Utility of Three Serum Biomarkers for Early Detection of Systemic Envenoming Following Viper Bites in Sri Lanka.

STUDY OBJECTIVE: Early detection of systemic envenoming is critical for early antivenom therapy to minimize morbidity and mortality from snakebite. We assessed the diagnostic utility of 3 serum biomarkers in the early detection of systemic envenoming in viper bites in rural Sri Lanka. METHODS: All confirmed snakebite patients admitted to Teaching Hospital Anuradhapura from July 2020 to June 2021 were included. On admission, blood was collected for venom concentrations, prothrombin time/international normalized ratio, fibrinogen concentration, serum creatinine concentration, and 3 serum biomarkers, namely secretory phospholipase A2 (sPLA2) activity, neutrophil gelatinase-associated lipocalin (sNGAL) concentration, and clusterin (sClu) concentration. Systemic envenoming was defined by the presence of venom-induced consumption coagulopathy, neurotoxicity, acute kidney injury, or the presence of nonspecific clinical effects. RESULTS: A total of 237 confirmed snakebite patients (Russell's viper, 72; hump-nosed viper, 80; nonvenomous snakes, 31; and unidentified bites, 54) with sufficient preantivenom serum samples were recruited [median age: 42 years (interquartile range [IQR] 29 to 53 years); 173 men (73%)]. Systemic envenoming occurred in 68 (94%) Russell's viper bites, 48 (60%) hump-nosed viper bites, and 45 (83%) unidentified bites. The median sPLA2 activity was 72 nmol/mL/min (IQR 30 to 164) for Russell's viper envenoming, 12 nmol/mL/min (IQR 9 to 16) for hump-nosed viper envenoming, and 11 nmol/mL/min (IQR 9 to 14) for nonvenomous bites. There was no difference in sNGAL and sCLu concentrations among the 3 groups. The median sPLA2 activity of patients with systemic envenoming was 16 nmol/min/mL (IQR 11 to 59) compared to 11 nmol/min/mL (IQR 9 to 14) in patients without systemic envenoming; the difference between medians was 5 nmol/min/mL (95% confidence interval [CI] 4 to 12). The area under the receiver operator characteristic curve (AUC-ROC) of admission sPLA2 activity was the best predictor of systemic envenoming in all snakebites (AUC-ROC 0.72, 95% CI 0.66 to 0.79), whereas sNGAL and sClu concentrations were poor predictors. sPLA2 activity was a better predictor of systemic envenoming in Russell's viper bites (AUC-ROC 0.90, 95% CI 0.76 to 1.00) and in those presenting within 2 hours of a bite. A sPLA2 activity more than 23.5 nmol/min/mL had a sensitivity of 41% (95% CI 34% to 49%), and a specificity of 97% (95% CI 91% to 99.5%) in predicting systemic envenoming. A sPLA2 activity of more than 46 nmol/min/mL on admission had a sensitivity of 67% (95% CI 55% to 77%) and a specificity of 100% (95% CI 51% to 100%) in predicting systemic envenoming in Russell's viper bites. CONCLUSIONS: sPLA2 activity is an early predictor of systemic envenoming following snakebite, particularly in Russell's viper bites and in those who present early.

Health issues and related health-seeking behaviours of snakebite patients after hospital discharge: A cohort study from rural Sri Lanka.

We aimed to assess the unresolved health issues experienced by a cohort of snakebite survivors and their health-seeking behaviours during the first three months after the snakebite. Patients from the Anuradhapura snakebite cohort admitted to the Teaching Hospital Anuradhapura, Sri Lanka, from July 2021 to June 2022 were recruited. Patients were interviewed over the telephone three weeks and three months post-bite to collect data on unresolved health problems post-discharge, patient's adherence to the review plan, newly experienced health issues, health-seeking behaviours and the effect on daily routine. Only snakebite survivors who could be contacted at both three weeks and three months were included. Of 710 eligible patients, 384 (54%) were contactable at both reviews. On discharge from the hospital, 248/384 (65%) had unresolved effects of the snakebite, including 224/384 (58%) who had local effects. The unresolved health problems were reported by patients bitten by H. hypnale (54%), D. russelii (23%), and unidentified snakes (19%). At three weeks and three months, 98/384 (26%) and 52/384 (14%) still had unresolved local effects of envenoming, respectively. Of 144/384 (38%) who were advised to attend review visits post-discharge, mostly to assess renal function, 118 (82%) complied. 112/384 (29%) patients reported self-motivated treatment seeking for unresolved effects of snakebite over the three months. Of them, 87 (78%) visited Sri Lankan indigenous medical practitioners. Patients missed a median of two working days (IQR: 2-4 days) post-discharge. 26 (6.7%) were unable to return to work at 3 weeks, and five patients at 3 months. In rural Sri Lanka, a significant number of viper bite patients leave hospital with mild persistent local effects, which commonly leads to them seeking further treatment. Despite that, almost all snakebite survivors had returned to work at three months post-bite.

Merrem's Hump-Nosed Viper (Hypnale hypnale) Bite on the Tongue of an Infant Leading to Upper Airway Obstruction: An Unusual Presentation.

Snakebites in unusual anatomical locations may lead to life-threatening consequences. Merrem's hump-nosed viper (Hypnale hypnale) is a medically important snake in Sri Lanka and India that causes many bites and envenomings. Their bites occur almost exclusively on upper and lower limbs and commonly result in local effects, with some patients developing systemic envenoming. No antivenom is available for treating envenoming by H. hypnale. We report an unusual case of H. hypnale bite on the tongue of a 10-month-old infant resulting in rapid local swelling of the tongue and floor of the mouth, requiring prompt intervention to prevent life-threatening upper airway obstruction. Early tracheostomy prevented upper airway obstruction and, along with supportive steroid therapy and antibiotics, led to a complete resolution of the local effects of the infant without permanent disability, despite the unavailability of antivenom.

Investigating skeletal muscle biomarkers for the early detection of Australian myotoxic snake envenoming: an animal model pilot study.

INTRODUCTION: Myotoxicity is an important toxidrome that can occur with envenoming from multiple Australian snake types. Early antivenom administration is an important strategy to reduce the incidence and severity of myotoxicity. The current gold standard biomarker, serum creatine kinase activity, does not rise early enough to facilitate early antivenom administration. Several other skeletal muscle biomarkers have shown promise in other animal models and scenarios. The aim of this study was to examine the predictive values of six skeletal muscle biomarkers in a rat model of Australian snake myotoxicity. METHODS: Sprague-Dawley rats were anaesthetised and administered either Pseudechis porphyriacus (red-bellied black snake) or Notechis scutatus (tiger snake) venom, or normal saline via intramuscular injection. Blood samples were collected. Assays were performed for serum creatine kinase skeletal muscle troponin-I concentration, skeletal muscle troponin-C concentration, myoglobin activity, skeletal muscle myosin light chain-1 concentration, and creatine kinase-MM activity. Serum markers were plotted against time, with comparison of area under the concentration (or activity)-time curve. The predictive values of six skeletal muscle biomarkers were examined using receiver operating characteristic curves. RESULTS: There was no difference in area under the serum creatine kinase activity-time curve between venom and control groups. Serum creatine kinase-MM activity rose early in the venom treated rats, which had a significantly greater area under the serum activity-time curve. No difference in area under the serum concentration-time curve was demonstrated for the other biomarkers. Creatine kinase-MM activity had a superior predictive values than creatine kinase activity at 0-4 hours and 0-10 hours after venom administration, as indicated by area under the receiver operating characteristic curves (95 per cent confidence intervals) of 0.91 (0.78-1.00) and 0.88 (0.73-1.00) versus 0.79 (0.63-0.95) and 0.66 (0.51-0.80). DISCUSSION: The limitations of serum creatine kinase activity in early detection of myotoxicity were demonstrated in this rat model. CONCLUSION: Serum creatine kinase-MM activity was superior for early detection of Australian myotoxic snake envenoming.

A Bite by Shaw's Short Sea Snake (Hydrophis curtus): A Case of Mild Myotoxicity or a Dry Bite?

Although sea snakes (Elapidae) are commonly encountered by fishermen, accurately authenticated envenomings by them are uncommon in clinical literature. We report an authenticated case of Shaw's short, or spine-bellied, sea snake (Hydrophis curtus) bite in a young fisherman from northern Sri Lanka. The patient had clinical and biochemical evidence of mild transient myotoxicity but no evidence of neuromuscular paralysis or significant renal injury. Consideration of the clinical manifestations suggests either a mild envenoming or a dry bite. The patient completely recovered without any antivenom therapy and was discharged on the fourth day. Prolonged observation may be beneficial to exclude complications of sea snake envenoming.

Approach to Study the Efficacy and Safety of New Complementary and Alternative Medicine Formulations: Lesson during COVID-19 from Sri Lanka.

COVID-19 affected Sri Lanka from early 2020, a time of considerable ignorance accompanied by wide media coverage of a devastating epidemic in Italy and Europe. Many were attracted to complementary and alternative medicine (CAM) or traditional medicine (TM) in this desperate situation. Several preparations were claimed to be effective against COVID-19 globally. Dammika Bandara Syrup© was one such preparation promoted for preventing and treating SARS-CoV-2 infection. It was based on bees' honey, pericarp and mace of Myristica fragrans (nutmeg), the seed of Foeniculum vulgare and fresh rhizome of Zingiber officinale, all believed to have anti-viral properties. Following an unpublished clinical study claiming efficacy, Dammika Bandara Syrup© gained wide media publicity and political patronage. The producer claimed of Goddess Kali revealing the formula added an anthropological, cultural, and religious complexity to the issue. The demand for the product increased rapidly as a debate raged both in public and in the parliament on utilizing such products in combating COVID-19. The Department of Ayurveda, which is statutorily responsible for regulating CAM/TM had to respond to the situation. The legislation to regulate such indigenous medicinal products was weak, and the crisis deepened as thousands converged to the production facility, defying mobility restrictions introduced to control COVID-19. This led to the Ministry of Health requesting academics to form a team and conduct a clinical trial to prove its efficacy. This paper outlines the process and issues faced during the regulatory approval for the trial in a polarized political environment. Some health professionals accused the researchers of bowing to political pressure and questioned the scientific justification for the trial. However, the team considered this as an opportunity to streamline a path for research into CAM/TM therapies in situations such as COVID-19. Several processes were identified and addressed, such as the provisional registration of CAM preparations, assessing the potential efficacy of a CAM product, confirmation of authenticity and safety, standardization and supervision of production respecting cultural identities, obtaining approval for human use, choice of comparators, and ethical issues. We believe the study has helped set standards and a benchmark for CAM and TM research in Sri Lanka.

Incidence of serum sickness following Indian polyvalent antivenom therapy in a cohort of snake-envenomed patients in rural Sri Lanka.

INTRODUCTION: Serum sickness is a poorly reported delayed adverse reaction following snake antivenom therapy. We aimed to assess the frequency of serum sickness associated with administering Indian polyvalent antivenom in Sri Lanka. METHODS: We recruited patients from the Anuradhapura snakebite cohort who were admitted to a rural tertiary care hospital in Sri Lanka over one year period. Patients were interviewed over the phone 21 to 28 days post-envenoming to collect data on clinical effects: fever/chills, arthralgia/myalgia, rash, malaise, headache, abdominal pain, and nausea/vomiting. The presence of three or more symptoms between the 5th to 20th days after snake envenoming was defined as serum sickness. RESULTS: We were able to contact 98/122 (80%) patients who received antivenom and 423/588 (72%) who did not receive antivenom during the study period. The treated patients received a median dose of 20 vials (interquartile range: 20-30) of Indian polyvalent antivenom and of them, 92 (92%) received premedication. However, 67/98 (68%) developed acute adverse reactions to antivenom, including 19/98 (19%) developing anaphylaxis. Only 4/98 (4%) who received antivenom met the criteria for serum sickness, compared to none who did not receive antivenom therapy. All patients who developed serum sickness were envenomed by Russell's vipers, were premedicated, and received VINS Bioproducts antivenom. Three of them were treated with hydrocortisone in the acute stage, as premedication or as a treatment for acute adverse reactions of antivenom. Although all four patients sought medical advice for their symptoms, only one was clinically suspected to be serum sickness and treated, while the others were treated for infections. CONCLUSIONS: We confirmed that Indian polyvalent antivenom use in Sri Lanka is associated with high rates of acute adverse reactions. In contrast to studies of other antivenoms only a small proportion of patients developed serum sickness.

Does snake envenoming cause chronic kidney disease? A cohort study in rural Sri Lanka.

BACKGROUND: There is limited information on the risk of chronic kidney disease (CKD) following snakebite and its relationship with chronic interstitial nephritis in agricultural communities (CINAC). We aimed to investigate CKD in patients with a confirmed snakebite in rural Sri Lanka. METHODS: Patients prospectively recruited to the Anuradhapura snakebite cohort with authenticated bites were followed up. Two groups of patients were followed up: 199 patients in group I with a snakebite (August 2013-October 2014), reviewed after 4 years, and 168 patients in group II with a snakebite (May 2017-August 2018), reviewed after one year, with serum creatinine (estimated glomerular filtration rate [eGFR]) and urinary albumin to creatinine ratio (ACR). RESULTS: There were 12/199 (6%) in group I and 9/168 (5%) in group II with AKI following snakebite; 3/12 in group I and 2/9 in group II had haemodialysis. On review after 1 and 4 years, no patient had CKD and all had an eGFR ≥60 mL/min/1.73m2. Of 234 patients with a creatinine measured on discharge, 17/140 in group I and 11/94 in group II had a low eGFR (<60mL/min/1.73m2). In group I, 14/17 had a normal eGFR after four years, including 11/12 who had AKI following snakebite, and the 3/17 with a low eGFR on review had CKD or co-morbidities for CKD. In group II, 10/11 had a normal eGFR after one year, including all nine patients with AKI following snakebite, and the one patient with a low eGFR on review had CKD. Fifty patients (25%) in group I and 43 (26%) in group II had a high urinary ACR on review, all but two in each group had microalbuminuria. Multivariate logistic regression showed in group I that only comorbidities for CKD were associated with high urinary ACR, and in group II comorbidities for CKD, snakebite associated AKI and snake type were associated with high urinary ACR. All nine patients from both groups with a low eGFR (CKD stages 3-5) had CKD prior to the snakebite or treatment for hypertension or diabetes. CONCLUSION: There was no significant association between snakebite-associated AKI and CKD in patients followed up from a snakebite cohort post-bite. Microalbuminuria was common in these patients but likely associated with hypertension, diabetes mellitus and CINAC in this rural farming population.

The accuracy of nucleic acid amplification tests (NAATs) in detecting human intestinal nematode infections: A protocol for a systematic review and meta-analysis.

Human intestinal nematode infections are a global public health issue as they can result in considerable morbidity in infected individuals, mainly in developing countries. These infections continue to go undiagnosed, as they tend to be mainly endemic in resource-poor communities where there is a shortage of experienced laboratory staff and relevant diagnostic technologies. This is further exacerbated by the nature of intermittent shedding of eggs and larvae by these parasites. Diagnostic methods range from simple morphological identification to more specialised high-throughput sequencing technologies. Microscopy-based methods, although simple, are labour-intensive and considerably less sensitive than molecular methods which are rapid and have high levels of accuracy. Molecular methods use nucleic acid amplification (NAA) to amplify the deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) fragments of the parasite to detect and determine its presence using different technologies (NAAT). They have increased the sensitivity of detection and quantitation of intestinal nematode infections, especially in low infection intensity settings. The absence of a gold standard test limits current diagnosis and, in turn, restricts intervention measures and effective control efforts. The objective of this review is to determine the accuracy of NAATs in detecting human intestinal nematode infections using Kato-Katz as the reference test for the most common soil-transmitted helminth (STH) infections and the scotch tape test for enterobiasis and Baermann method for strongyloidiasis. Relevant studies will be identified by searches in electronic databases. Two reviewers will independently screen the literature against eligibility criteria. The methodological quality of studies will then be appraised by two reviewers using the Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) tool. Discrepancies will be addressed by a third reviewer. The true positives, false positives, true negatives and false negatives of all the studies will be extracted into contingency tables. In paired forest plots, study-specific sensitivity and specificity with a 95 per cent confidence interval will be displayed. The systematic review of this protocol will report the diagnostic accuracy of currently available NAATs for the detection of human intestinal nematode infections. This will help healthcare providers and administrators determine the diagnostic method to be used in different clinical and preventive settings. Trial registration: PROSPERO registration number for this protocol is CRD42022315730.

Comparison of bedside clotting tests for detecting venom-induced consumption coagulopathy following Sri Lankan viper envenoming.

BACKGROUND: The whole blood clotting test (WBCT) is commonly used for diagnosing venom-induced consumption coagulopathy (VICC) in resource-poor settings. We aimed to investigate the diagnostic accuracy of the WBCT and capillary blood clotting test (CBCT) for detecting VICC in viper envenoming in Sri Lanka. METHODS: All confirmed snakebites admitted to Teaching Hospital Anuradhapura from July 2020 to June 2021 were included. On admission, WBCTs after 15, 20 and 25 min observation times (WBCT-15, WBCT-20 and WBCT-25) and CBCT observed in 30 s intervals (CBCT-t), 5 and 10 min CBCT (CBCT-5 and CBCT-10) were done. Blood was collected simultaneously for prothrombin time (PT)/international normalized ratio (INR) and plasma fibrinogen. We defined VICC as an INR >1.5 (Incomplete VICC = INR>1.5 and complete VICC = ≥3.0). RESULTS: A total of 272 confirmed snakebites (Russell's viper[76], hump-nosed viper[89], non-venomous snakes[51] and unidentified bites[56]) were recruited (median age: 42 y [interquartile range: 30- 53 y]; 189 males [69%]). On admission, 82 (30%) had incomplete VICC (INR >1.5 and <3) and 77 (28%) had complete VICC (INR ≥3). Sixteen (6%) developed clinically apparent bleeding. The WBCT-15 had the best sensitivity of 47% for detecting VICC and 68% for complete VICC. The sensitivities of the WBCT-20, WBCT-25, CBCT-5 and CBCT-10 was 30-35%. The sensitivities of all tests were better in detecting complete VICC, VICC in Russell's viper bites and more than 2 h post-bite. The WBCT-15 test had a sensitivity of 76% for VICC in confirmed Russell's viper bites. For detection of VICC, CBCT-t had an an excellent sensitivity of 97%, but a poor specificity of 35% for an optimal cut-off of >6.25 min. CONCLUSION: WBCTs are poorly diagnostic for VICC in Russell's viper and hump-nosed viper envenoming, missing up to two-thirds of patients for some tests. The WBCT-15 was the best test, improving for more severe VICC and greater than 2 h post-bite.

Long-term health effects perceived by snakebite patients in rural Sri Lanka: A cohort study.

The acute effects of snakebite are often emphasized, with less information on long-term effects. We aimed to describe the long-term health effects perceived by patients followed up after confirmed snakebites. Two groups of snakebite patients (>18y) from the Anuradhapura snakebite cohort were reviewed: Group I had a snakebite during August 2013-October 2014 and was reviewed after 4 years, and group II had a snakebite during May 2017-August 2018, and was reviewed after one year. Patients were invited by telephone, by sending letters, or doing home visits, including 199 of 736 patients (27%) discharged alive from group I and 168 of 438 patients (38%) from group II, a total of 367 followed up. Health effects were categorised as musculoskeletal, impact on daily life, and medically unexplained. Health issues were attributed to snakebite in 107/199 patients (54%) from group I and 55/168 patients (33%) from group II, suggesting the proportion with health issues increases with time. Sixteen patients (all viperine bites) had permanent musculoskeletal problems, none with a significant functional disability affecting daily routine. 217/367 reported being more vigilant about snakes while working outdoors, but only 21/367 were using protective footwear at review. Of 275 farmers reviewed, only six (2%) had restricted farming activities due to fear of snakebite, and only one stopped farming. 104/199 (52%) of group I and 42/168 (25%) of group II attributed non-specific symptoms (fatigue, body aches, pain, visual impairment) and/or oral cavity-related symptoms (avulsed teeth, loose teeth, receding gums) to the snakebite, which cannot be explained medically. In multivariate logistic regression, farming, type of snake, antivenom administration, and time since snakebite were associated with medically unexplained symptoms. The latter suggests medically unexplained effects increased with time. Based on two groups of snakebite patients reviewed one and four years post-bite, we show that long-term musculoskeletal disabilities are uncommon and not severe in snakebite survivors in rural Sri Lanka. However, a large portion of patients complain of various non-specific general and oral symptoms, not explainable based on the known pathophysiology of snakebite. These perceived effects of snakebite were more common in patients with systemic envenoming, and were more frequent the longer the time post-bite.

Kounis syndrome following an anaphylactic reaction to antivenom in a patient with Russell's viper (Daboia russelii) bite: A case report.

Kounis syndrome is the occurrence of acute coronary syndrome associated with mast cell and platelet activation in the setting of allergic or anaphylactic insults. Kounis syndrome has been previously reported following snake envenoming rarely, with or without antivenom therapy. We report a case of inferolateral ST elevation myocardial infarction 32 hours from a confirmed Russell's viper bite. He also had an anaphylactic reaction soon after antivenom. The absence of underlying atheromatous coronary artery disease during subsequent cardiac imaging was suggestive of a diagnosis of a type I variant of Kounis syndrome. Chest pain completely resolved by day 6 following initiation of standard treatment for acute coronary syndrome. Concurrence of allergic features and acute coronary syndrome in a snakebite patient following antivenom therapy should alert clinicians to the possibility of Kounis syndrome, which should be diagnosed with a high degree of clinical suspicion.

In Vitro Neutralization of the Myotoxicity of Australian Mulga Snake (Pseudechis australis) and Sri Lankan Russell's Viper (Daboia russelii) Venoms by Australian and Indian Polyvalent Antivenoms.

We studied the neutralisation of Sri Lankan Russell's viper (Daboia russelii) and Australian mulga snake (Pseudechis australis) venom-induced myotoxicity by Indian (Vins and Bharat) and Australian (Seqirus) polyvalent antivenoms, using the in vitro chick biventer skeletal muscle preparation. Prior addition of Bharat or Vins antivenoms abolished D. russelii venom (30 µg/mL)-mediated inhibition of direct twitches, while Australian polyvalent antivenom was not protective. Bharat antivenom prevented, while Vins and Australian polyvalent antivenoms partially prevented, the inhibition of responses to exogenous KCl. Myotoxicity of Mulga venom (10 µg/mL) was fully neutralised by the prior addition of Australian polyvalent antivenom, partially neutralised by Vins antivenom but not by Bharat antivenom. Although the myotoxicity of both venoms was partially prevented by homologous antivenoms when added 5 min after the venom, with an increasing time delay between venom and antivenom, the reversal of myotoxicity gradually decreased. However, antivenoms partially prevented myotoxicity even 60 min after venom. The effect of antivenoms on already initiated myotoxicity was comparable to physical removal of the toxins by washing the bath at similar time points, indicating that the action of the antivenoms on myotoxicity is likely to be due to trapping the toxins or steric hindrance within the circulation, not allowing the toxins to reach target sites in muscles.

The Effect of Australian and Asian Commercial Antivenoms in Reversing the Post-Synaptic Neurotoxicity of O. hannah, N. naja and N. kaouthia Venoms In Vitro.

Despite antivenoms being the only established specific treatment for neuromuscular paralysis arising from snake envenoming, their ability to reverse the post-synaptic neurotoxicity in snake envenoming is poorly understood. We investigated the ability of five commercial antivenoms i.e., King cobra monovalent, Thai cobra monovalent, Thai neuro polyvalent, Indian polyvalent and Australian polyvalent antivenoms to reverse neurotoxicity induced by the venoms of King cobra (Ophiophagus hannah, 3 µg/mL), Indian cobra (Naja naja, 5 µg/mL) and Thai cobra (Naja kaouthia, 3 µg/mL) using the in vitro chick-biventer cervicis nerve-muscle preparation. All three venoms displayed post-synaptic neurotoxicity, which was prevented by all tested antivenoms (40 µL/mL) added to the bath prior to venom. All antivenoms partially reversed the established post-synaptic neuromuscular block after the addition of the three venoms during a 180 min observation period, but to varying degrees and at different rates. The neurotoxic effects of O. hannah venom recovered to a greater magnitude (based on twitch height restoration) and faster than the neurotoxicity of N. kaouthia venom, which recovered to a lower magnitude more slowly. The recovery of post-synaptic neurotoxicity by N. naja venom was hindered due to the likely presence of cytotoxins in the venom, which cause direct muscle damage. The observations made in this study provide further evidence that the commercial antivenoms are likely to actively reverse established α-neurotoxin-mediated neuromuscular paralysis in snake envenoming, and there is cross-neutralisation with different antivenoms.

Isolation and Characterization of Two Postsynaptic Neurotoxins From Indian Cobra (Naja Naja) Venom.

The Indian Cobra (Naja naja) is among the "Big Four" responsible for most of the snakebite envenoming cases in India. Although recent proteomic studies suggest the presence of postsynaptic neurotoxins in N. naja venom, little is known about the pharmacology of these toxins. We isolated and characterized α-Elapitoxin-Nn2a (α-EPTX-Nn2a; 7020 Da) and α-Elapitoxin-Nn3a (α-EPTX-Nn3a; 7807 Da), a short-chain and long-chain postsynaptic neurotoxin, respectively, which constitute 1 and 3% of N. naja venom. α-EPTX-Nn2a (100-300 nM) and α-EPTX-Nn3a (100-300 nM) both induced concentration-dependent inhibition of indirect twitches and abolished contractile responses of tissues to exogenous acetylcholine and carbachol, in the chick biventer cervicis nerve-muscle preparation. The prior incubation of tissues with Indian polyvalent antivenom (1 ml/0.6 mg) prevented the in vitro neurotoxic effects of α-EPTX-Nn2a (100 nM) and α-EPTX-Nn3a (100 nM). The addition of Indian polyvalent antivenom (1 ml/0.6 mg), at the t90 time point, could not reverse the in vitro neurotoxicity of α-EPTX-Nn2a (100 nM). The in vitro neurotoxicity of α-EPTX-Nn3a (100 nM) was partially reversed by the addition of Indian polyvalent antivenom (1 ml/0.6 mg), as well as repeated washing of the tissue. α-EPTX-Nn2a displayed non-competitive antagonism of concentration-response curves to carbachol, with a pA2 of 8.01. In contrast, α-EPTX-Nn3a showed reversible antagonism of concentration-response curves to carbachol, with a pA2 of 8.17. De novo sequencing of α-EPTX-Nn2a and α-EPTX-Nn3a showed a short-chain and long-chain postsynaptic neurotoxin, respectively, with 62 and 71 amino acids. The important observation made in this study is that antivenom can reverse the neurotoxicity of the clinically important long-chain neurotoxin, but not the short-chain neurotoxin, from N. naja venom.

Isolation and Pharmacological Characterization of α-Elapitoxin-Oh3a, a Long-Chain Post-Synaptic Neurotoxin From King Cobra (Ophiophagus hannah) Venom.

The King Cobra (Ophiophagus hannah) is the world's largest venomous snake and has a widespread geographical distribution throughout Southeast Asia. Despite proteomic studies indicating the presence of postsynaptic neurotoxins in O. hannah venom, there are few pharmacological investigations of these toxins. We isolated and characterized α-elapitoxin-Oh3a (α-EPTX-Oh3a; 7,938 Da), a long-chain postsynaptic neurotoxin, which constitutes 5% of O. hannah venom. α-EPTX-Oh3a (100-300 nM) caused concentration-dependent inhibition of indirect twitches and inhibited contractile responses of tissues to exogenous acetylcholine and carbachol, in the chick biventer cervicis nerve-muscle preparation. The prior incubation of tissues with Thai Red Cross Society King Cobra antivenom (1 ml/0.8 mg) prevented the in vitro neurotoxic effects of α-EPTX-Oh3a (100 nM). The addition of Thai Red Cross Society King Cobra antivenom (1 ml/0.8 mg), at the t90 time point partially reversed the in vitro neurotoxicity of α-EPTX-Oh3a (100 nM). Repeatedly washing the tissue did not allow significant recovery from the in vitro neurotoxic effects of α-EPTX-Oh3a (100 nM). α-EPTX-Oh3a demonstrated pseudo-irreversible antagonism of concentration-response curves to carbachol, with a pA2 of 8.99. De novo sequencing of α-EPTX-Oh3a showed a long-chain postsynaptic neurotoxin with 72 amino acids, sharing 100% sequence identity with Long neurotoxin OH-55. In conclusion, the antivenom is useful for reversing the clinically important long-chain α-neurotoxin-mediated neuromuscular paralysis.

Indian Polyvalent Antivenom Accelerates Recovery From Venom-Induced Consumption Coagulopathy (VICC) in Sri Lankan Russell's Viper (Daboia russelii) Envenoming.

Background: Venom-induced consumption coagulopathy (VICC) is an important clinical consequence of Russell's viper (Daboia russelii) envenoming. There is limited evidence for antivenom effectiveness in resolving VICC. We aimed to compare the recovery of VICC in patients who received and did not receive antivenom following Russell's viper envenoming. Patients and Methods: This was a non-randomized observational study comparing patients with VICC from Russell's viper envenoming given antivenom for systemic envenoming and those not given antivenom. Antivenom administration was decided by the treating physicians. We included 44 patients with confirmed Russell's viper bites with one or more International Normalized Ratio (INR) value ≥ 1.5 (VICC). We compared five patients who did not receive antivenom with 39 patients who did receive antivenom. The primary outcome was the proportion of patients with an INR < 1.5 by 48 h post-bite. Results: The antivenom group had higher peak serum venom concentrations [median (IQR) = 272 (96-1,076) ng/mL versus 21 (8-58) ng/mL] and more severe VICC compared to the no antivenom group. Twenty seven of 39 patients (69%) in the antivenom group had an INR < 1.5 at 48 h post-bite compared to none of the five patients (0%) in the no antivenom group (absolute difference: 69%; 95%CI: 13 to 83%; p = 0.006; Fisher's exact test). The fibrinogen recovered in 32 of 39 patients (82%) in the antivenom group compared to one of five patients (20%) in the no antivenom group (absolute difference 62%; 95% CI: 28 to 95%; p = 0.001; Fisher's exact test). Both INR and fibrinogen were significantly improved between 24 and 48 h post-bite in the antivenom group compared to the no antivenom group. Conclusion: Antivenom accelerated the recovery of VICC in patients with Russell's viper envenoming, compared to no recovery in a smaller group of patients with milder VICC not receiving antivenom. This supports the efficacy of antivenom in patients with VICC.

Mild venom-induced consumption coagulopathy associated with thrombotic microangiopathy following a juvenile Russell's viper (Daboia russelii) envenoming: A case report.

Russell's viper envenoming causes venom-induced consumption coagulopathy (VICC) within hours of a bite, which is associated with thrombotic microangiopathy (TMA) and acute kidney injury (AKI) in a proportion of cases. We report a juvenile Russell's viper bite in which a patient developed mild VICC after the usual 24-h observation period, which was subsequently associated with severe AKI due to TMA. This shows the clinical importance of detecting and treating mild VICC, which may be delayed or not detected with bedside clotting tests.

Effect of Indian Polyvalent Antivenom in the Prevention and Reversal of Local Myotoxicity Induced by Common Cobra (Naja naja) Venom from Sri Lanka In Vitro.

Bites by many Asiatic and African cobras (Genus: Naja) cause severe local dermonecrosis and myonecrosis, resulting in permanent disabilities. We studied the time scale in which two Indian polyvalent antivenoms, VINS and Bharat, remain capable of preventing or reversing in vitro myotoxicity induced by common cobra (Naja naja) venom from Sri Lanka using the chick biventer cervicis nerve-muscle preparation. VINS fully prevented while Bharat partially prevented (both in manufacturer recommended concentrations) the myotoxicity induced by Naja naja venom (10 µg/mL) when added to the organ baths before the venom. However, both antivenoms were unable to reverse the myotoxicity when added to organ baths 5 and 20 min post-venom. In contrast, physical removal of the venom from the organ baths by washing the preparation 5 and 20 min after the venom resulted in full and partial prevention of the myotoxicity, respectively, indicating the lag period for irreversible cellular injury. This suggests that, although the antivenoms contain antibodies against cytotoxins of the Sri Lankan Naja naja venom, they are either unable to reach the target sites as efficiently as the cytotoxins, unable to bind efficiently with the toxins at the target sites, or the binding with the toxins simply fails to prevent the toxin-target interactions.