Green pit viper (Trimeresurus albolabris and T. macrops) venom antigenaemia and kinetics in humans

Green pit viper bite is a common public health problem in Southeast Asia. Although most patients experience only local swelling, some may suffer from severe systemic bleeding that can be delayed. Venom antigenaemia was measured by enzyme-linked immunosorbent assay and correlated with clinical findings in 42 patients. Initial venom antigenaemia was not predictive enough for clinical uses. A kinetic study (n = 27) showed highest levels at presentation and, then, progressive decline. The average half-life was 27.5 h during the first three days and over 50 h on days 5-7 after bite. Two small subsets (7.4% each) showed persistently detectable venom on day 14 and a subsequent rise in venom antigenaemia. They were associated with prolonged thrombocytopaenia and coagulopathy, respectively. These data demonstrated the long half-life of the venom, suggesting that waiting for spontaneous resolution of coagulopathy is not preferable. In addition, the delayed venom disappearance, not the initial values, was correlated with haemostatic disorders.     

Dark green pit viper (Trimeresurus popeorum) bite: clinical and serial coagulation profiles in 51 cases

Trimeresurus popeorum, a dark green pit viper, is commonly found in Southeast Asia. This study describes the clinical picture and blood studies of 51 patients bitten by this snake. Affected limbs were swollen; and hemorrhagic blebs in fingers and toes were found in 12 patients. Lymphangitis was observed in 4 instances. Six individuals exhibited hypofibrinogenemia of 0-84 mg/dl, and 2 cases developed thrombocytopenia and bleeding. The presence of venom in the blood of these patients was demonstrated. Positive fibrin degradation products of 40-320 micrograms/ml were observed in 6 cases with hypofibrinogenemia, and in 8 other cases. Nineteen patients had short euglobulin lysis times of 51.8 +/- 24.7 min. Hyperfibrinogenemia of 626.7 +/- 288.9 mg/dl was found in 18 cases. Apart from bleeding, there were no systemic symptoms. Hypofibrinogenemia became normal in 3-12 days. The clinical course in all patients was uneventful, and none received antivenin.     

Bites by the white-lipped pit viper (Trimeresurus albolabris) and other species in Hong Kong. A survey of 4 years' experience at the Prince of Wales Hospital

The case records of 242 snake bite victims admitted to the Prince of Wales Hospital in Hong Kong between September 1984 and October 1988 were studied retrospectively. When the snake was identified, the White-lipped pit viper (Trimeresurus albolabris) was by far the commonest species involved. In addition to local oedema and inflammation, evidence of a significant blood clotting disturbance was present in at least 10% of cases, defibrination and thrombocytopenia being the commonest findings. Since such abnormality was not always sought the true figure is likely to be higher. Three fatalities occurred, one of which was secondary to a probable White-lipped pit viper bite, one to a bite by Chinese cobra and one to a bite by Russell's viper.     

The species of green pit viper in Bangkok

The aims of this study were to delineate the species of Green pit viper and clinical patterns in Bangkok areas. This study was a blind independent comparison. Among 188 Green pit vipers collected only two species, i.e. Trimeresurus alborabris and T. macrops were found. There were no differences in snake sizes and in clinical patterns of snake bite. The majority of the clinical cases (90.1%) was classified as of mild degree of severity and severe envenomation was observed only in T. alborabris victims. The ratio between T. alborabris and T. macrops victims in Bangkok residents was 1.7:1 but the ratio was reversed in Thonburi residents.     

Cross neutralization of Hypnale hypnale (hump-nosed pit viper) venom by polyvalent and monovalent Malayan pit viper antivenoms in vitro and in a rodent model

Hypnale hypnale (hump-nosed pit viper) is a medically important venomous snake in Sri Lanka and Southwestern India. Bite of this snake may result in hemostatic dysfunction, acute kidney injury and death. Clinical studies indicated that the locally available polyvalent antivenoms produced in India are not effective against hump-nosed pit viper envenoming. Hence, there is an urgent need to search for effective antivenom. In this paper, we examined the ability of Calloselasma rhodostoma (Malayan pit viper) monovalent antivenom and the Hemato polyvalent antivenom (both produced by Thai Red Cross Society, TRCS) to neutralize the lethality and toxic effects of H. hypnale venom, as C. rhodostoma is considered a sister taxon of H. hypnale. In vitro neutralization studies showed that the Hemato polyvalent antivenom effectively neutralized the lethality of H. hypnale venom (1.52 mg venom/mL antivenom) as well as the hemorrhagic, procoagulant and necrotic activities of the venom. The monovalent C. rhodostoma antivenom could also neutralize the lethality and toxic activities of the venom, but the potency was lower. The Hemato polyvalent antivenom also effectively protected mice from the lethal and local effects of H. hypnale venom in an in vivo rodent model of envenoming. Furthermore, the polyvalent antivenom could also effectively neutralize the venom of Daboia russelii (2.50 mg venom/mL antivenom), another common cause of snake bites in Sri Lanka and South India. These findings suggested that the Hemato polyvalent antivenom may be beneficial in the antivenom treatment of H. hypnale envenoming.     

Alleviation of viper venom induced platelet apoptosis by crocin (Crocus sativus): implications for thrombocytopenia in viper bites

Viper envenomations are characterized by prominent local and systemic manifestations including hematological alterations. Snake venom metalloproteinases (SVMPs) and phospholipase A₂ (PLA₂) plays crucial role in the pathophysiology of hemorrhage by targeting/altering the platelets function which may result in thrombocytopenia. Platelets undergo the classic events of mitochondria-mediated apoptotic pathway due to augmented endogenous reactive oxygen species (ROS) levels. The observed anticoagulant effects during viper envenomations could be due to exacerbated platelet apoptosis and thrombocytopenia. Moreover, antivenin treatments are ineffective against the venom-induced oxidative stress; therefore, it necessitates an auxiliary therapy involving antioxidants which can effectively scavenge the endothelium-generated/endogenous ROS and protect the platelets. The present study explored the effects of viper venom on platelet apoptosis and its amelioration by a phytochemical crocin. The study evaluated the Vipera russelli venom-induced apoptotic events including endogenous ROS generation, intracellular Ca²⁺ mobilization, mitochondrial membrane depolarization, cyt-c translocation, caspase activation and phosphatidylserine externalization which were effectively mitigated when the venom was pre-treated with crocin. The study highlights one of the less studied features of venom-induced secondary complications i.e. platelet apoptosis and sheds light on the underlying basis for venom-induced thrombocytopenia, systemic hemorrhage and in vivo anticoagulant effect.     

Clinical significance of venom antigen levels in patients envenomed by the Malayan pit viper (Calloselasma rhodostoma)

Serial venom antigen levels were measured by enzyme-linked immunosorbent assay (ELISA) in 46 patients with systemic envenoming by the Malayan pit viper (Calloselasma rhodostoma), a major cause of snake bite in Southeast Asia. The principal effects of the venom are defibrination, hemorrhage and local tissue necrosis. Admission venom levels, which varied between 0 and 595 ng/ml, correlated with the incidence of spontaneous systemic bleeding, blood incoagulability and concentrations of plasma fibrinogen and serum fibrin degradation products. The presence or absence of nonclotting blood also correlated with the time elapsed between the bite and hospital admission. The development of nonclotting blood may be delayed by up to 72 hr after the bite even though circulating venom and raised FDP may be detected at presentation. This is probably explained by a temporary equilibrium between synthesis and consumption of fibrinogen. Venom antigenemia recurred in 12 patients (26%) suggesting continuous absorption of venom from the wound or saturation of extravascular binding sites. Admission venom levels also correlated with the extent of local swelling and the occurrence of tissue necrosis at the site of the bite. Venom was detected in 87% of wound aspirates and 88% of urine specimens taken on admission. Tourniquets, of the type used in rural Thailand, did not delay the absorption of venom into the circulation.     

TA-2, a thrombin-like enzyme from the Chinese white-lipped green pitviper (Trimeresurus albolabris): isolation, biochemical and biological characterization

Through three chromatographic steps, a new thrombin-like enzyme (TLE), named TA-2, from the venom of the Chinese white-lipped green pitviper (Trimeresurus albolabris) has been isolated and purified to homogeneity. TA-2 was a single-chain glycoprotein with about 6% sugar, pI 3.9 and a molecular weight of 38.8 kD. Its N-terminal sequence (VVGGDECNIN) showed high sequence conformity with many other TLEs. In vitro, it coagulated bovine fibrinogen (108.6 NIH units/mg) and cleaved the Aα and Bβ chains of bovine fibrinogen-releasing fibrinopeptide A and B, but did not degrade bovine fibrin; displayed high stability at different temperature, pH, and presence of several divalent cations and inhibitors; also exhibited strong activity towards casein (192.3 units/mg) and high esterase activity upon Nα-p-tosyl-L-arginine methyl ester (11 units/mg); and behaved as a promoter to platelet aggregation induced by ADP or collagen. In vivo, TA-2 caused dose-dependent prolongation of bleeding time in mice, but had no hemorrhagic and edema-inducing activities even at high concentrations.     

Immunological properties of Hypnale hypnale (hump-nosed pit viper) venom: antibody production with diagnostic and therapeutic potentials

Envenomation by hump-nosed pit viper (Hypnale hypnale, Hh) in Sri Lanka has caused significant morbidity and mortality, attributed to 35% of total venomous snakebites. In Southwestern India (Kerala), H. hypnale was increasingly identified as a dangerous and common source of envenomation, second to the Russell's viper but ahead of the cobra bites. Unfortunately, there is still no specific antivenom to date. This study aims to investigate the immunological properties of the venom and to assess the feasibility of specific Hh antivenom production as well as the development of a diagnostic assay. Hh venom elicited satisfactory titers of anti-Hh IgG in rabbits after 3rd immunization. The anti-Hh IgG, isolated with caprylic acid precipitation method, was effective in neutralizing the venom lethality (potency=48 LD(50) per ml IgG) as well as its procoagulant, hemorrhagic and necrotic effects, indicating the possibility to produce the specific antivenom using the common immunization regime. Cross-reactivity studies using indirect ELISA showed that anti-Hh IgG cross-reacted extensively with several Asiatic crotalid venoms, particularly that of Calloselasma rhodostoma (73.6%), presumably due to the presence of venom antigens common to both snakes. Levels of immunological cross-reactivity were vastly reduced with double-sandwich ELISA. Further work demonstrated that the assay was able to distinguish and quantify venoms of H. hypnale, Daboia russelii and Echis carinatus sinhaleyus (three common local viperid) used to spike human sera at various concentrations. The assay hence may be a useful investigating tool for diagnosing biting species and studying the time course profile of venom concentrations in blood.     

Stability of Russell's viper venom toxoid (lyophilized form) on storage

A previously developed Russell's viper venom toxoid in Myanmar is in a liquid form that shows reversion in the form of a reduced number of formaldehyde linkages and toxicity during storage at 37 degrees C and at room temperature. In order to have a safe, potent and stable toxoid, a lyophilized form was prepared in the present study from the liquid toxoid through the use of a freeze dryer. Both the liquid and lyophilized forms were then stored at 4 degrees C and at room temperature, and in addition to safety and immunogenicity tests, biochemical parameters such as the protein content, the activity of venom enzymes (proteinase, phospholipase A, phosphodiesterase, and arginine esterase), and the released free formalin amounts were assessed at 3-month intervals over a period of 1 year. The results indicate that under both conditions, the lyophilized toxoid shows minimum changes in enzyme activity, a reduced tendency toward formaldehyde linkage, no toxicity, and more immunogenicity in comparison with the respective liquid toxoids. It could therefore be hypothesized that Russell's viper venom toxoid in a lyophilized form is more promising in terms of efficacy and stability for prophylactic use in human immunization than the conventional toxoid in a liquid form.     

The Russell viper venom time (RVVT) test for investigation of lupus anticoagulant (LA)

Lupus anticoagulants (LAs) are a laboratory representation of the clinical syndrome of antiphospholipid syndrome (APS), and can also arise in other pathological states. Laboratory testing for LA is complex and three separate recent guidelines have been published. One test, the Russell viper venom time (RVVT), is the mandated laboratory test for inclusion in LA identification/exclusion in all three guidance documents. This is because the the RVVT is recognized to have great sensitivity for LA, with this generally recognized to be greater than that of most other LA screening assays. However, the RVVT is also very sensitive to the presence of many anticoagulant drugs, which diminishes its specificity for LA. Various strategies can be used to improve LA specificity and reduce anticoagulant assay interference.     

The action of Russell''s viper venom on fibrin formation and fibrinolysis in vivo

Envenoming by Russell's viper caused a marked rise in FPA, B beta 15-42 fragment and fibrin derived cross-linked D-dimer fragment indicative of a consumptive coagulopathy with hyperfibrinolysis. There was no increase in tPA or tPA-I levels post envenoming, which suggests that the increase in fibrinolytic activity was not due to venom-induced release of tPA from the vessel walls but may have been attributable to a direct effect of the venom or to a secondary physiological response to fibrin deposition. The effectiveness of the antivenom is demonstrated by its ability to prevent further cleavage of fibrinogen and the return to normal fibrinogen levels by 24 h. A secondary rise in FPA at this time indicates that the initial dose of antivenom may have been too small. The antivenom alone or in combination with the venom causes the release of tPA, tPA-I and vWF by the vessel walls. This may be a consequence of the severe anaphylactic reactions seen in some patients.     

花鲈感染简单异尖线虫及扫描电镜观察

目的简单异尖线虫(Anisakis simplex)是一类寄生于海水鱼类及哺乳类动物,可引起人类异尖线虫病(anisaki-osis)的食源性人兽共患寄生虫。作者从浙江宁波患病花鲈(Latealabrax japonicus)体内检出简单异尖线虫,检出率为100%(11/11)。患病鲈鱼体表无异常症状,解剖鱼体在肝脏、胃壁、肠壁、肠系膜等部位均有简单异尖线虫寄生,线虫盘成螺旋状包于包囊内,病鱼感染线虫强度为20～30条/尾。感染严重病鱼出现胃穿孔、胃壁出血、肝脏具白色结节等病症。光学显微镜观察线虫虫体为长圆筒形,两端较细,肌质食道,腺体的胃位于食管后面,黑色不透明,和肠连接处有一斜的分界线。扫描电镜观察虫体体表具致密环纹,前端钝圆,具1个钻齿和4个钝角状乳突,排泄口位于腹侧两个乳突之间,尾部短而圆,末端具一明显尾突。     

Fatal diffuse thrombotic microangiopathy after a bite by the "Fer-de-Lance" pit viper (Bothrops lanceolatus) of Martinique

In Martinique, a man bitten two days earlier by a pit viper (Bothrops lanceolatus) was hospitalized with impaired consciousness and tetraplegia. Investigations confirmed cerebral and myocardial infarctions. Resolving thrombocytopenia was associated with virtually normal blood prothrombin time/activated partial thromboplastin time but increasing hyperfibrinogenemia. Despite specific antivenom treatment, he developed fatal left ventricular failure six days after the bite. At autopsy, multiple cerebral, myocardial and mesenteric infarctions were found. Rupture of mitral chordae tendinae was the likely cause of death. Histopathologic examination showed multi-focal thrombotic microangiopathy with intimal-medial dissection by thrombi extending from foci of endothelial damage in small cerebral, myocardial, pulmonary, mesenteric, and interlobular renal arteries and arterioles. These findings were the causes of infarctions. There was intense angiogenesis in organizing cerebral infarcts. Immunohistochemical analysis showed platelet aggregates and endothelial cells within microthrombi. Viperidae venoms contain vascular endothelial toxins, notably metalloproteinase hemorrhagins, but von Willebrand factor activators or vascular endothelial growth factor-type factors are more likely to have been implicated in this case.     

Use of a simplified dilute Russell's viper venom time (DRVVT) confirms heterogeneity among 'lupus anticoagulants'

A simplified dilute Russell's viper venom time (DRVVT) test--in which the venom, trace phospholipid and calcium were combined into a single reagent--was evaluated for the detection of lupus anticoagulants (LA) in 28 plasma samples containing non-specific circulating anticoagulants. In agreement with previous studies, the DRVVT was found to be insensitive to defects in contact and haemophilic factors and was only marginally affected by antibodies directed against factor VIII. Thus, the use of a DRVVT test in investigations of anticoagulants reduces the risk of confusing a haemorrhagic inhibitor of factor VIII with a non-haemorrhagic LA. In comparisons of sensitivity against activated partial thromboplastin time tests (APTT-Actin FSL and Organon-Teknika reagents) the simplified DRVVT was prolonged slightly more than the APTT in most of the test plasmas containing various non-specific circulating anticoagulants. Three anticoagulants affecting APTTs more than the DRVVT were found to be associated with anticardiolipin IgMs. APTT-prolonging anticoagulants, whether prolonging DRVVT tests or not, showed similar 'correction' of their APTTs by the addition of platelets or phospholipid. Thus, phospholipid-dependent or LA show heterogeneity. Those affecting only the APTT and not DRVVT should perhaps be classified differently.