laboratory data, and management approaches were compared in victims with and without wound necrosis. Results: A total of 185 patients were evaluated: three patients (1.6%) were asymptomatic; whereas tissue swelling and pain, local ecchymosis, wound necrosis, coagulopathy, thrombocytopenia, rhabdomyolysis, and renal impairment were present in 182, 53, 13, 15, 10, 1, and 3 patients, respectively. One patient died from coagulopathy and hemorrhagic shock. Antivenom was administered to all envenomed patients at a median time of 1.8 h after the bite. The median total dose of antivenom was five vials. Chi-square analysis showed that bitten fingers, using cold packs during first aid, presence of bullae or blisters, lymphangitis or lymphadenitis, local numbness and suspected infection to be significantly associated with wound necrosis. After adjustment using a multivariate logistic regression model, only cold packs as first aid, bulla or blister formation, and wound infection remained significant. Conclusions: The main effects of T. s. stejnegeri envenomation are tissue swelling, pain, and local ecchymosis. We do not recommend the use of cold packs during first aid to reduce wound pain, as this may be a risk factor for wound necrosis. In addition, patients with bulla or blister formation should be carefully examined for subsequent wound necrosis. Antiplatelet use may worsen systemic bleeding. No severe rhabdomyolysis or renal failure was observed in this large case series, we therefore considered that they were not prominent effects of T. s. stejnegeri bite.(AU)