Venom Concentrations and Clotting Factor Levels in a Prospective Cohort of Russell's Viper Bites with Coagulopathy.
ABSTRACT: BACKGROUND:Russell's viper envenoming is a major problem in South Asia and causes venom induced consumption coagulopathy. This study aimed to investigate the kinetics and dynamics of venom and clotting function in Russell's viper envenoming. METHODOLOGY/PRINCIPAL FINDINGS:In a prospective cohort of 146 patients with Russell's viper envenoming, we measured venom concentrations, international normalised ratio [INR], prothrombin time (PT), activated partial thromboplastin time (aPTT), coagulation factors I, II, V, VII, VIII, IX and X, and von Willebrand factor antigen. The median age was 39 y (16-82 y) and 111 were male. The median peak INR was 6.8 (interquartile range [IQR]: 3.7 to >13), associated with low fibrinogen [median,<0.01 g/L; IQR: <0.01-0.9 g/L), low factor V levels [median,<5%; IQR: <5-4%], low factor VIII levels [median,40%; IQR: 12-79%] and low factor X levels [median, 48%; IQR: 29-67%]. There were smaller reductions in factors II, IX and VII over time. All factors recovered over 48 h post-antivenom. The median INR remained >3 at 6 h post-antivenom but had reduced to <2, by 24 h. The aPTT had also returned to close to normal (<50 sec) at 24 h. Factor VII, VIII and IX levels were unusually high pre-antivenom, median peak concentrations of 393%, 307% and 468% respectively. Pre-antivenom venom concentrations and the INR (r = 0.20, p = 0.02) and aPTT (r = 0.19, p = 0.03) were correlated (non-parametric Spearman analysis). CONCLUSIONS:Russell's viper coagulopathy results in prolonged aPTT, INR, low fibrinogen, factors V, VIII and X which recover over 48 h. Severity of clotting abnormalities was associated with venom concentrations.
Project description:<h4>Background</h4>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.<h4>Methodology/principal findings</h4>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).<h4>Conclusion</h4>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.
Project description:Essentials Russell's viper envenoming is a major health issue in South Asia and causes coagulopathy. We studied the effect of fresh frozen plasma and two antivenom doses on correcting coagulopathy. Fresh frozen plasma did not hasten recovery of coagulopathy. Low-dose antivenom did not worsen coagulopathy. SUMMARY:Background Russell's viper (Daboia russelii) envenoming is a major health issue in South Asia and causes venom-induced consumption coagulopathy (VICC). Objectives To investigate the effects of fresh frozen plasma (FFP) and two antivenom doses in correcting VICC. Methods We undertook an open-label randomized controlled trial in patients with VICC at two Sri Lankan hospitals. Patients with suspected Russell's viper bites and coagulopathy were randomly allocated (1 : 1) to high-dose antivenom (20 vials) or low-dose antivenom (10 vials) plus 4 U of FFP. The primary outcome was the proportion of patients with an International Normalized Ratio (INR) of < 2 at 6 h after antivenom administration. Secondary outcomes included anaphylaxis, major hemorrhage, death, and clotting factor recovery. Results From 214 eligible patients, 141 were randomized: 71 to high-dose antivenom, and 70 to low-dose antivenom/FFP; five had no post-antivenom blood tests. The groups were similar except for a delay of 1 h in antivenom administration for FFP patients. Six hours after antivenom administration, 23 of 69 (33%) patients allocated to high-dose antivenom had an INR of < 2, as compared with 28 of 67 (42%) allocated to low-dose antivenom/FFP (absolute difference 8%; 95% confidence interval - 8% to 25%). Fifteen patients allocated to FFP did not receive it. Severe anaphylaxis occurred equally frequently in each group. One patient given FFP developed transfusion-related acute lung injury. Three deaths occurred in low-dose antivenom/FFP patients, including one intracranial hemorrhage. There was no difference in recovery rates of INR or fibrinogen, but there was more rapid initial recovery of factor V and FX in FFP patients. Conclusion FFP after antivenom administration in patients with Russell's viper bites did not hasten recovery of coagulopathy. Low-dose antivenom/FFP did not worsen VICC, suggesting that low-dose antivenom is sufficient.
Project description:BACKGROUND:Sri Lankan Russell's viper (Daboia russelii) envenoming is reported to cause myotoxicity and neurotoxicity, which are different to the effects of envenoming by most other populations of Russell's vipers. This study aimed to investigate evidence of myotoxicity in Russell's viper envenoming, response to antivenom and the toxins responsible for myotoxicity. METHODOLOGY AND FINDINGS:Clinical features of myotoxicity were assessed in authenticated Russell's viper bite patients admitted to a Sri Lankan teaching hospital. Toxins were isolated using high-performance liquid chromatography. In-vitro myotoxicity of the venom and toxins was investigated in chick biventer nerve-muscle preparations. Of 245 enrolled patients, 177 (72.2%) had local myalgia and 173 (70.6%) had local muscle tenderness. Generalized myalgia and muscle tenderness were present in 35 (14.2%) and 29 (11.8%) patients, respectively. Thirty-seven patients had high (>300 U/l) serum creatine kinase (CK) concentrations in samples 24h post-bite (median: 666 U/l; maximum: 1066 U/l). Peak venom and 24h CK concentrations were not associated (Spearman's correlation; p = 0.48). The 24h CK concentrations differed in patients without myotoxicity (median 58 U/l), compared to those with local (137 U/l) and generalised signs/symptoms of myotoxicity (107 U/l; p = 0.049). Venom caused concentration-dependent inhibition of direct twitches in the chick biventer cervicis nerve-muscle preparation, without completely abolishing direct twitches after 3 h even at 80 ?g/ml. Indian polyvalent antivenom did not prevent in-vitro myotoxicity at recommended concentrations. Two phospholipase A2 toxins with molecular weights of 13kDa, U1-viperitoxin-Dr1a (19.2% of venom) and U1-viperitoxin-Dr1b (22.7% of venom), concentration dependently inhibited direct twitches in the chick biventer cervicis nerve-muscle preparation. At 3 ?M, U1-viperitoxin-Dr1a abolished twitches, while U1-viperitoxin-Dr1b caused 70% inhibition of twitch force after 3h. Removal of both toxins from whole venom resulted in no in-vitro myotoxicity. CONCLUSION:The study shows that myotoxicity in Sri Lankan Russell's viper envenoming is mild and non-life threatening, and due to two PLA2 toxins with weak myotoxic properties.
Project description:<b>Background: </b>Detection and quantification of snake venom in envenomed patients' blood is important for identifying the species responsible for the bite, determining administration of antivenom, confirming whether sufficient antivenom has been given, detecting recurrence of envenoming, and in forensic investigation. Currently, snake venom detection is not available in clinical practice in Sri Lanka. This study describes the development of enzyme immunoassays (EIA) to differentiate and quantify venoms of Russell's viper (Daboia russelii), saw-scaled viper (Echis carinatus), common cobra (Naja naja), Indian krait (Bungarus caeruleus), and hump-nosed pit viper (Hypnale hypnale) in the blood of envenomed patients in Sri Lanka.<br><br><b>Methodology / principal findings: </b>A double sandwich EIA of high analytical sensitivity was developed using biotin-streptavidin amplification for detection of venom antigens. Detection and quantification of D. russelii, N. naja, B. caeruleus, and H. hypnale venoms in samples from envenomed patients was achieved with the assay. Minimum (less than 5%) cross reactivity was observed between species, except in the case of closely related species of the same genus (i.e., Hypnale). Persistence/ recurrence of venom detection following D. russelii envenoming is also reported, as well as detection of venom in samples collected after antivenom administration. The lack of specific antivenom for Hypnale sp envenoming allowed the detection of venom antigen in circulation up to 24 hours post bite.<br><br><b>Conclusion: </b>The EIA developed provides a highly sensitive assay to detect and quantify five types of Sri Lankan snake venoms, and should be useful for toxinological research, clinical studies, and forensic diagnosis.
Project description:In vitro antivenom efficacy studies were compared to rodent lethality studies to test two Indian snake antivenoms (VINS and BHARAT) against four Sri Lankan snakes. In vitro efficacy was tested at venom concentrations consistent with human envenoming. Efficacy was compared statistically for one batch from each manufacturer where multiple vials were available. In binding studies EC50 for all VINS antivenoms were less than BHARAT for D. russelii [553 μg/mL vs. 1371 μg/mL;p = 0.016), but were greater for VINS antivenoms compared to BHARAT for N. naja [336 μg/mL vs. 70 μg/mL;p < 0.0001]. EC50 of both antivenoms was only slighty different for E. carinatus and B. caeruleus. For procoagulant activity neutralisation, the EC50 was lower for VINS compared to BHARAT - 60 μg/mL vs. 176 μg/mL (p < 0.0001) for Russell's viper and 357 μg/mL vs. 6906μg/mL (p < 0.0001) for Saw-scaled viper. Only VINS antivenom neutralized in vitro neurotoxicity of krait venom. Both antivenoms partially neutralized cobra and didn't neutralize Russell's viper neurotoxicity. Lethality studies found no statistically significant difference in ED50 values between VINS and BHARAT antivenoms. VINS antivenoms appeared superior to BHARAT at concentrations equivalent to administering 10 vials antivenom, based on binding and neutralisation studies. Lethality studies were inconsistent suggesting rodent death may not measure relevant efficacy outcomes in humans.
Project description:Russell's vipers (RVs) envenoming is an important public health issue in South-East Asia. Disseminated intravascular coagulopathy, systemic bleeding, hemolysis, and acute renal injury are obvious problems that develop in most cases, and neuromuscular junction blocks are an additional problem caused by western RV snakebite. The complex presentations usually are an obstacle to early diagnosis and antivenom administration. Here, we tried to produce highly specific antibodies in goose yolks for use in a paper-based microfluidic diagnostic kit, immunochromatographic test of viper (ICT-Viper), to distinguish RVs from other vipers and even cobra snakebite in Asia. We used indirect ELISA to monitor specific goose IgY production and western blotting to illustrate the interaction of avian or mammal antibody with venom proteins. The ICT-Viper was tested not only in prepared samples but also in stored patient serum to demonstrate its preliminary efficacy. The results revealed that specific anti-Daboia russelii IgY could be raised in goose eggs effectively without inducing adverse effects. When it was collocated with horse anti-Daboia siamensis antibody, which broadly reacted with most of the venom proteins of both types of Russell's viper, the false cross-reactivity was reduced, and the test showed good performance. The limit of detection was reduced to 10 ng/ml in vitro, and the test showed good detection ability in clinical snake envenoming case samples. The ICT-Viper performed well and could be combined with a cobra venom detection kit (ICT-Cobra) to create a multiple detection strip (ICT-VC), which broadens its applications while maintaining its detection ability for snake envenomation identification. Nonetheless, the use of the ICT-Viper in the South-East Asia region is pending additional laboratory and field investigations and regional collaboration. We believe that the development of this practical diagnostic tool marks the beginning of positive efforts to face the global snakebite issue.
Project description:The venom proteome of Siamese Russell's viper from Taiwan, alongside complementary in vivo lethality neutralization assay and in vitro third-generation antivenomics assessment of the preclinical efficacy of the homologous antivenom manufactured in Taiwan CDC's Vaccine Center, are here reported. Taiwanese Russell's viper venom proteome comprised 25 distinct gene products, with the heterodimeric PLA₂ viperotoxin-F representing the most abundant toxin (47.5% of total venom proteome). Coagulation FV-activating serine proteinase (RVV-V, 14%), the PIV-SVMP activator of FX (RVV-FX, 8.5%), and less abundant toxins from nine protein families, make up its venom proteome. Venom composition-pathology correlations of D. siamensis envenomings in Taiwan are discussed. The lethal effect of Taiwanese D. siamensis venom was 0.47 mg/g mouse. Antivenomics-guided assessment of the toxin recognition landscape of the Taiwanese Russell's viper antivenom, in conjunction with complementary in vivo neutralization analysis, informed the antivenom's maximal toxin immunorecognition ability (14 mg total venom proteins/vial), neutralization capacity (6.5 mg venom/vial), and relative content of lethality neutralizing antibodies (46.5% of the toxin-binding F(ab')₂ antibodies). The antivenomics analysis also revealed suboptimal aspects of the CDC-Taiwan antivenom. Strategies to improve them are suggested.
Project description:<h4>Background</h4>Snake envenoming is a major clinical problem in Sri Lanka, with an estimated 40,000 bites annually. Antivenom is only available from India and there is a high rate of systemic hypersensitivity reactions. This study aimed to investigate whether the rate of infusion of antivenom reduced the frequency of severe systemic hypersensitivity reactions.<h4>Methods and findings</h4>This was a randomized comparison trial of two infusion rates of antivenom for treatment of non-pregnant adult patients (>14 y) with snake envenoming in Sri Lanka. Snake identification was by patient or hospital examination of dead snakes when available and confirmed by enzyme-immunoassay for Russell's viper envenoming. Patients were blindly allocated in a 11 randomisation schedule to receive antivenom either as a 20 minute infusion (rapid) or a two hour infusion (slow). The primary outcome was the proportion with severe systemic hypersensitivity reactions (grade 3 by Brown grading system) within 4 hours of commencement of antivenom. Secondary outcomes included the proportion with mild/moderate hypersensitivity reactions and repeat antivenom doses. Of 1004 patients with suspected snakebites, 247 patients received antivenom. 49 patients were excluded or not recruited leaving 104 patients allocated to the rapid antivenom infusion and 94 to the slow antivenom infusion. The median actual duration of antivenom infusion in the rapid group was 20 min (Interquartile range[IQR]:20-25 min) versus 120 min (IQR:75-120 min) in the slow group. There was no difference in severe systemic hypersensitivity reactions between those given rapid and slow infusions (32% vs. 35%; difference 3%; 95%CI:-10% to +17%;p = 0.65). The frequency of mild/moderate reactions was also similar. Similar numbers of patients in each arm received further doses of antivenom (30/104 vs. 23/94).<h4>Conclusions</h4>A slower infusion rate would not reduce the rate of severe systemic hypersensitivity reactions from current high rates. More effort should be put into developing better quality antivenoms.<h4>Trial registration</h4>www.slctr.lk SLCTR/2007/005.
Project description:BACKGROUND: Snake bite is one of the most neglected public health issues in poor rural communities worldwide. In addition to the clinical effects of envenoming, treatment with antivenom frequently causes serious adverse reactions, including hypersensitivity reactions (including anaphylaxis) and pyrogenic reactions. We aimed to investigate the immune responses to Sri Lankan snake envenoming (predominantly by Russell's viper) and antivenom treatment. METHODOLOGY/PRINCIPAL FINDINGS: Plasma concentrations of Interleukin (IL)-6, IL-10, tumor necrosis factor ? (TNF?), soluble TNF receptor I (sTNFRI), anaphylatoxins (C3a, C4a, C5a; markers of complement activation), mast cell tryptase (MCT), and histamine were measured in 120 Sri Lankan snakebite victims, both before and after treatment with antivenom. Immune mediator concentrations were correlated with envenoming features and the severity of antivenom-induced reactions including anaphylaxis. Envenoming was associated with complement activation and increased cytokine concentrations prior to antivenom administration, which correlated with non-specific systemic symptoms of envenoming but not with coagulopathy or neurotoxicity. Typical hypersensitivity reactions to antivenom occurred in 77/120 patients (64%), satisfying criteria for a diagnosis of anaphylaxis in 57/120 (48%). Pyrogenic reactions were observed in 32/120 patients (27%). All patients had further elevations in cytokine concentrations, but not complement activation, after the administration of antivenom, whether a reaction was noted to occur or not. Patients with anaphylaxis had significantly elevated concentrations of MCT and histamine. CONCLUSIONS/SIGNIFICANCE: We have demonstrated that Sri Lankan snake envenoming is characterized by significant complement activation and release of inflammatory mediators. Antivenom treatment further enhances the release of inflammatory mediators in all patients, with anaphylactic reactions characterised by high levels of mast cell degranulation but not further complement activation. Anaphylaxis is probably triggered by non allergen-specific activation of mast cells and may be related to the quality of available antivenom preparations, as well as a priming effect from the immune response to the venom itself.
Project description:BACKGROUND:Daboia siamensis (Eastern Russell's viper) is a medically important snake species found widely distributed across Southeast Asia. Envenomings by this species can result in systemic coagulopathy, local tissue injury and/or renal failure. While administration of specific antivenom is an effective treatment for Russell's viper envenomings, the availability of, and access to, geographically-appropriate antivenom remains problematic in many rural areas. In this study, we determined the binding and neutralizing capability of antivenoms manufactured by the Thai Red Cross in Thailand against D. siamensis venoms from four geographical locales: Myanmar, Taiwan, China and Thailand. METHODOLOGY/PRINCIPLE FINDINGS:The D. siamensis monovalent antivenom displayed extensive recognition and binding to proteins found in D. siamensis venom, irrespective of the geographical origin of those venoms. Similar immunological characteristics were observed with the Hemato Polyvalent antivenom, which also uses D. siamensis venom as an immunogen, but binding levels were dramatically reduced when using comparator monovalent antivenoms manufactured against different snake species. A similar pattern was observed when investigating neutralization of coagulopathy, with the procoagulant action of all four geographical venom variants neutralized by both the D. siamensis monovalent and the Hemato Polyvalent antivenoms, while the comparator monovalent antivenoms were ineffective. These in vitro findings translated into therapeutic efficacy in vivo, as the D. siamensis monovalent antivenom was found to effectively protect against the lethal effects of all four geographical venom variants preclinically. Assessments of in vivo nephrotoxicity revealed that D. siamensis venom (700 ?g/kg) significantly increased plasma creatinine and blood urea nitrogen levels in anaesthetised rats. The intravenous administration of D. siamensis monovalent antivenom at three times higher than the recommended scaled therapeutic dose, prior to and 1 h after the injection of venom, resulted in reduced levels of markers of nephrotoxicity and prevented renal morphological changes, although lower doses had no therapeutic effect. CONCLUSIONS/SIGNIFICANCE:This study highlights the potential broad geographical utility of the Thai D. siamensis monovalent antivenom for treating envenomings by the Eastern Russell's viper. However, only the early delivery of high antivenom doses appears to be capable of preventing venom-induced nephrotoxicity.