In Vitro and In Vivo Neutralizing Activity of Uvaria chamae Leaves Fractions on the Venom of Naja nigricollis in Albino Rat and Bovine Blood.

BACKGROUND: Snakebite envenomation is a global priority ranked top among other neglected tropical diseases. There is a folkloric claim that Uvaria chamae is beneficial for the management of snakebite and wounds in African ethnobotanical surveys. Besides, there are many registered patents asserting the health benefits of U. chamae. OBJECTIVE: This study aimed to investigate U. chamae's potentials and identify candidates for the development of tools for the treatment and management of N. nigricollis envenomation. METHODS: Freshly collected U. chamae leaves were air-dried, powdered, and extracted in methanol. The median lethal dose of the extract was determined and further fractionated with n-hexane, n-butanol and ethyl acetate. Each fraction was tested for neutralizing effect against venom-induced haemolytic, fibrinolytic, hemorrhagic, and cytotoxic activities. RESULTS: U. chamae fractions significantly (p<0.05) neutralized the haemolytic activity of N. nigricollis venom in n-butanol; 31.40%, n-hexane; 33%, aqueous residue; 39.60% and ethyl acetate; 40.70% at the concentration of 100mg/ml of each fraction against 10mg/ml of the snake venom when compared to the positive control. The fibrinolytic activity of N. nigricollis venom was significantly (p<0.05) neutralized in n-hexane at 73.88%, n-butanol; 72.22% and aqueous residue; 72.22% by the fractions of U. chamae. In addition, haemorrhagic activity of N. nigricollis venom was significantly (p<0.05) neutralized by U. chamae fractions at the concentrations of 100mg/ml, 200mg/ml and 400mg/ml except for n-butanol and aqueous residues at 400 mg/ml. CONCLUSION: U. chamae leaves fractions possess a high level of protection against N. nigricollis venoms-induced lethality and thus validate the pharmacological rationale for its usage in the management of N. nigricollis envenomation.

Pets in peril: The relative susceptibility of cats and dogs to procoagulant snake venoms.

Snakebite is a common occurrence for pet cats and dogs worldwide and can be fatal. In Australia the eastern brown snake (Pseudonaja textilis) is responsible for an estimated 76% of reported snakebite cases to domestic pets nationally each year, with the primary pathology being venom-induced consumptive coagulopathy. While only 31% of dogs survive P. textilis bites without antivenom, cats are twice as likely to survive bites (66%). Even with antivenom treatment, cats have a significantly higher survival rate. The reason behind this disparity is unclear. Using a coagulation analyser (Stago STA R Max), we tested the relative procoagulant effects of P. textilis venom-as well as 10 additional procoagulant venoms found around the world-on cat and dog plasma in vitro, as well as on human plasma for comparison. All venoms acted faster upon dog plasma than cat or human, indicating that dogs would likely enter coagulopathic states sooner, and are thus more vulnerable to procoagulant snake venoms. The spontaneous clotting time (recalcified plasma with no venom added) was also substantially faster in dogs than in cats, suggesting that the naturally faster clotting blood of dogs predisposes them to being more vulnerable to procoagulant snake venoms. This is consistent with clinical records showing more rapid onset of symptoms and lethal effects in dogs than cats. Several behavioural differences between cats and dogs are also highly likely to disproportionately negatively affect prognosis in dogs. Thus, compared to cats, dogs require earlier snakebite first-aid and antivenom to prevent the onset of lethal venom effects.

Detection of Snake Venom in Post-Antivenom Samples by Dissociation Treatment Followed by Enzyme Immunoassay.

Venom detection is crucial for confirmation of envenomation and snake type in snake-bite patients. Enzyme immunoassay (EIA) is used to detect venom, but antivenom in samples prevents venom detection. We aimed to detect snake venom in post-antivenom samples after dissociating venom-antivenom complexes with glycine-HCl (pH 2.2) and heating for 30 min at 950 °C. Serum samples underwent dissociation treatment and then Russell's viper venom or Australian elapid venom measured by EIA. In confirmed Russell's viper bites with venom detected pre-antivenom (positive controls), no venom was detected in untreated post-antivenom samples, but was after dissociation treatment. In 104 non-envenomed patients (negative controls), no venom was detected after dissociation treatment. In suspected Russell's viper bites, ten patients with no pre-antivenom samples had venom detected in post-antivenom samples after dissociation treatment. In 20 patients with no venom detected pre-antivenom, 13 had venom detected post-antivenom after dissociation treatment. In another 85 suspected Russell's viper bites with no venom detected pre-antivenom, 50 had venom detected after dissociation treatment. Dissociation treatment was also successful for Australian snake envenomation including taipan, mulga, tiger snake and brown snake. Snake venom can be detected by EIA in post-antivenom samples after dissociation treatment allowing confirmation of diagnosis of envenomation post-antivenom.

Predictors of the development of post-snakebite compartment syndrome.

BACKGROUND: To identify the factors associated with the development of post-snakebite compartment syndrome (PSCS) in snakebite patients and to analyze the clinical prognosis of these patients. METHODS: We retrospectively reviewed the medical records of patients who presented to our institution with snakebites from March 2009 to December 2012. The clinical data, hospital course and outcome were all recorded. RESULTS: A total of 136 patients were included in the present study. Nine patients developed PSCS and underwent fasciotomy. Relative to the non-PSCS group, the PSCS group demonstrated a significant increase in the white blood cell count (WBC, p = 0.006), segment form (Seg, p ≤ 0.001), aspartate aminotransferase level (AST, p = 0.002) and alanine aminotransferase level (ALT, p = 0.008). Elevated WBC count and AST level were identified as independent risk factors for PSCS (p = 0.028 and 0.037, respectively) in a multivariate analysis. CONCLUSIONS: Snakebite patients have a high likelihood of developing locoregional complications such as PSCS. Symptomatic snakebite patients should be observed for at least 48 h, and increased WBC counts and AST levels are risk factors for PSCS.

Detection of venom after antivenom is not associated with persistent coagulopathy in a prospective cohort of Russell's viper (Daboia russelii) envenomings.

BACKGROUND: Venom recurrence or persistence in the circulation after antivenom treatment has been documented many times in viper envenoming. However, it has not been associated with clinical recurrence for many snakes, including Russell's viper (Daboia spp.). We compare the recovery of coagulopathy to the recurrence or persistence of venom in patients with Russell's viper envenoming. METHODOLOGY/PRINCIPAL FINDINGS: The study included patients with Russell's viper (D. russelii) envenoming presenting over a 30 month period who had Russell's viper venom detected by enzyme immunoassay. Demographics, information on the snake bite, and clinical effects were collected for all patients. All patients had serum collected for venom specific enzyme immunoassay and citrate plasma to measure fibrinogen levels and prothrombin time (international normalised ratio; INR). Patients with venom recurrence/persistence were compared to those with no detectable recurrence of venom. There were 55 patients with confirmed Russell's viper envenoming and coagulopathy with low fibrinogen concentrations: 31 with venom recurrence/persistence, and 24 with no venom detected post-antivenom. Fibrinogen concentrations increased and INR decreased after antivenom in both the recurrence and non-recurrence patients. Clinical features, laboratory parameters, antivenom dose and length of hospital were similar for both groups. Pre-antivenom venom concentrations were higher in patients with venom recurrence/persistence with a median venom concentration of 385 ng/mL (16-1521 ng/mL) compared to 128 ng/mL (14-1492 ng/mL; p = 0.008). CONCLUSION: Recurrence of Russell's viper venom was not associated with a recurrence of coagulopathy and length of hospital stay. Further work is required to determine if the detection of venom recurrence is due to the venom specific enzyme immunoassay detecting both venom-antivenom complexes as well as free venom.

Death adder envenoming causes neurotoxicity not reversed by antivenom--Australian Snakebite Project (ASP-16).

BACKGROUND: Death adders (Acanthophis spp) are found in Australia, Papua New Guinea and parts of eastern Indonesia. This study aimed to investigate the clinical syndrome of death adder envenoming and response to antivenom treatment. METHODOLOGY/PRINCIPAL FINDINGS: Definite death adder bites were recruited from the Australian Snakebite Project (ASP) as defined by expert identification or detection of death adder venom in blood. Clinical effects and laboratory results were collected prospectively, including the time course of neurotoxicity and response to treatment. Enzyme immunoassay was used to measure venom concentrations. Twenty nine patients had definite death adder bites; median age 45 yr (5-74 yr); 25 were male. Envenoming occurred in 14 patients. Two further patients had allergic reactions without envenoming, both snake handlers with previous death adder bites. Of 14 envenomed patients, 12 developed neurotoxicity characterised by ptosis (12), diplopia (9), bulbar weakness (7), intercostal muscle weakness (2) and limb weakness (2). Intubation and mechanical ventilation were required for two patients for 17 and 83 hours. The median time to onset of neurotoxicity was 4 hours (0.5-15.5 hr). One patient bitten by a northern death adder developed myotoxicity and one patient only developed systemic symptoms without neurotoxicity. No patient developed venom induced consumption coagulopathy. Antivenom was administered to 13 patients, all receiving one vial initially. The median time for resolution of neurotoxicity post-antivenom was 21 hours (5-168). The median peak venom concentration in 13 envenomed patients with blood samples was 22 ng/mL (4.4-245 ng/mL). In eight patients where post-antivenom bloods were available, no venom was detected after one vial of antivenom. CONCLUSIONS/SIGNIFICANCE: Death adder envenoming is characterised by neurotoxicity, which is mild in most cases. One vial of death adder antivenom was sufficient to bind all circulating venom. The persistent neurological effects despite antivenom, suggests that neurotoxicity is not reversed by antivenom.

Tiger snake (Notechis spp) envenoming: Australian Snakebite Project (ASP-13).

OBJECTIVES: To describe the clinical syndrome associated with definite tiger snake (Notechis spp) envenoming and to examine the ability of tiger snake antivenom (TSAV) to bind free venom in vivo. DESIGN, SETTING AND PARTICIPANTS: We conducted a prospective cohort study within the Australian Snakebite Project, reviewing all definite tiger snake envenoming cases between October 2004 and June 2011. Definite cases were identified by venom-specific enzyme immunoassay or expert snake identification. MAIN OUTCOME MEASURES: Clinical effects of tiger snake envenoming; peak venom concentrations; number of vials of antivenom administered. RESULTS: Fifty-six definite tiger snake envenomings were identified. Clinical effects included venom-induced consumption coagulopathy (VICC) (n = 53), systemic symptoms (n = 45), myotoxicity (n = 11) and neurotoxicity (n = 17). Thrombotic microangiopathy occurred in three patients, all of whom developed acute renal failure. There were no deaths. A bite-site snake venom detection kit test was done in 44 patients, but was positive for tiger snake in only 33 cases. Fifty-three patients received TSAV and eight of these patients had immediate hypersensitivity reactions, severe enough in one case to satisfy diagnostic criteria for severe anaphylaxis. The median peak venom concentration in 50 patients with pretreatment blood samples available was 3.2 ng/mL (interquartile range [IQR], 1-12 ng/mL; range 0.17-152 ng/mL). In 49 patients with post-treatment blood samples available, no venom was detected in serum after the first antivenom dose. Ten patients were given 1 vial of TSAV; the median dose was 2 vials (range, 1-4 vials). Pretreatment serum venom concentrations did not vary significantly between patients given 1 vial of TSAV and those given 2 or more vials. CONCLUSION: Tiger snake envenoming causes VICC, systemic symptoms, neurotoxicity and myotoxicity. One vial of TSAV, the dose originally recommended when the antivenom was first made available, appears to be sufficient to bind all circulating venom.

A phase I study of continuous infusion cilengitide in patients with solid tumors.

BACKGROUND: Cilengitide (EMD121974) is a cyclized pentapeptide that is a potent and selective integrin antagonist which has shown activity in malignant gliomas. In all previous studies, cilengitide has been administered in an intermittent fashion. However, cilengitide has a short half-life of 3-5 h with no evidence of drug accumulation. These data prompted the initiation of this phase I study of continuous infusion cilengitide. METHODS: Cilengitide was administered as a continuous infusion without break in 4-week cycles. Plasma samples for pharmacokinetic studies were obtained weekly in cycle 1 immediately prior to and 2 h after infusion bag change. RESULTS: Thirty-five patients were treated (median age 56; 23 males) at dose levels of 1, 2, 4, 8, 12, 18, 27, and 40 mg/h. Toxicities were limited to grade ≤ 2 and showed no relation to dose. Fatigue was most common (17%), while all other toxicities were reported in <10% of patients. No dose-limiting toxicities were observed, and therefore the maximum tolerated dose was not reached. Pharmacokinetic analysis showed that values for clearance and volume of distribution were comparable across dose levels, and the steady-state concentration increased proportionally with dose. CONCLUSIONS: Cilengitide can be safely administered as a continuous infusion at doses up to at least 40 mg/h, which represents the maximum feasible dose due to drug solubility and delivery limitations. The pharmacokinetics of continuous infusion cilengitide are linear and consistent with the results obtained using a twice weekly infusion.

Mild snake envenomation.

Snake bite envenomation typically requires treatment with effective first aid and antivenom. There is a spectrum of envenomation seen, which includes mild envenomation, but this has not been reported previously. We report two cases of mild envenomation and describe the changes in laboratory coagulation values. The patients had a benign clinical course without receiving antivenom. We strongly recommend that if clinicians are considering not treating any envenomated patients with antivenom, they do so only on expert advice.

The role of thromboelastography in the management of children with snake-bite in southern Africa.

In the absence of a direct laboratory test of envenomation, there is a need for an alternative mechanism for the early recognition of envenomation following snake-bite in children. A severe clinical diathesis may result either from envenomation or from the release of an inappropriate tourniquet applied as 'first-aid' often several hours before presentation to hospital. Abnormalities of clotting are associated with both events. A normal thromboelastogram (TEG) provides early recognition of patients in whom the clinical course is likely to be benign (sensitivity = 94%). An abnormal TEG identifies patients of whom 50% will develop a severe clinical diathesis. A TEG is a more accurate predictor of disease severity than International Normalized Ratio alone. The TEG does not supplant clinical observation in the management of snake-bite in children but allows stratification into high- and low-risk categories.

First clinical experiences with specific sheep Fab fragments in snake bite. Report of a multicentre study of Vipera berus envenoming.

OBJECTIVES: To evaluate the efficacy and safety of specific, ovine Fab fragments in the treatment of envenoming by the common adder, Vipera berus. DESIGN: Open study with historical controls. SETTING: Multicentre study involving patients (n = 30) with V. berus envenoming, treated in 18 Swedish hospitals during 1991-94. MAIN OUTCOME MEASURES: Initial symptoms, clinical course after treatment, duration of hospital stay and adverse effects of the antivenom were evaluated. Two earlier studied patient groups, given either equine F(ab)2 antivenom (n = 30) or no antivenom (n = 16), were used as controls. RESULTS: Specific ovine Fab fragments influenced favourably the acute symptomatology as well as the long term clinical course. Acute symptoms such as hypotension, shock, vomiting, diarrhoea and CNS-depression resolved quickly. The incidence of extensive swelling involving the trunk and the length of hospital stay were both reduced significantly compared to nontreated patients (23 vs. 88% and 3.5 vs. 6 days). Also the incidence of anaemia was reduced (23 vs. 44%). These results were consistent with those obtained with equine F(ab')2 antivenom, but with ovine Fab there were no immediate anaphylactic reactions or serum sickness. CONCLUSION: Specific Fab fragments produced from sheep immunized with V. berus venom were safe and effective in counteracting the effects of V. berus bite in humans. These results justify further studies of this new treatment for snake envenoming.

An objective approach to antivenom therapy and assessment of first-aid measures in snake bite.

Treatment of systemic envenoming in snake-bite victims has, in the past, depended almost entirely on the individual clinician's experience in assessing the severity of envenoming. The efficacy of treatment is obviously related to the neutralizing potency of the antivenom used, the route by which it is administered and the dose. The development of enzyme immunoassays has permitted a more scientific appraisal, allowing estimation of circulating specific venom and antivenom concentrations at any time after the bite in the patient's blood. It is therefore possible to measure accurately the efficacy of antivenom in the neutralization and clearance of venom antigen. In Brazil, it appears that clinicians treat patients with excessive amounts of highly efficient antivenoms and this results in an unacceptably high incidence of reactions. In Sri Lanka, the use of imported, Indian antivenom is relatively ineffective in neutralizing the venoms of Sri Lankan snakes, demonstrating the real problem of venom variability within individual species. In West Africa, the improved clearance of venom following treatment of Echis victims with a monospecific as opposed to a polyspecific antivenom has been demonstrated, and new, smaller fragment, Fab antivenoms have been developed and are now under clinical assessment. Such clinically-based immunological studies should result in more efficient and controlled use of expensive antivenoms for treatment of systemic envenoming and the accurate assessment of newly designed products. Such studies also emphasise the importance of individual countries producing their own antivenoms for treatment of systemic envenoming. Likewise, the use of such objective systems now enables the use of first-aid measures such as tourniquets to be properly assessed.

Effects of three fractions obtained from Naja haje venom on hemolysis and lipid metabolism in rabbits

Three fractions (F1, F2 and F3) were obtained from the venom of the Egyptian cobra Naja haje by gel filtration. F1 and F2 had indirect hemolytic effects on rabbit erytrocytes. The HU50 (the amount of the fraction causing 50 per cent hemolysis) values of F1 and F2 were 12.04 ñ 3.89 and 36.57 ñ 2.20 ug, respectively. The effects of the three cobra venom fractions on lipid metabolism were studied after 1, 2 3 and 4 hours. Serum total lipids and cholesterol were significantly decreased at almost all tested times, but variable changes were observed in the serum triglycerides. Serum inorganic phosphorus levels were significantly increased with F1 and F2 administration more than that with the F3 fraction. These results suggest that F1 and F2 possess phospholipase A2-like activities and their effects on the lipid metabolism is more intense than that of F3.

Snake envenomation in cats and its detection by rapid immunoassay.

OBJECTIVE: To determine the usefulness of a snake venom detection kit (SVDK) in the management of envenomed cats. DESIGN: A clinical study. ANIMALS: Twenty-two cats were investigated. PROCEDURE: Cats injected subcutaneously with approximately 0.25 or 1.0 lethal dose (LD) of tiger snake venom or 1 or 4 LD of brown snake venom were observed for clinical symptoms of envenomation at intervals over the ensuring 24 to 48 hours(h). Blood and urine samples were taken at regular intervals and assayed in a quantitative laboratory assay for snake venoms. Selected samples were assayed in parallel in a rapid, semi-quantitative SVDK. RESULTS: The studies showed that it was important to estimate the elapsed time from envenomation to presentation. If this time was less than 8 h, blood was the most appropriate sample and a negative result should exclude serious envenomation. If the elapsed time exceeded 8 h, it was essential that urine be sampled. Venom levels in urine were high at 8 h and approached the level of test sensitivity over 24 to 48 h; however by this time clinical signs were obvious in endangered cats. CONCLUSIONS: Careful use of the SVDK is a valuable aid in the management of a potentially envenomed cat.

Neurotoxicity and haemostatic disturbances in patients envenomed by the Papuan black snake (Pseudechis papuanus).

Among 335 patients presenting with snakebites in Central Province, Papua New Guinea, nine were proved by enzyme immunoassay to have been bitten by Papuan black snakes (Pseudechis papuanus). Seven showed clinical evidence of envenoming. Early symptoms included vomiting and tender local lymph nodes. Five patients had neurotoxic signs and one required mechanical ventilation. Spontaneous systemic bleeding occurred in two patients. Coagulation studies in four patients showed thrombocytopenia, prolongation of prothrombin time, mild defibrination and depletion of other clotting factors with elevated fibrin(ogen) degradation products and other evidence of fibrinolysis. One patient developed mild renal dysfunction. There was no evidence of intravascular haemolysis or rhabdomyolysis. These clinical observations, which do not distinguish victims of P. papuanus from those of taipans (Oxyuranus scutellatus canni), suggest that the venom contains neurotoxic, haemorrhagic and mild procoagulant activities. Only two other cases of proven envenoming by this species have been reported. There appears to have been a decline in the abundance of this species, and hence its medical importance, over the last 25 years.

Diagnosis and treatment of confirmed and suspected snake bite. Implications from an analysis of 46 paediatric cases.

OBJECTIVE: To identify reliable predictors of envenomation in suspected snake bite and to examine the current standard of treatment in envenomed patients. DESIGN: Retrospective cohort analysis of children presenting with suspected or confirmed snake bite in southern mainland Australia. Detection of snake venom in urine or blood was taken as proof of envenomation. SETTING: Intensive Care Unit, Royal Children's Hospital, Melbourne. PATIENTS: Forty-six children presenting between 1979 and 1990. MAIN OUTCOME MEASURES: Positive and negative clinical evidence, venom tests, and coagulation tests. RESULTS: Twenty-seven children (59%) had suspected bites; 10 (22%) were bitten but not envenomed; 9 (19%) were envenomed. Two died of coagulopathy. Headache, abdominal pain or vomiting were moderately predictive of envenomation (positive predictive values of 63%, 57% and 64% respectively). Coagulopathy was a highly sensitive, specific and reliably predictive (100%) indicator of envenomation. The pressure-immobilisation bandage was used in 28% of cases at the scene of the bite and in 41% on contact with medical or paramedical services. Twelve patients received antivenom; of these, six received adrenaline as premedication. CONCLUSION: Headache, abdominal pain, nausea or vomiting, or abnormal coagulation tests accurately predict envenomation by snakes in southern mainland Australia. More or better education on first aid and clinical management of snake bite is needed.

Diagnosis of snake venoms by a reverse latex agglutination test.

A reverse latex agglutination test using protein A column purified rabbit antivenom IgG-sensitized latex particles was developed for the detection of the six medically important snake venoms of Thailand. The detection limit of the reverse latex agglutination test was 0.16 to 1.2 micrograms/mL of crude venoms. Cross-reactions with heterologous venoms were observed at concentrations 460 to 16000 times that of homologous venoms. Detection of various snake venoms in clinical specimens was carried out by the reverse latex agglutination test. The sensitivity was 52.5% of the 59 serum samples. There was one (1.69%) false positive sample. The positive detection of venom in wound swabs (26 cases) was 38.5% and was not statistically different from that observed in paired serum samples. Interference from human plasma, serum and urine on the reverse latex agglutination test could be eliminated by adsorption with normal rabbit IgG-coated latex suspension or by heat inactivation at 56 degrees C for 30 min. Prozone effect observed in some sera was eliminated by heat inactivation at 56 degrees C for 30 min. The sensitized latex particles were stable at 4 degrees C and -20 degrees C for at least 3 months. Cycles of freezing-thawing and lyophilization did not change their reactivities. The total test time was about 40 min.

Detection of venom by enzyme linked immunosorbent assay (ELISA) in patients bitten by snakes in Thailand.

The ability of an enzyme linked immunosorbent assay (ELISA) to detect venom was evaluated in 251 patients bitten by four of the commonest poisonous snakes in Thailand. Serum was tested only from patients who brought the snakes that had bitten them. About one third of all bitten patients had detectable venom antigenaemia, though a smaller proportion were symptomatic. Serum venom concentrations on admission correlated with the severity of clinical manifestations. The test was sensitive and specific even for specimens that had been collected and stored under suboptimal conditions. The technique is suitable for forensic use in cases of suspected snakebite. The combination of snake identification and venom antigen detection should be a more reliable means of studying the epidemiology of snakebite than the measurement of venom antibodies in a population.

Capillary enzyme immunoassay field kits for the detection of snake venom in clinical specimens: a review of two years' use.

A capillary enzyme immunoassay kit for the detection of snake venom in specimens from envenomed patients has now been in use for two summer periods. Results from 130 questionnaires completed after the use of a kit are tabulated. Positive detection and identification of the snake involved was reported in 37 cases of patients in whom some clinical signs of envenoming were apparent. In 58 patients in whom no clinical signs of envenoming were observed, no venom was detected. Performance characteristics of the test were very good and could be improved if bite site swabbings or urine samples were used as the specimen for test in preference to serum or blood.