Antivenom Research Articles

Complete Ophthalmoplegia with Areflexia Following Snake Bite can Mimic Brain Death

Ocular complications after snake bite are well reported.External ophthalmoplegia is an established association with neurotoxic envenomation, but, combination of internal and external ophthalmoplegia(complete ophthalmoplegia) can mimic brain death and pose a dilemma to the doctors treating such patients regarding continuation of therapy. We here report a case of a 8 years boy who was playing and something has pierced in his right hand. He thendeveloped pain , swelling in his right hand and forearm and weakness of limbs and subsequently other systemic symptoms and signs.On examinationtwo clean puncture wounds were identified on his right index finger. A provisional diagnosis of snake bite with severe envenomation was made and anti snake venom was started. Over a period of 3-4 hourshis weakness progressed and became areflexic.Subsequently the patient developed internal and external ophthalmoplegia and loss of other brain stem reflexes mimicking brain death.

Ovine Crotalid Antivenin Improves Symptoms After Copperhead Snake Envenomation

Copperhead snake envenomation carries less morbidity than other crotaline envenomations, and there is uncertainty as to whether antivenin is indicated.

Assessment of Serum Biochemical Indices of Albino Rats Induced with Naja nigricollis Venom and Treated with Parkia biglobosa Stem Bark

Assessments of Serum Biochemical Indices of Albino Rats induced with Naja nigricollis venom treated with Parkia biglobosa stem bark were conducted and evaluated. A total of 42 healthy adult Albino rats of both sexes and average weight 153 -275g were used for the experiment. The venom was collected in low light condition at ambient temperature by using a short acting general anesthesia (Halothane) and kept at 4oC. The rats were injected with venom at 0.04 mg followed by conventional anti venom (1 ml) and the plant extract were administered at different doses of 300, 500 and 700 mg/kg respectively. The animals were scarified, and 5ml of blood samples were collected from each rat within 24hrs and tested for ALP, ALT, AST, total protein and Albumin. The LD50 was determined using Lorke’s method and found to be greater than 500mg/kg per oral. The results showed increased and decreased in ALT, ALP and AST while total protein and Albumin were normal. The stem bark extract of Parkia biglobosaa can be said to possess anti-snake venom activity which neutralize the toxic effects of N. nigricollis venom. The potent Snake venom neutralizing capacity of this plant extract is recommended for therapeutic purpose in case of snake bite.

Phase II Clinical Trial to Establish Efficacy of a Locally Appropriate Bivalent Anti Snake Venom in Pakistan

Objective: This study was conducted to determine the efficacy of Snake anti-venom Immunoglobulin [IgG] manufactured by Anti-Snake Venom [ASV]/Anti-Rabies [ARV] Serology Laboratory, Health Department, Government of Sindh. Methods: The prospective, observational single arm study was conducted after the approval of IRB. Study included six patients with viper [Echis carinatus sochureki] snakebites referred to the emergency ward of Peoples University of Medical & Health Sciences Hospital, Nawabshah and District Headquarter Hospital Mithi, Sindh, Pakistan with consultation of Clinical and Principal investigator. The study was conducted over a period of three months [August 2015 to November 2015]. All patients were given IV infusion of 10 mL [1 vial] investigational ASV diluted in 100 mL normal saline except one patient who received 5 mL management dose and 5 mL subsequent dose for the recovery of coagulopathy. The efficacy was assessed by Primary and secondary efficacy endpoints, i.e. the dose at which maximum no of patients were treated [permanent restoration of normal blood coagulation tested by 20-minute whole blood clotting test [20-minute WBCT] with minimum toxicity. Results: All patients recovered from coagulopathy after receiving IV infusion of 10 mL investigational ASV diluted in 100 mL normal saline tested by 20-minute WBCT. Mean Recovery time was 9:15 ± 3:25 hours. Conclusion: Safety and efficacy was assessed for the Bivalent Anti venom Immunoglobulin-NQ1 [IgG] manufactured by ASV/ARV Serology Laboratory, Health Department, Government of Sindh.

Inside the venom factory

Nick Casewell extracts venom from the world's most lethal snakes. And he is developing an antivenom that will be nothing short of revolutionary

Snake bite: Medico-legal significance

Introduction: Most of the snake bite deaths reported were due to delay in giving antivenom or non-availability of species specific antivenom and the relatives of deceased allege against doctors that proper care is not given. Forensic pathologist is expected not only to give cause of death but also give opinions about potential negligence in such cases. Case report: A middle aged woman died while being transported from a local hospital to a tertiary care hospital following a snake bite. She was brought to the hospital with symptoms suggestive of neurotoxin poisoning and anti-snake venom was given at the local hospital. The patient developed shortness of breath and she was transferred to a tertiary-care hospital for further ventilator support, but the patient was pronounced dead on the way after 2 hours of the bite. Bite mark with two fangs was found on left upper part of the breast. Inflammatory signs were found around the bite mark with necrosis and blister formation. Autopsy revealed no hematological manifestations. Conclusions: The COD was envenomation of neurotoxin due to cobra bite. The allegation of medical negligence may be challenged, because death could occur rapidly within 30 minutes to two hours in cobra bites and delay in transport may also have played a vital role especially being a rural area. Role of non-availability of species specific antivenom and efficacy of common anti snake venom should be further studied before giving opinions regarding potential negligence.

Antivenomics of a European vipera antivenom

Antivenomics of a European vipera antivenom

Preclinical evaluation of three polyspecific antivenoms against the venom of Echis ocellatus: Neutralization of toxic activities and antivenomics

Snakebite envenoming has a heavy burden in the public health in sub-Saharan Africa. The viperid species Echis ocellatus (carpet viper or saw-scaled viper) is the medically most important snake in the savannahs of western sub-Saharan Africa. Several antivenoms are being distributed and used in this region for the treatment of envenomings by E. ocellatus, but the preclinical efficacy of some of these antivenoms has not been assessed. The present study evaluated the preclinical efficacy against E. ocellatus venom of three polyspecific antivenoms: (a) Snake Venom Antiserum (Pan Africa), manufactured by Premium Serums and Vaccines (India); (b) Snake Venom Antiserum (Africa), manufactured by VINS Bioproducts (India); and (c) Antivipmyn® Africa, manufactured by Instituto Bioclon (Mexico). Antivenomics analysis revealed the ability of the three antivenoms to immunocapture the majority of components of the venoms of E. ocellatus from Cameroon, Nigeria and Mali, although their maximal immunocapturing capability varied. Bioclon and Premium Serums antivenoms were effective in the neutralization of lethal, hemorrhagic and in vitro coagulant activities of the venom of E. ocellatus from Cameroon, albeit with different potencies. VINS antivenom neutralized hemorrhagic activity of this venom, but failed to neutralize lethality at the highest antivenom dose tested, and had a low neutralizing efficacy against in vitro coagulant effect.

The antivenom index: An online, systematic response to non-native envenomations in the U.S.

The antivenom index: An online, systematic response to non-native envenomations in the U.S.

A Simple and Novel Strategy for the Production of a Pan-specific Antiserum against Elapid Snakes of Asia

Snakebite envenomation is a serious medical problem in many tropical developing countries and was considered by WHO as a neglected tropical disease. Antivenom (AV), the rational and most effective treatment modality, is either unaffordable and/or unavailable in many affected countries. Moreover, each AV is specific to only one (monospecific) or a few (polyspecific) snake venoms. This demands that each country to prepare AV against its local snake venoms, which is often not feasible. Preparation of a ‘pan-specific’ AV against many snakes over a wide geographical area in some countries/regions has not been possible. If a ‘pan-specific’ AV effective against a variety of snakes from many countries could be prepared, it could be produced economically in large volume for use in many countries and save many lives. The aim of this study was to produce a pan-specific antiserum effective against major medically important elapids in Asia. The strategy was to use toxin fractions (TFs) of the venoms in place of crude venoms in order to reduce the number of antigens the horses were exposed to. This enabled inclusion of a greater variety of elapid venoms in the immunogen mix, thus exposing the horse immune system to a diverse repertoire of toxin epitopes, and gave rise to antiserum with wide paraspecificity against elapid venoms. Twelve venom samples from six medically important elapid snakes (4 Naja spp. and 2 Bungarus spp.) were collected from 12 regions/countries in Asia. Nine of these 12 venoms were ultra-filtered to remove high molecular weight, non-toxic and highly immunogenic proteins. The remaining 3 venoms were not ultra-filtered due to limited amounts available. The 9 toxin fractions (TFs) together with the 3 crude venoms were emulsified in complete Freund’s adjuvant and used to immunize 3 horses using a low dose, low volume, multisite immunization protocol. The horse antisera were assayed by ELISA and by in vivo lethality neutralization in mice. The findings were: a) The 9 TFs were shown to contain all of the venom toxins but were devoid of high MW proteins. When these TFs, together with the 3 crude venoms, were used as the immunogen, satisfactory ELISA antibody titers against homologous/heterologous venoms were obtained. b) The horse antiserum immunologically reacted with and neutralized the lethal effects of both the homologous and the 16 heterologous Asian/African elapid venoms tested. Thus, the use of TFs in place of crude venoms and the inclusion of a variety of elapid venoms in the immunogen mix resulted in antiserum with wide paraspecificity against elapid venoms from distant geographic areas. The antivenom prepared from this antiserum would be expected to be pan-specific and effective in treating envenomations by most elapids in many Asian countries. Due to economies of scale, the antivenom could be produced inexpensively and save many lives. This simple strategy and procedure could be readily adapted for the production of pan-specific antisera against elapids of other continents.

Cost-Effectiveness of Antivenoms for Snakebite Envenoming in 16 Countries in West Africa

BackgroundSnakebite poisoning is a significant medical problem in agricultural societies in Sub Saharan Africa. Antivenom (AV) is the standard treatment, and we assessed the cost-effectiveness of making it available in 16 countries in West Africa.MethodsWe determined the cost-effectiveness of AV based on a decision-tree model from a public payer perspective. Specific AVs included in the model were Antivipmyn, FAV Afrique, EchiTab-G and EchiTab-Plus. We derived inputs from the literature which included: type of snakes causing bites (carpet viper (Echis species)/non-carpet viper), AV effectiveness against death, mortality without AV, probability of Early Adverse Reactions (EAR), likelihood of death from EAR, average age at envenomation in years, anticipated remaining life span and likelihood of amputation. Costs incurred by the victims include: costs of confirming and evaluating envenomation, AV acquisition, routine care, AV transportation logistics, hospital admission and related transportation costs, management of AV EAR compared to the alternative of free snakebite care with ineffective or no AV. Incremental Cost Effectiveness Ratios (ICERs) were assessed as the cost per death averted and the cost per Disability-Adjusted-Life-Years (DALY) averted. Probabilistic Sensitivity Analyses (PSA) using Monte Carlo simulations were used to obtain 95% Confidence Intervals of ICERs.ResultsThe cost/death averted for the 16 countries of interest ranged from $1,997 in Guinea Bissau to $6,205 for Liberia and Sierra Leone. The cost/DALY averted ranged from $83 (95% Confidence Interval: $36-$240) for Benin Republic to $281 ($159–457) for Sierra-Leone. In all cases, the base-case cost/DALY averted estimate fell below the commonly accepted threshold of one time per capita GDP, suggesting that AV is highly cost-effective for the treatment of snakebite in all 16 WA countries. The findings were consistent even with variations of inputs in 1—way sensitivity analyses. In addition, the PSA showed that in the majority of iterations ranging from 97.3% in Liberia to 100% in Cameroun, Guinea Bissau, Mali, Nigeria and Senegal, our model results yielded an ICER that fell below the threshold of one time per capita GDP, thus, indicating a high degree of confidence in our results.ConclusionsTherapy for SBE with AV in countries of WA is highly cost-effective at commonly accepted thresholds. Broadening access to effective AVs in rural communities in West Africa is a priority.

Prospective evaluation of pain, swelling, and disability from copperhead envenomation

Context: In light of the existing controversy regarding antivenin treatment for copperhead envenomation, a more detailed analysis of the disability from this species is needed. Objective: Our objective was to prospectively determine the duration of pain, swelling, and functional disability, i.e., residual venom effects, in patients with copperhead envenomation. Methods: Patients with venomous snakebite reported to the North Texas Poison Center between April 2009 and November 2011 were assessed. Patients with confirmed envenomations were contacted by a specialist in poison information. Day zero was the day of the bite and verbal phone consent for study enrollment was obtained at that time. The patient (or their guardian) was contacted by phone daily thereafter, and asked to rate their pain, edema/swelling, and disability using the modified DASH and LEFS scales. Patients were followed to resolution of all symptoms or return to baseline. Results: About 104 cases of venomous snakebite were followed; of which 17 were excluded due to being a dry bites (5) or for having insufficient data during follow-up (11) or due to coagulopathy (1). Overall, residual venom effects from copperhead bites for most patients last between 7 and 13 days. Median time to complete pain resolution was 7 days (mean = 10.7 days). Median length of time to resolution of swelling was 10 days (mean = 13 days) and median length of time to resolution of functional disability was 9 days (mean = 12.2 days). Discussion: Residual venom effects from copperhead envenomation in this study had a slightly shorter duration than some other studies. Data are skewed due to outliers where residual venom effects lasted for up to 89 days. Initial reoccurrence of some symptoms may be seen. Antivenom (AV) is currently being used for a large percentage of patients with copperhead envenomation. Finally, no differences in duration of venom effects were seen based on age or location of bite. Conclusion: Our study suggests that residual venom effects from copperhead species persist for between 10 and 13 days but may persist for months. Future studies are necessary to identify risk factors for severe/prolonged injury and to define the benefit of AV in patients with copperhead envenomation.

Pre-clinical assessment of the effectiveness of modified polyvalent antivenom in the neutralization of Naja naja venom toxicity.

The potency of conventional antivenom (AV) conjugated to soy protein nanoparticles (NPs) (C-AV) was compared with the free AV in neutralizing the systemic toxicity of Naja naja venom. The effective dose (ED50 ) of AV and C-AV to neutralize the venom-induced toxicity in mice was found to be 19.89 and 9.50mg, respectively. The histopathological examination of heart, liver, and kidney indicated that the systemic toxicity induced by the venom was effectively neutralized by lower concentrations of C-AV than compared with AV. In addition, C-AV was found to be more effective in neutralizing the edema forming activity of N. naja venom compared with the AV. Thus, the results of this study indicate that the potency of commercially available AV could be improved by conjugating it to soy protein NPs.

Costs of antivenoms higher in US than Mexico

Costs of antivenoms higher in US than Mexico

Leukemoid Reaction in Patients with <i>Echis carinatus</i> Bite:A Two-Case Report

Snake bite is a major environmental hazard in the rural areas. Venom induced coagulopathy is a significant cause of both morbidity and mortality in these patients. Leukemoid reaction is a rare phenomenon after snake bite. Here we report two cases who developed venom induced coagulopathy and leukemoid reaction after viperine bite. These patients treated with anti snake venom, and completely recovered after 14 days. To the best of our knowledge, these are the first two cases report to do so.

An instructive case of presumed brown snake (Pseudonaja spp.) envenoming

Context. Several species of medically important Australian elapid snakes are frequently involved in human envenoming. The brown snake group (Pseudonaja spp., 9 species) is most commonly responsible for envenoming including life-threatening or fatal cases. Several Pseudonaja spp. can inflict human envenoming that features minor local effects, but may cause serious systemic venom disease including defibrination coagulopathy, thrombocytopenia, micro-angiopathic hemolytic anemia (MAHA) and, rarely, paralysis. Pseudonaja envenoming is typically diagnosed by history, clinical assessment including occasional active clinical bleeding noted on physical examination (e.g. from venipuncture sites, recent cuts, etc.), and laboratory detection of coagulopathy (prolonged activated partial thromboplastin time [APTT]/INR, elevated D-dimer, afibrinogenemia and thrombocytopenia). Lack of verified identity of the envenoming snake species is a common problem in Australia and elsewhere. Identification and confirmation of the envenoming Australian snake taxon is often attempted with enzyme sandwich immunoassay venom detection kits (SVDKs). However, the SVDK has limited utility due to unreliable specificity and sensitivity when used to detect venoms of some Australian elapids. Antivenom (AV) remains the cornerstone of treatment, although there is debate concerning the recommended dose (1 vs. 2 or more vials) necessary to treat serious Pseudonaja envenoming. Envenomed patients receiving timely treatment uncommonly succumb, but a proportion of seriously envenomed patients may exhibit clinical or laboratory evidence of myocardial insult. Case details. An 88-year-old woman presented her dog to a veterinarian after it had sustained a bite by a witnessed snake, reportedly an eastern brown snake (Pseudonaja textilis, Elapidae). The woman became suddenly confused, and lost consciousness at the veterinary office. After transport to hospital, she denied any contact with the snake, but developed large haematomas at intravenous (i.v.) catheter insertion sites; blood tests revealed a severe defibrination coagulopathy, consistent with envenoming by a brown snake. An SVDK-tested urine sample was negative. A non-contrast CT of her head showed a minor subacute infarction of the left corona radiata. A twelve-lead ECG was normal, but her troponins were mildly elevated (39 ng/L). A diagnosis of brown snake envenoming was made and she received 2 vials of brown snake AV i.v., without adverse incident. Thirty min post AV her Glasgow Coma Score (GCS) had improved from 13 to 15 (normal). At 3.5 h post AV all bleeding from i.v. sites ceased, although her troponin T level peaked at 639 ng/L, supporting a diagnosis of non-ST elevated myocardial infarction (NSTEMI). Discussion. Severe brown snake envenoming may occur in the absence of a perceived bite, and AV is temporally associated with improvement in clinical findings and coagulopathy. However, severe envenoming by this species can be complicated by cardiovascular events that in the circumstance of incomplete or absent history may confuse the primary diagnosis and affect patient outcome.

Snakebites and scorpion stings in the Brazilian Amazon: identifying research priorities for a largely neglected problem.

Envenomings by snakebites and scorpion stings impose a high burden worldwide and result in considerable social and economic impact [1]. It is estimated that snakebite rates are as high as over 1.8 million cases per year, with associated deaths reaching more than 90,000 cases annually [2]. However, snakebites are a neglected condition with no associated WHO programmes for control and prevention. The countries most affected by snakebites are located in the intertropical zone in areas with high rates of field use for agriculture where the main affected populations are adult men working in agricultural activities [1]. Approximately two billion people are living in areas at risk for scorpion stings, with over one million accidents occurring annually worldwide [3]. However, the true burden of snakebites and scorpion stings is probably higher and difficult to estimate since only a few countries have a reliable system for epidemiological surveillance of these events. In Brazil, the Ministry of Health implemented the National Program for Snakebites Control in 1986, extended to other poisonous animals in 1988. Since then, antivenom (AV) production has been standardized and all the AV production from the three national laboratories (Instituto Butantan, Fundacao Ezequiel Dias, and Instituto Vital Brazil) was acquired by the Ministry of Health for distribution free of charge to patients. Five types of snake AVs are currently available: Bothrops AV (main one), Crotalus AV, Bothrops-Crotalus AV, Bothrops-Lachesis AV, and Micrurus AV. For scorpion stings, there are two types of AVs available in Brazil: Tityus scorpion AV and a polyvalent AV against Loxosceles and Phoneutria spiders and the Tityus scorpion. Table 1 summarizes information on snake and scorpion AVs produced in Brazil. Table 1 Venom pools used for snake and scorpion AVs production in Brazil. In 2013, 27,181 and 78,091 cases of snakebites and scorpion stings were reported by the Brazilian Ministry of Health, respectively [4]. The highest incidence was in the North region (52.6 cases/100,000 inhabitants) followed by the Midwest (16.4/100,000). These values, expected to be higher in remote areas of the Brazilian Amazon [5], may be underestimated due to considerable underreporting. Fig 1 presents the spatial distribution of snakebites and scorpion stings in the Brazilian Amazon. Fig 1 Spatial distribution of snakebites and scorpion stings in the Brazilian Amazon in 2013.

Cytotoxiciy of Naja nubiae (Serpentes: Elapidae) and Echis ocellatus (Serpentes: Viperidae) Venoms from Sudan

In Sudan, as in many African countries, no local specific antivenom is manufactured resulting in snake bite victims being treated by antivenoms imported from abroad. In the present work we measured the cytotoxic effect of the recently described spitting cobra (Naja nubiae) and the carpet viper (Echis ocellatus) snake venoms using a cell based assay. We also investigated the efficacy of four antivenoms CSL (Australia), SAIMR (South Africa), snake venom antiserum (India), and EchiTAb-Plus-ICP (Cost Rica) to neutralize the cytotoxic effect of the two venoms. The venoms resulted in a remarkable inhibition of cell viability with N. nubiae being more cytotoxic than E. ocellatus. The four antivenoms studied were effective in neutralizing N. nubiae cytotoxicity. However, only partial efficacy in neutralizing the cytotoxic effect of E. ocellatus was achieved using CSL (Australia) and SVA (India) antivenoms. Based on the cross neutralization by the four antivenoms, the Sudanese N. nubiae venom most likely has homologous epitopes with similar snakes from Australia, South Africa, India, and Cost Rica, while E. ocellatus venom from Sudan shares little homology with similar snakes from other countries.

Effectiveness of A Clinical Protocol Implemented To Standardize Snakebite Management In Iran: Initial Evaluation

This study was designed to evaluate the effectiveness of a new protocol implemented to standardize snakebite management in Iran. In this study, 27 patients treated according to the new protocol in 2012 (P+) were compared with 22 patients treated according to the previous modality in the year before implementation of the protocol (P-) in Mashhad Medical Toxicology Centre (MTC). Demographic characteristics and treatment details of all patients were recorded prospectively. Envenomation severity of each victim was assessed according to snakebite severity score (SSS). After implementation of the protocol, a smaller percentage of patients received antivenom (AV) therapy (78% vs 95%; P=.079). In spite of no significant difference in baseline severity of envenomation between the 2 groups (SSS [mean±SD], 34.8±18.1 vs 35.5±17.4; P=.801), the P+ group received significantly fewer AV vials (8.4±6.8 vs 12.1±5.6 vials; P=.042) and had a significantly shorter length of hospital stay (2.2±1.5 vs 3.2±1.8 days; P=.027). Moreover, smaller proportion of P+ patients experienced recurrence of venom-induced effects; however, the difference was not significant (18.5% vs 36%; P=.159). The reduction in use of antiallergy treatments to prevent or treat acute hypersensitivity reactions approached statistical significance (41% vs 68%; P=.051). These findings denote a reduction in AV use of approximately 4 vials and a reduction in hospital stay of 1 day for each patient, which translates to approximately $196/patient in healthcare cost savings. Implementation of a snakebite management protocol at MTC reduced overall antivenom usage, use of antiallergy interventions, and length of hospital stay.

Quality Control Testing for Tracking Endotoxin-Producing Gram-Negative Bacteria during the Preparation of Polyvalent Snake Antivenom Immunoglobulin.

Snake bites represent a serious public health problem, particularly in rural areas worldwide. Antitoxic sera preparations are antibodies from immunized animals and are considered to be the only treatment option. The purification of antivenom antibodies should aim at obtaining products of consistent quality, safety, efficacy, and adherence to good manufacturing practice principles. Endotoxins are an integral component of the outer cell surface of Gram-negative bacteria. They are common contaminates of the raw materials and processing equipment used in the manufacturing of antivenoms. In this work, and as a part of quality control testing, we establish and examine an environmental monitoring program for identification of potential sources of endotoxin-producing Gram-negative bacteria throughout the whole steps of antivenom preparation. In addition, we follow all the steps of preparation starting from crude plasma till finished product using a validated sterility and endotoxin testing.Samples from air, surface, and personnel were collected and examined through various stages of manufacturing for the potential presence of Gram-negative bacteria. A validated sterility and endotoxin test was carried out in parallel at the different production steps. The results showed that air contributed to the majority of bacterial isolates detected (48.43%), followed by surfaces (37.5%) and then personnel (14%). The most common bacterial isolates detected were Achromobacter xylosoxidans, Ochrobactrum anthropi, and Pseudomonas aeruginosa, which together with Burkholderia cepacia were both also detected in cleaning water and certain equipment parts. A heavy bacterial growth with no fungal contamination was observed in all stages of antivenom manufacturing excluding the formulation stage. All samples were positive for endotoxin including the finished product.Implementation and continued evaluation of quality assurance and quality improvement programs in aseptic preparation is essential in ensuring the safety and quality of these products. Antitoxic sera preparations are the only treatment option for snake bites worldwide. They are prepared by immunizing animals, usually horses, with snake venom and collecting horse plasma, which is then subjected to several purification steps in order to finally prepare the purified immunoglobulins. Components of the bacterial cell wall known as endotoxins can constitute a potential hazardous contamination known as pyrogen in antisera, which can lead to fever and many other adverse reactions to the person subjected to it.In this work, we monitored the environment associated with the different steps of production and purification of snake antivenom prepared from immunized horses. We examined the air quality, surface, and personnel for possible sources of contamination, particularly the presence of Gram-negative bacteria, which is the major source of endotoxin presence. We also monitored all stages of preparation by sterility and endotoxin testing. Our results showed that air contributed to the majority of bacterial isolates. Sterility testing revealed the presence of bacterial contamination in all the intermediate steps, as only the final preparation after filtration was sterile. Endotoxin was present in all tested samples and the final product. Good manufacturing practice procedures are essential in any facility involved in antisera production.