Neotropical Rattlesnake (Crotalus simus) Venom Pharmacokinetics in Lymph and Blood Using an Ovine Model.

The most abundant protein families in viper venoms are Snake Venom Metalloproteases (SVMPs), Snake Venom Serine Proteases (SVSPs) and Phospholipases (PLA2s). These are primarily responsible for the pathophysiology caused by the bite of pit-vipers; however, there are few studies that analyze the pharmacokinetics (PK) of whole venom (WV) and its protein families. We studied the pathophysiology, PK profile and differential absorption of representative toxins from venom of Neotropical Rattlesnake (Crotalus simus) in a large animal model (ovine). Toxins studied included crotoxin (the main lethal component), which causes moderate to severe neurotoxicity; SVSPs, which deplete fibrinogen; and SVMPs, which cause local tissue damage and local and systemic hemorrhage. We found that Whole Venom (WV) was highly bioavailable (86%) 60 h following intramuscular (IM) injection, and extrapolation suggests that bioavailability may be as high as 92%. PK profiles of individual toxins were consistent with their physicochemical properties and expected clinical effects. Lymph cannulated animals absorbed 1.9% of WV through lymph during the first 12 h. Crotoxin was minimally detectable in serum after intravenous (IV) injection; however, following IM injection it was detected in lymph but not in blood. This suggests that crotoxin is quickly released from the blood toward its tissue targets.

Antiophidic solanidane steroidal alkaloids from Solanum campaniforme.

Three new solanidane alkaloids bearing a 22,23-epoxy ring (1-3) and four known compounds were isolated from leaves of Solanum campaniforme. The structures were determined using spectroscopic techniques, including 1D and 2D NMR, and HRESIMS experiments. The antiophidic activity of the alkaloids was tested against Bothrops pauloensis venom. Compounds 1-3 completely inhibited myotoxicity without inhibiting phospholipase A2 activity of the venom, while hemorrhage and skin necrosis were significantly reduced in the presence of alkaloids 1 and 2.

Compartment syndrome after Bothrops jararaca snakebite: monitoring, treatment, and outcome.

OBJECTIVE: To report the outcome of a patient who developed compartment syndrome after Bothrops jararaca snakebite. CASE REPORT: A 39-year-old male was admitted 5 h after being bitten on the lower right leg. Physical examination revealed tense swelling, ecchymosis, hypoesthesia, and intense local pain that worsened after passive stretching, limited right foot dorsiflexion, and gingival bleeding. The case was classified as moderate/severe and eight vials of bothropic antivenom (AV) were infused 1 h postadmission. The main laboratory findings upon admission were incoagulable blood (incoagulable PT, aPTT, and INR), thrombocytopenia, serum creatine kinase (CK) of 580 U/L (reference value < 170 U/L), and a serum venom level of 33.7 ng/mL (ELISA; cutoff = 2.3 ng/mL). High anterior compartment pressure (60 mmHg) was identified 8 h post bite, with progressively lower pressures after AV administration and limb elevation (36 mmHg; 19 h post bite). However, moderate pain and limited foot dorsiflexion persisted. In addition, there was a progressive increase in serum CK (6,729 U/L; 45 h post bite), as well as marked edema and hemorrhage of the anterior compartment detected by magnetic resonance imaging (MRI) at 48 h post bite. A fasciotomy done after a further increase in intracompartmental pressure (66 mmHg, 57 h post bite) revealed hemorrhage/necrosis of the anterior tibial muscle that subsequently required partial resection. The patient developed a local infection (day 15 post bite) and a permanent fibular palsy. CONCLUSION: Compartment syndrome is an unusual but severe complication of snakebites. MRI, in conjunction with subfascial pressure measurements, may be useful in the diagnosis of compartment syndrome after snakebites.

Efficacy and safety of two whole IgG polyvalent antivenoms, refined by caprylic acid fractionation with or without beta-propiolactone, in the treatment of Bothrops asper bites in Colombia.

The efficacy and safety of two whole IgG polyvalent antivenoms (A and B) were compared in a randomised, blinded clinical trial in 67 patients systemically envenomed by Bothrops asper in Colombia. Both antivenoms were fractionated by caprylic acid precipitation and had similar neutralising potencies, protein concentrations and aggregate contents. Antivenom B was additionally treated with beta-propiolactone to lower its anticomplementary activity. Analysing all treatment regimens together, there were no significant differences between the two antivenoms (A=34 patients; B=33 patients) in the time taken to reverse venom-induced bleeding and coagulopathy, to restore physiological fibrinogen concentrations and to clear serum venom antigenaemia. Blood coagulability was restored within 6-24 h in 97% of patients, all of whom had normal coagulation and plasma fibrinogen levels 48 h after the start of antivenom treatment. Two patients (3.0%) had recurrent coagulopathy and eight patients suffered recurrence of antigenaemia within 72 h of treatment. None of the dosage regimens of either antivenom used guaranteed resolution of venom-induced coagulopathy within 6 h, nor did they prevent recurrences. A further dose of antivenom at 6 h also did not guarantee resolution of coagulopathy within 12-24 h in all patients. The incidence of early adverse reactions (all mild) was similar for both antivenoms (15% and 24%; P>0.05).

Kinetics of venom and antivenom serum and clinical parameters and treatment efficacy in Bothrops alternatus envenomed dogs.

Dogs envenomed with non-lethal doses of Bothrops alternatus venom received standard antivenom therapy, im injections of flunixin meglumine, or topical treatmentwith aqueous Curcuma longa plant extract. Biodistribution of the venom and antivenom were determined by ELISA. There was no significant difference in the efficacy of antivenom and plant extract on local effects; flunixin treatment had lower efficacy. Distribution of the venom was similar with all 3 treatments. Serum levels of the antivenom reached maximum 2-4 h after administration and were not detected after the 5th d.

Coagulopathy following lethal and non-lethal envenoming of humans by the South American rattlesnake (Crotalus durissus) in Brazil.

The South American tropical rattlesnake (Crotalus durissus subspp) is responsible for approximately 10% of bites from venomous snakes in Brazil. We studied 24 victims of bites by this species over 3 years, in south-eastern Brazil, particularly investigating haemostatic alterations. Thirteen patients were defined as moderately envenomed and 11 as severe. There were two deaths, which were not attributed to venom-induced haemostatic disturbances. However, envenoming by C. durissus is frequently associated with haemostatic disorders, which are probably attributable mainly to the action of the thrombin-like enzyme, with possible additional effects secondary to the powerful myotoxic activity of the venom.

A randomized, blinded, comparative trial of one pepsin-digested and two whole IgG antivenoms for Bothrops snake bites in Uraba, Colombia. The Regional Group on Antivenom Therapy Research (REGATHER).

The therapeutic efficacy and the incidence of early antivenom reactions (EARs) were compared in a clinical trial performed in 79 patients bitten by Bothrops sp. in Urabá, Colombia. Patients were randomized into three groups according to the antivenom administered: A (n = 30, Butantan polyspecific, pepsin-digested Bothrops antivenom); B (n = 27, Butantan polyspecific, whole IgG Bothrops antivenom); and C (n = 22, Colombian commercial, monovalent, whole IgG Bothrops antivenom). The groups were comparable in all clinical and epidemiologic aspects; 33 patients had mild, 22 moderate, and 24 severe envenoming. At the doses used (two, four, and six vials [10 ml/vial] for mild, moderate, and severe envenomings, respectively) there were no differences between the antivenoms in restoring normal hemostatic parameters within 24 hr. The evolution of local envenoming was comparable in the three groups. Serum venom/antivenom kinetics determined by ELISA showed a complete clearance of venom levels 1 hr after treatment in mild/moderate envenomings. In severe cases, venom levels remained detectable up to 24 hr and recurrence of antigenemia was observed in some cases. Antivenom concentrations remained at high levels up to 24 hr of treatment. The incidence of EARs was significantly different in the groups: A (36.7%), B (11.1.%), and C (81.8%). There were no life-threatening anaphylactic reactions. We conclude that the efficacy of the three antivenoms was similar in neutralizing human Bothrops envenomings and that the production of whole IgG antivenoms by caprylic acid fractionation is a good alternative for reducing the incidence of EARs.

ELISA assays for the detection of Bothrops lanceolatus venom in envenomed patient plasmas.

A double antibody sandwich enzyme linked immunosorbant assay (ELISA) was carried out to detect Bothrops Ianceolatus venom in plasma from envenomed patients at various time intervals (0, 6, 12, 18 and 24 hrs). The test could detect Bothrops lanceolatus levels up to 12 ng/mL of envenomed patient plasmas. Elaboration of an easy, fast and species-diagnostic based on this ELISA technique useful to physicians is discussed.

Time factor in the detection of circulating whole venom and crotoxin and efficacy of antivenom therapy in patients envenomed by Crotalus durissus.

Thirty-seven patients envenomed by Crotalus durissus were classified into three groups according to the interval between the bite and hospital admission (delta T): group 1 (n = 14, delta T < 4 hr), group 2 (n = 14, delta T > 4 hr < 8 hr) and group 3 (n = 9, delta T > 8 hr). Venous blood from these patients was sampled for biochemical and hematological analysis and for whole venom, crotoxin and antivenom enzyme-linked immunosorbent assays before antivenom treatment (T0) and at 1 hr (T1), 6 hr (T6), 12 hr (T12) and 24 hr (T24) after the start of antivenom therapy. The patients were treated with 100-200 ml (10-20 ampules) of C. durissus antivenom. Whole venom and crotoxin were detected in 13 (92.8%) and 11 (78.6%) of 14 group 1 patients, respectively, in 11 (78.6%) and six (42.9%) of 14 group 2 patients, respectively, and in two (22.2%) and one (11.1%) of nine group 3 patients, respectively, before antivenom treatment. Data from this study show that whole venom and crotoxin were not detected in most of patients when the time elapsed between the bite and hospital admission was greater than 8 hr, and crotoxin was not detected in most of the patients who were admitted to the hospital at times ranging from 4 to 8 hr after the snakebite. Plasma whole venom, crotoxin and antivenom levels measured over time in these patients show the efficacy of antivenom treatment, since circulating venom and crotoxin were no longer detected 1 hr after antivenom therapy and high antivenom titers persisted for at least 24 hr after serotherapy.

Clinical indicators of envenoming and serum levels of venom antigens in patients bitten by Bothrops lanceolatus in Martinique. Research Group on Snake Bites in Martinique.

An enzyme-linked immunosorbent assay was developed to measure venom antigen levels in the serum of 40 patients bitten by Bothrops lanceolatus. The grading system used for the severity of envenomation (grades 1 to 4, minor to major) was predominantly based on the presence of local signs. Serum venom levels increased with the grade of severity (P < 0.001, by Spearman's rank correlation test); they were 6 +/- 6 ng/mL (mean +/- SD) in clinically non-envenomed patients (grade 1, n = 3), 7.6 +/- 11.7 (n = 17), 44.3 +/- 41.8 (n = 17), and 80.3 +/- 34.1 ng/mL (n = 3) in patients diagnosed as grade 2, 3 and 4 respectively. However, venom antigens could not be detected in the serum of 54% of patients who showed clinical signs of envenomation. Most patients diagnosed as grade 2, 3 or 4 were given 20, 40 and 60 mL of a monospecific F(ab')2 antivenom, respectively. Venom concentrations > or = 15 ng/mL were observed in all patients with progressive aggravation of swelling despite the use of early antivenom therapy. No venom was detectable in blood samples taken after completion of serotherapy. All patients recovered. These results confirm the efficacy of both the clinical severity scoring system used and the therapeutic regimen.

Bothrops alternatus envenoming in young dogs

Bothrops alternatus venom was intramuscularly inoculated (3mg/kg) into 12 dogs, 30 to 65 days old. Spontaneou bleeding commenced twenty minutes later. Blood samples obtained 3 and 20 minutes after venom inoculation presented spontaneuous clotting formation. Plasmatic fibrinogen decreased within 3 minutes. Partial thromboplastin time (PTT) and one-stage prothrombin time (PT) were found. Plasma did not coagulate 40 minutes after inoculation. Platelet counts did not vary but their function was altered. Histopathology pointed out severe muscular necrosis and massive hemorrhage in the inoculation area. Regional ganglia showed intense hemorrhage. The 45 and 65-day-old animals showed alveolar thickening of the septum and generalized congestion, but the 30-day-old animals showed thrombosis of small arteries and arterioles. Renal lesions were different with the age. Cortical tubular necrosis was present in puppies, and intense cortical tubular hydropic degeneration was present in adult dogs. Thymus hemorrhage and necrosis were present.

A randomized double-blind clinical trial of two antivenoms in patients bitten by Bothrops atrox in Colombia. The Regional Group on Antivenom Therapy Research (REGATHER).

A randomized double-blind clinical trial in 39 patients envenomed by Bothrops atrox in Antioquia and Chocó, Colombia, was performed to compare the efficacy and safety of 2 equine-derived antivenoms prepared at Instituto Clodomiro Picado, University of Costa Rica. Twenty patients received a monovalent anti-B. atrox antivenom (group A) and 19 patients were treated with a polyvalent (Crotalinae) antivenom (group B). Both antivenoms were equally efficient in the neutralization of the most relevant signs of envenoming (haemorrhage and blood clotting time alteration). Fourteen patients (36%) presented early adverse reactions to antivenoms and no significant difference between the 2 groups was observed. Urticaria (18%) was the most frequent early adverse reaction and there was no life-threatening anaphylactic reaction. Based on clinical criteria and serum venom levels, estimated by an enzyme immunoassay, 15 patients were classified into 2 groups: mild and moderate/severe envenoming. With the antivenom doses used in this study (3, 6 and 9 vials for mild, moderate and severe envenoming, respectively), both antivenoms were equally efficient in clearing serum venom levels within the first hour of treatment, and the levels remained below the lower limit of venom detection for 24 h. Antivenom concentration in serum remained high for up to 24 h after antivenom infusion, suggesting that an excess of antibody in relation to circulating antigen had been administered.

Venomous snake bite without clinical envenoming ('dry-bite'). A neglected problem in Brazil.

Venomous snake bite without envenoming ('dry-bite') has been recognized throughout the world, but neglected in the Brazilian literature. Forty cases of patients bitten by venomous snakes of the genera Bothrops (lance-headed vipers) and Crotalus (South American rattlesnakes), confirmed by identification of the captured or dead snake, were seen in a 34-month prospective study carried out in a teaching hospital in southeastern Brazil. Out of the 33 cases of lance-headed viper and 7 of rattlesnake bite, respectively 10 (30.3%) and 3 (42.9%) had no clinical or laboratory evidence of local or systemic envenoming. Both within the Bothrops and Crotalus groups, there was no significant difference in sex and age of the patients, and time between bite and medical assistance between the patients who had from those who did not have clinical envenoming. The high prevalence of 'dry-bite' in this study may have several possible explanations, and has implications on the indication of antivenom for the treatment of venomous snake bite. Antivenom administration may be postponed or even not indicated for victims of snake bite presenting no manifestations of local or systemic envenoming.

Different clotting mechanisms of Bothrops jararaca snake venom on human and rabbit plasmas.

Bothrops jararaca venom is approximately 3.5 times more effective at coagulating rabbit plasma than human plasma. To investigate this difference B. jararaca venom was treated with several enzymatic inhibitors and the minimum coagulant dose was determined both on plasma anticoagulated with sodium citrate or a mixture of sodium citrate and heparin, and on fibrinogen (both human and rabbit). On human plasma, the thrombin-like component of the venom accounted for c. 60% of the coagulant activity, such activity was negligible on rabbit plasma. The venom had little clotting activity on rabbit fibrinogen. The factor II- and X-activator components could be inhibited by EDTA, EGTA and 2-mercaptoethanol, whereas the thrombin-like activity was inhibited by PMSF. These differences show that (using human plasma) B. jararaca clotting activity is mainly due to the thrombin-like component, whereas the factor II- and X-activator components are more important on rabbit plasma. The delayed action of the thrombin-like enzyme on rabbit fibrinogen may be attributed to the difference between rabbit and human fibrinopeptide A. Thus, the increased coagulant activity on rabbit plasma may be due to a faster rate of activation of factor X, V or II by snake venom enzymes.

Use of enzyme immunoassays to compare the effect and assess the dosage regimens of three Brazilian Bothrops antivenoms. The Butantan Institute Antivenom Study Group (BIASG).

The effect of the three main Brazilian polyspecific antivenoms on venom clearance was assessed in 118 moderately envenomed victims of bites by Bothrops species (mainly B. jararaca) in Sao Paulo State, Brazil. Serum samples taken from patients at intervals during their stay in the hospital and at followup approximately four weeks later were tested by enzyme immunoassay for the presence of whole venom and therapeutic antivenom. Results indicated that in patients treated with the standard regimen of either four (40 ml) or eight (80 ml) ampules of each antivenom, venom was cleared from the circulation within four days of antivenom administration. However, high concentrations of antivenom persisted for approximately 10 days and remained detectable until 30-50 days after administration. This suggests that patients may be being treated with excessive amounts of antivenom in Brazil. This practice increases the national cost of antivenom therapy and may contribute to the high frequency of antivenom reactions. Clinically, there was no obvious difference in the efficacy between the three antivenoms.

Serum levels of bothropic venom in patients without antivenom intervention.

Serum kinetics of bothropic venom were evaluated in eight snakebite patients, who due to a national shortage, received no specific antivenom therapy. The cases were clinically classified as mild, moderate, and severe. Patients were bled sequentially and serum levels of venom were assayed by ELISA. Venom level ranges differed among the groups, with peak levels of less than 13 ng/ml, 32 ng/ml, and 120 ng/ml for the mild, moderate, and severe groups, respectively. There was no clear pattern of kinetics in the groups. Regression analysis involving the variables severity and peak venom levels yielded a statistically significant correlation (rs = 0.80, P less than 0.05). These data indicate that different amounts of circulating venom correlate with clinical severity, even in highly complex venoms, and stress the importance of careful clinical classification in the proper management of bothropic incidents.