Efficacy of a low dose of antivenom for severe neuroparalysis in Bungarus caeruleus (common krait) envenomation: a pilot study.

Introduction: Despite the widespread use of antivenom for the treatment of snakebite envenoming in the Indian subcontinent, the ideal dose of antivenom has been a point of contention. Low-dose regimens can economize on a scarce resource in low- and middle-income countries. This study assessed the effectiveness of a low-dose (10 vials) antivenom regimen compared to the usual 20 vials in patients with krait bite neuroparalysis requiring mechanical ventilation. Methods: This study was a prospective controlled pilot study conducted in a tertiary-care hospital in north India. Participants were eligible if they were ≥12 years old, had krait bite neurotoxicity, showed severe paralysis requiring mechanical ventilation, and had access to antivenom therapy within 24 h of the bite. The primary outcome was the duration of mechanical ventilation, and the secondary outcomes were the length of hospital stay and in-hospital survival. Results: Fifteen patients received 10 vials of antivenom, and 25 received 20 vials. The two treatment groups had similar baseline demographics, clinical and laboratory features, snakebite severity scores, and median time from snakebite to initiation of antivenom therapy. The low-dose regimen was as effective as the standard dose concerning the median duration of mechanical ventilation (41 h vs. 55 h, P = 0.094), the median length of stay (78 h vs. 85.5 h, P = 0.360), and in-hospital deaths (1 vs. 3, P = 1.000). The incidence of ventilator-associated pneumonia was similar between the two groups (1 vs 3, P = 1.000). Conclusion: A low dose of antivenom effectively treats patients with severe krait bite neuroparalysis.

Development of a Biosensor to Detect Venom of Malayan Krait (Bungarus candidus).

Malayan krait (Bungarus candidus) envenoming is a cause of significant morbidity and mortality in many Southeast Asian countries. If intubation and specific antivenom administration are delayed, the most significant life-threatening outcome may be the inhibition of neuromuscular transmission and subsequent respiratory failure. It is recommended that krait-envenomed victims without indications of neurotoxicity, e.g., skeletal muscle weakness or ptosis, immediately receive 10 vials of antivenom. However, the administration of excess antivenom may lead to hypersensitivity or serum sickness. Therefore, monitoring venom concentrations in patients could be used as an indicator for snake antivenom treatment. In this study, we aimed to develop a screen-printed gold electrode (SPGE) biosensor to detect B. candidus venom in experimentally envenomed rats. The gold electrodes were coated with monovalent Malayan krait IgG antivenom and used as venom detection biosensors. Electrochemical impedance spectrometry (EIS) and square wave voltammetry (SWV) measurements were performed to detect the electrical characterization between B. candidus venom and monovalent IgG antivenom in the biosensor. The EIS measurements showed increases in charge transfer resistance (Rct) following IgG immobilization and incubation with B. candidus venom solution (0.1-0.4 mg/mL); thus, the antibody was immobilized on the electrode surface and venom was successfully detected. The lowest current signal was detected by SWV measurement in rat plasma collected 30 min following B. candidus experimental envenoming, indicating the highest level of venom concentration in blood circulation (4.3 ± 0.7 µg/mL). The present study demonstrates the ability of the SPGE biosensor to detect B. candidus venom in plasma from experimentally envenomed rats. The technology obtained in this work may be developed as a detection tool for use along with the standard treatment of Malayan krait envenoming.

A retrospective analysis of epidemiology, clinical features of envenomation, and in-patient management of snakebites in a model secondary hospital of Assam, North-east India.

Assam, a Northeastern State of India, is inhabited by several venomous snake species causing substantial morbidity and mortality. The data on the epidemiology of snakebites and their management is underreported in this region. Hence, a secondary health-based retrospective study was carried out at Demow Model Hospital, Sivasagar, Assam, to evaluate the clinical and epidemiological profile of snakebite cases reported in this rural hospital and their management. Snakebites occurring between April 2018 to August 2022 were reviewed based on socio-demographic details of the patient, clinical symptoms, and treatment using a standard questionnaire. Out of the 1011 registered snakebite cases, 139 patients (13.7%) counted for venomous bites, among which 92 patients (66.19%) accounted for viper bites (green pit viper and Salazar's pit viper), and 30 patients (21.5%) were bitten by elapid snakes (Indian monocled Cobra, banded krait, and greater/lesser black krait). A maximum number of snakebite cases (80.5%) were reported from the interior rural villages and documented from July to September (51.3%). Elapid snake envenomed patients, except one, were successfully treated with commercial antivenom, neostigmine, and glycopyrrolate. Because commercial polyvalent antivenom against "Big Four" venomous snakes of India showed poor neutralization of pit-vipers envenomation; therefore, pit-viper bite patients were treated with repurposed drugs magnesium sulfate and glycerin compression dressing. Adverse serum reactions were reported only in 3 (11.1%) cases. The preventive measures and facilities adopted at the Demow Model Hospital significantly reduce snakebite death and morbidity; therefore, they can be s practised across various states in India as a prototype.

The Potassium Channel Blocker ß-Bungarotoxin from the Krait Bungarus multicinctus Venom Manifests Antiprotozoal Activity.

Protozoal infections are a world-wide problem. The toxicity and somewhat low effectiveness of the existing drugs require the search for new ways of protozoa suppression. Snake venom contains structurally diverse components manifesting antiprotozoal activity; for example, those in cobra venom are cytotoxins. In this work, we aimed to characterize a novel antiprotozoal component(s) in the Bungarus multicinctus krait venom using the ciliate Tetrahymena pyriformis as a model organism. To determine the toxicity of the substances under study, surviving ciliates were registered automatically by an original BioLaT-3.2 instrument. The krait venom was separated by three-step liquid chromatography and the toxicity of the obtained fractions against T. pyriformis was analyzed. As a result, 21 kDa protein toxic to Tetrahymena was isolated and its amino acid sequence was determined by MALDI TOF MS and high-resolution mass spectrometry. It was found that antiprotozoal activity was manifested by ß-bungarotoxin (ß-Bgt) differing from the known toxins by two amino acid residues. Inactivation of ß-Bgt phospholipolytic activity with p-bromophenacyl bromide did not change its antiprotozoal activity. Thus, this is the first demonstration of the antiprotozoal activity of ß-Bgt, which is shown to be independent of its phospholipolytic activity.

Delayed Neurological Manifestation in Krait Bites Despite Anti-snake Venom Therapy.

A severe medical emergency that poses a life-threatening risk is envenomation from a snake bite. Among the several snake families, krait bites are known to result in neurological symptoms, including ptosis, headache, and sweating. A 12-year-old adolescent boy who had been bitten by a krait appeared in this instance. The patient showed neurological symptoms after receiving anti-snake venom (ASV). He had three rounds of ASV and made a full recovery. To the best of our knowledge, there have not yet been any reports of this kind of delayed neurological signs after a krait bite, despite getting ASV in the adolescent population.

Bilateral Simultaneous Optic Neuritis Following Envenomations by Indian Cobra and Common Krait.

In India, most snakebite envenomation (SBE) incidents are caused by the "Big Four" snakes which include Russell's viper, common krait, Indian cobra, and saw-scaled viper. Their common envenomation effects include neurotoxicity, myotoxicity, and coagulopathy. However, they also induce rare complications such as priapism, pseudoaneurysm, and sialolithiasis. Ocular manifestations such as optic neuritis develop rarely following envenomations by non-spitting snakes and they may cause temporary vision changes and blindness if untreated. While optic neuritis following Indian cobra envenomation has been reported previously, this was not encountered in victims of common kraits. Hence, for the first time, we report optic neuritis developed in a victim following envenomation by a common krait and compare its clinical features and diagnostic and therapeutic methods used with another case of optic neuritis in a victim of an Indian cobra bite. Both patients received antivenom treatment and made an initial recovery; however, optic neuritis developed several days later. The condition was diagnosed using ophthalmic examination together with computed tomography and/or magnetic resonance imaging methods. Due to very similar clinical features, both patients received intravenous corticosteroids which restored their vision and successfully treated optic neuritis. This case report suggests that the optic neuritis developed in a common krait envenomation is comparable to the one developed following a cobra bite, and therefore, the same diagnostic and therapeutic approaches can be used. This study also raises awareness of this rare complication and provides guidance for the diagnosis and treatment of SBE-induced optic neuritis.

A Life-Threatening Incidence of Neurotoxic Indian Krait Snake Bite: A Case Report.

The Indian Krait delivers one of the most lethal venoms compared to other Asian snakes. The venom of the common Krait comprises substantial neurotoxins that cause muscular paralysis. Significant snake bite incidence occurs in rural areas. The significant death rate caused by snake bites is seldomly reported in the medical literature. A 14-year-old adolescent girl was brought by her parents to the emergency department (ED) in an unconscious state. The patient reported swelling on her right hand with fang marks of a snake bite, sweating, and increased salivation. The primary therapeutic intervention was given to the patient and she was treated with intravenous anti-snake venom serum, antibiotics, and anti-epileptics during hospitalisation.

Hirata's disease (insulin autoimmune syndrome) following envenomation by a common krait.

Snakebite envenomation is known to cause local as well as systemic haematological, myotoxic and neurological effects. Adverse effects on the endocrine system following envenomation are rarely reported. Hirata's disease, also known as insulin autoimmune syndrome (IAS) is a rare disorder that causes hypoglycaemia due to excessive production of insulin autoantibodies. This report describes a rare case of IAS which developed in a snakebite victim following envenomation by a common krait and antivenom treatment. The patient was initially treated with dextrose and corticosteroids, although plasmapheresis was required to reduce the concentration of insulin antibodies and normalise the patient's glucose level. The patient then made an uneventful recovery without permanent sequelae. This report demonstrates the impacts of envenomation by a common krait on developing Hirata's disease and creates awareness among clinicians who treat snakebite envenomation.

Comparative Study of the Effect of Snake Venoms on the Growth of Ciliates Tetrahymena pyriformis: Identification of Venoms with High Antiprotozoal Activity.

To search for compounds with antiprotozoal activity, effects of snake venoms on the ciliates Tetrahymena pyriformis was studied. T. pyriformis from subkingdom of Protozoa, including the protozoal pathogens, was used as a model organism to select the venoms that are the most active against parasitic protozoans. Various concentrations of venoms were added to the cells, and the cells that survived after 24 h were counted. Among the six snake species from the Viperidae family, the venom of the viper Vipera berus, which completely killed the cells at 49 µg/mL, was the most active. Among four species from the Elapidae family, the previously studied cobra venoms containing cytotoxins with strong antiprotozoal activity as well as the venom of krait Bungarus multicinctus (10 µg/mL) were the most active. The venoms of the pit vipers and Nikolsky's viper did not show any activity at 12.5 mg/mL. Thus, the venoms of V. berus and B. multicinctus are promising for the isolation of new antiprotozoal compounds.

Fijian sea krait behavior relates to fine-scale environmental heterogeneity in old-growth coastal forest: The importance of integrated land-sea management for protecting amphibious animals.

The importance of terrestrial coastal ecosystems for maintaining healthy coral reef ecosystems remains understudied. Sea kraits are amphibious snakes that require healthy coral reefs for foraging, but little is known about their requirements of terrestrial habitats, where they slough their skin, digest prey, and breed. Using concurrent microclimate measurements and behavior surveys, we show that a small, topographically flat atoll in Fiji with coastal forest provides many microhabitats that relate to the behaviors of Yellow Lipped Sea Kraits, Laticauda colubrina. Microclimates were significantly related to canopy cover, leaf litter depth, and distance from the high-water mark (HWM). Sea kraits were almost exclusively observed in coastal forest within 30 m of the HWM. Sloughing of skins only occurred within crevices of mature or dying trees. Resting L. colubrina were significantly more likely to occur at locations with higher mean diurnal temperatures, lower leaf litter depths, and shorter distances from the HWM. On Leleuvia, behavior of L. colubrina therefore relates to environmental heterogeneity created by old-growth coastal forests, particularly canopy cover and crevices in mature and dead tree trunks. The importance of healthy coastal habitats, both terrestrial and marine, for L. colubrina suggests it could be a good flagship species for advocating integrated land-sea management. Furthermore, our study highlights the importance of coastal forests and topographically flat atolls for biodiversity conservation. Effective conservation management of amphibious species that utilize land- and seascapes is therefore likely to require a holistic approach that incorporates connectivity among ecosystems and environmental heterogeneity at all relevant scales.

Dramatic neuromuscular paralysis following occult snakebites: An awareness for the primary care physician.

Neurotoxic snakebites are a common emergency in tropical countries and account for significant morbidity and mortality worldwide. Manifestations vary from mild ptosis and ophthalmoplegia to severe flaccid paralysis with ventilatory failure. At times, the neuromuscular paralysis may be severe enough for patients to be misdiagnosed as a locked-in syndrome or brain dead. Occult snakebites, wherein patients are unaware of the bite and fang marks are absent, have been reported in kraits, an endemic neurotoxic snake belonging to the Elapidae family. We report a series of three cases in which young males presented with dramatic neuromuscular paralysis and were likely suffering from elapid snake bites. Each of these patients presented an intriguing clinical challenge and had different in-hospital outcomes. Primary care physicians in the emergency department are usually the first respondents to such patients. Owing to a lack of snake bite history and unavailability of specific diagnostic tests, severe envenomation presents a challenge for physicians, unless they are aware of it and a high level of suspicion is maintained.

Complex target SELEX-based identification of DNA aptamers against Bungarus caeruleus venom for the detection of envenomation using a paper-based device.

Complex target SELEX always have been an intriguing approach to the scientific community, as it offers the potential discovery of novel biomarkers. We herein successfully performed SELEX on Bungarus caeruleus venom to develop a panel of highly affine aptamers that specifically recognizes the B. caeruleus (common krait) venom and was able to discriminate the B. caeruleus venom from Cobra, Russell's, and Saw-scaled viper's venom. The aptamers generated against the crude venom also lead to the identification of the specific component of the venom, which is ß-Bungarotoxin, a toxin uniquely present in the B. caeruleus venom. The best performing aptamer candidates were used as a molecular recognition element in a paper-based device and were able to detect as low as 2 ng krait venom in human serum background. The developed aptamer-based paper device can be used for potential point-of-care venom detection applications due to its simplicity and affordability.

Paediatric cases of Ceylon krait (Bungarus ceylonicus) bites and some similar looking non-venomous snakebites in Sri Lanka: Misidentification and antivenom administration.

Ceylon krait (Bungarus ceylonicus) of the family Elapidae is a highly venomous endemic species inhabiting in the wet zone and some parts of the intermediate climatic zones of Sri Lanka. Clinical records of its bites are rare and limited to five case reports in the literature. It is of interest to note that there are several non-venomous snakes in Sri Lanka having similar morphological appearance to kraits causing identification difficulties which lead to unnecessary and unindicated administration of antivenom. We report two paediatric cases of proven Ceylon krait bites and three adult patients with similar looking non-venomous snakebites. These children were 1½ and 13 years old and developed neuroparalysis without progressing to respiratory failure and recovered. Both the children were administered Indian polyvalent antivenom which has not developed against endemic Ceylon krait venom. The two adult patients also received antivenom due to the misidentification without clinical and laboratory evidence of envenoming.

Plasticity matches phenotype to local conditions despite genetic homogeneity across 13 snake populations.

In a widespread species, a matching of phenotypic traits to local environmental optima is generally attributed to site-specific adaptation. However, the same matching can occur via adaptive plasticity, without requiring genetic differences among populations. Adult sea kraits (Laticauda saintgironsi) are highly philopatric to small islands, but the entire population within the Neo-Caledonian Lagoon is genetically homogeneous because females migrate to the mainland to lay their eggs at communal sites; recruits disperse before settling, mixing up alleles. Consequently, any matching between local environments (e.g. prey sizes) and snake phenotypes (e.g. body sizes and relative jaw sizes (RJSs)) must be achieved via phenotypic plasticity rather than spatial heterogeneity in gene frequencies. We sampled 13 snake colonies spread along an approximately 200 km northwest-southeast gradient (n > 4500 individuals) to measure two morphological features that affect maximum ingestible prey size in gape-limited predators: body size and RJS. As proxies of habitat quality (HQ), we used protection status, fishing pressure and lagoon characteristics (lagoon width and distance of islands to the barrier reef). In both sexes, spatial variation in body sizes and RJSs was linked to HQ; albeit in different ways, consistent with sex-based divergences in foraging ecology. Strong spatial divergence in morphology among snake colonies, despite genetic homogeneity, supports the idea that phenotypic plasticity can facilitate speciation by creating multiple phenotypically distinct subpopulations shaped by their environment.

Unusually prolonged neuromuscular weakness caused by krait (Bungarus caeruleus) bite: Two case reports.

Snakebites are common in India and the most common neurotoxic snakebites in India are due to Common krait (Bungarus caeruleus) and cobra (Naja naja). Severe envenomation may mimic brain death or a locked-in state with flaccid paralysis in a descending manner and total ophthalmoplegia. Usually, patients who receive timely antivenom and ventilator support recover completely without any sequalae. We are reporting two cases of krait bite with an unusually long period of flaccid paralysis, which required prolong ventilation. While case 1 required 10 days of mechanical ventilation followed by 5 days of non-invasive ventilation, case 2 required 11 days of mechanical ventilation followed by 5 days of non-invasive ventilation. Both the cases had delayed recovery and residual weakness at 3-month follow up. These case reports suggest that krait bite may cause prolong neuromuscular weakness in children, which has implications for both acute and chronic management.

A Wolf in Another Wolf's Clothing: Post-Genomic Regulation Dictates Venom Profiles of Medically-Important Cryptic Kraits in India.

The Common Krait (Bungarus caeruleus) shares a distribution range with many other 'phenotypically-similar' kraits across the Indian subcontinent. Despite several reports of fatal envenomings by other Bungarus species, commercial Indian antivenoms are only manufactured against B. caeruleus. It is, therefore, imperative to understand the distribution of genetically distinct lineages of kraits, the compositional differences in their venoms, and the consequent impact of venom variation on the (pre)clinical effectiveness of antivenom therapy. To address this knowledge gap, we conducted phylogenetic and comparative venomics investigations of kraits in Southern and Western India. Phylogenetic reconstructions using mitochondrial markers revealed a new species of krait, Romulus' krait (Bungarus romulusi sp. nov.), in Southern India. Additionally, we found that kraits with 17 mid-body dorsal scale rows in Western India do not represent a subspecies of the Sind Krait (B. sindanus walli) as previously believed, but are genetically very similar to B. sindanus in Pakistan. Furthermore, venom proteomics and comparative transcriptomics revealed completely contrasting venom profiles. While the venom gland transcriptomes of all three species were highly similar, venom proteomes and toxicity profiles differed significantly, suggesting the prominent role of post-genomic regulatory mechanisms in shaping the venoms of these cryptic kraits. In vitro venom recognition and in vivo neutralisation experiments revealed a strong negative impact of venom variability on the preclinical performance of commercial antivenoms. While the venom of B. caeruleus was neutralised as per the manufacturer's claim, performance against the venoms of B. sindanus and B. romulusi was poor, highlighting the need for regionally-effective antivenoms in India.

Fight or Flee: An Interesting Case of Snakebite With Delayed Recovery.

Snakebite is a neglected tropical disease, which is very common in the Indian subcontinent. The severity of respiratory muscle paralysis and the delay in recovery depend upon the dose of the venom injected, the severity of the venom, the species of the snake, the duration of presentation to the hospital, and the time and dose of administration of anti-snake venom (ASV). The reasons for this delayed neuromuscular recovery still remain an enigma. We highlight such a case of a young adult who had delayed neuromuscular recovery and prolonged ventilatory support following a neurotoxic snakebite.

Phospholipase A2 from krait Bungarus fasciatus venom induces human cancer cell death in vitro.

BACKGROUND: Snake venoms are the complex mixtures of different compounds manifesting a wide array of biological activities. The venoms of kraits (genus Bungarus, family Elapidae) induce mainly neurological symptoms; however, these venoms show a cytotoxicity against cancer cells as well. This study was conducted to identify in Bungarus fasciatus venom an active compound(s) exerting cytotoxic effects toward MCF7 human breast cancer cells and A549 human lung cancer cells. METHODS: The crude venom of B. fasciatus was separated by gel-filtration on Superdex HR 75 column and reversed phase HPLC on C18 column. The fractions obtained were screened for cytotoxic effect against MCF7, A549, and HK2 cell lines using colorimetric assay with the tetrazolium dye MTT- 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. The primary structure of active protein was established by ultra high resolution LC-MS/MS. The molecular mechanism of the isolated protein action on MCF7 cells was elucidated by flow cytometry. RESULTS: MTT cell viability assays of cancer cells incubated with fractions isolated from B. fasciatus venom revealed a protein with molecular mass of about 13 kDa possessing significant cytotoxicity. This protein manifested the dose and time dependent cytotoxicity for MCF7 and A549 cell lines while showed no toxic effect on human normal kidney HK2 cells. In MCF7, flow cytometry analysis revealed a decrease in the proportion of Ki-67 positive cells. As Ki-67 protein is a cellular marker for proliferation, its decline indicates the reduction in the proliferation of MCF7 cells treated with the protein. Flow cytometry analysis of MCF7 cells stained with propidium iodide and Annexin V conjugated with allophycocyanin showed that a probable mechanism of cell death is apoptosis. Mass spectrometric studies showed that the cytotoxic protein was phospholipase A2. The amino acid sequence of this enzyme earlier was deduced from cloned cDNA, and in this work it was isolated from the venom as a protein for the first time. It is also the first krait phospholipase A2 manifesting the cytotoxicity for cancer cells.

Quantitative proteomic analysis of venom from Southern India common krait (Bungarus caeruleus) and identification of poorly immunogenic toxins by immune-profiling against commercial antivenom.

OBJECTIVES: To study the venom proteome composition of Southern India (SI) Common Krait (Bungarus caeruleus) and immunological cross-reactivity between venom against commercial antivenom. METHODS: Proteomic analysis was done by nano LC-MS/MS and toxins were quantitated by label-free analysis. The immunological cross-reactivity of venom towards polyvalent antivenom (PAV) was assessed by ELISA, Immunoblotting, and immuno-chromatographic methods. RESULTS: A total of 57 enzymatic and non-enzymatic proteins belonging to 12 snake venom protein families were identified. The three finger toxins (3FTx) (48.3%) and phospholipase A2 (PLA2) (37.6%) represented the most abundant non-enzymatic and enzymatic proteins, respectively. ß-bungarotoxin (12.9%), a presynaptic neurotoxin, was also identified. The venom proteome composition is well correlated with its enzymatic activities, reported pharmacological properties, and clinical manifestations of krait envenomation. Immuno-cross-reactivity studies demonstrated better recognition of high molecular weight proteins (>45 kDa) of this venom by PAVs compared to low molecular weight (<15 kDa) toxins such as PLA2 and 3FTxs. CONCLUSION: The poor recognition of <15 kDa mass SI B. caeruleus venom proteins is of grave concern for the successful treatment of krait envenomation. Therefore, emphasis should be given to improve the immunization protocols and/or supplement of antibodies raised specifically against the <15 kDa toxins of this venom.

Acute neuromuscular paralysis, rhabdomyolysis and long lasting neurological deficits in Ceylon krait (Bungarus ceylonicus) bites: Two authentic cases from a serpentarium in Sri Lanka.

The Ceylon krait (Bungarus ceylonicus) is a highly venomous elapid snake endemic to Sri Lanka. Its bites are rare and only seven reports are found in the literature. Therefore, the clinical manifestations and natural history of envenoming of Ceylon krait are not well studied yet. Neuroparalysis is the main clinical manifestation of their bites. We report two cases of proven Ceylon krait bites of two young snake keepers working in a serpentarium. They developed acute neuroparalysis, abdominal pain and a period of amnesia. The first patient developed myalgia and increased level of serum creatine kinase suggestive of rhabdomyolysis. One was treated with Indian polyvalent antivenom and both recovered with some long-lasting clinical disabilities namely impairment of sensation of the bitten arm and persistent refraction errors in the eyes in the first patient. The second patient had persistent marked nystagmus.