Venomous snakes and snakebite victims are typical tropical diseases in developing agricultural countries like Vietnam, where every year there are up to 30,000 (Thirty thousand) envenomed victims. However, this pathology has been forgotten. As a result, snakebite victims suffered a high rate of death (19.5%), deformity, loss of function, and limb amputation (14.%). The main reason was because there were no specific anti-venoms available in clinic practice.

Since 1990, the Unit for Research and Manufacturing of Antivenoms at Cho Ray hospital (predecessor of the Vietnam Society of Toxicology -VST & Vietnam Institute of Toxicology- VIT) has researched and manufactured specific antivenoms for clinical application very effectively. On March 23, 1993, Vietnam Ministry of Health issued Decision No. 1888/K2DT allowing ” Research on the production of anti-venoms, research on clinical characteristics of snakebite patients and clinical treatment application.”. On November 14, 1998, the Minister, Ministry of Health issued decision No: 3203 ” Allowing clinical treatment applications of Naja kaouthia antivenom and Calloselasma rhodostoma antivenom “. (Fig.1).

Figure.1: Decision No.: 1888 & 3203 of the Minister, Ministry of Health allowing research to produce antivenoms for clinical treatment application.

2. RESEARCH RESULTS IDENTIFYING VENOMOUS SNAKE SPECIES IN VIETNAM, LAOS & CAMBODIA

The Vietnam Institute of Toxicology (Trinh Xuan Kiem and his colleagues) with the help of Prof. David Warrell (Oxford University), 1989 and Thorpe RC, Wuster.W, Malhotra (Aberdeen University)-UK, 2003, determined that Vietnam has three main venomous snake families of medical importance: Elapidae family (09 species), Viperidae family (03 species), and Hydrophiidae (Sea snake) family (17 species). They are the cause of up to 30,000 snakebite victims each year in Vietnam.

Images of 03 families with 14 venomous snake species of medical importance in Vietnam, Laos & Cambodia.

Elapidae family:

1.Chinese cobra (Naja naja / Naja atra): mainly in Northern Vietnam & Laos.

2. Monocellate cobra (Naja kaouthia):in the Southwestern VN & Cambodia.

3. Spitting cobra (Naja siamensis):in the Eastern-South central regions VN & Cambodia.

4.Yellow king cobra (Ophiophagus hannah): in the Southwest VN & Cambodia.

5. Black king cobra (Ophiophagus hannah):in Central & Northern VN, Laos & Cambodia.

6. Banded krait (Bungarus fasciatus):found in all regions of the three countries:  Vietnam, Laos & Cambodia.

7. Southern krait, (Malayan krait) (Bungarus candidus):only found in Southern VN & Cambodia.

8. Northern krait (Chinese or many banded krait) (Bungarus multicinctus):in Northern VN &Laos.

9. Red-headed krait (Bungarus flaviceps) and Red River Krait (Bungarus slowinskii) have been discovered in Vietnam. However, the consequences of these two snake species are not many, and their epidemiology has not been determined.

Viperidae family:​

1. White-lipped green pit viper (Trimeresurus albolabris): mainly red-tailed green viper, found in all regions of Vietnam, Laos & Cambodia.
2. Malayan pit viper (Agkistrodon rhodostoma or Calloselasma rhodostoma): mainly in the Southeast VN & Cambodia.
3. (Chinese habu, Taiwanese pit viper), (T. mucrosquamatus):found in the Northern border provinces of Vietnam.

Hydrophiidae Sea snakes:

17 species of Hydrophiidae (sea snakes), the most typical sea snakes are Enhydrina schistosa & Hydrophis cyanocinctus.

Images of snakes of medical importance in Vietnam, Laos & Cambodia:

Elapidae family:

Fig.2: Naja naja -Atra

Fig.3: Naja kaouthia

Fig.4 Back of Naja siamensis.

Fig.5: Front of Naja siamensis

Fig. 6:Yellow King Cobra (Ophiophagus hannah)	Fig.7: Black King Cobra (Ophiophagus hannah)

Fig.8: B.candidus in the South of VN	Fig.9: B.multicinctus in the North of VN.	Fig.10: Bungarus fasciatus

Viperidae family:

Fig.11:  Trimeresurus albolabris	Fig.12:Calloselasma rhodostoma	Fig.13. Trimeresurus mucrosquamatus

Hydrophiidae (Sea snake) family:

Fig.14: Hydrophis cyanocinctus	Fig.15: Enhydrina schistosa

3. CHARACTERISTIC CLINICAL SYNDROMES IN PATIENTS INFECTED WITH SNAKE VENOMS IN VIETNAM, LAO & CAMBODIA

3.1. In-spot – Fang marks: Venomous snake bites always leave fang marks on the spot (Except for Bungarus candidus and B.multicinctus snakes, which do not leave any fang marks). From the fang marks of the Elapidae family, purple-black necrotic tissue always appears and spreads quickly around. The fang marks of the Viperidae family always bleed uncontrollably… On the contrary, the teeth marks of healthy snakes are just scratches.

Note: Not all patients who are bitten by venomous snakes and have fang marks at the site of the bites are envenomed.

3.2. Clinical characteristics of envenomed patients with Elapidae family:

The neurotoxic syndrome  is characteristic of the Elapidae family with the most prominent clinical manifestation being neuromuscular paralysis: The earliest symptoms are vomiting, heavy eyelids, blurred vision, drowsiness, numbness of the mouth – tongue – lips, headache, dizziness… Ptosis caused by external ophthalmoplegia that appears a few minutes after being bitten by Elapidae snake. This is the first symptom of muscle paralysis. Paralysis is soft, uniform, rapidly progressing throughout the body. Particularly life-threatening is respiratory muscle paralysis afterwards acute respiratory failure, leading to death if not treated properly and promptly

Note : There is never focal paralysis. Peripheral nerve-muscle paralysis while the central nervous system remains normal and the hemostatic coagulation system remains normal.

3.3. Clinical characteristics of envenomed patients with Viperidae family

At the site of the bite: Uncontrolled bleeding from a snake bitten wound is a common symptom in all Viperidae envenomed patients, accompanied by tissue necrosis, pink blisters, cloudiness due to hemorrhage…

Whole body symptoms: Coagulation and hemostasis disorders appear in all envenomed patients with Viperidae venom. Spontaneous, multi-modal bleeding (spots, hemorrhagic plaques…) in the skin, mucous membranes of the whole body (gastrointestinal, genitals tract,..), especially dangerous is cerebral hemorrhage and pregnant women.

Lab-Test: All coagulation and hemostasis factors are reduced: Fibrinogen only remains trace (Normal: 200-400 mg/dl). Platelets < 50 G/l (Normal: 150-300 G/l).

3.4. Pathogenesis of snake venom

4. RESULTS OF RESEARCH IN MANUFACTURING ANTIVENOMS AND CLINICAL TREATMENT APPLICATION

4.1. Role of Antivenom:

In 1971, WHO recommended: ” Antivenom is the most effective medicine to treat envenomed patients and popular worldwide.”

Vietnam is honored to be the first place to invent the Antivenom  (AV) by Dr. A. Calmette right at the Sai gon – Pasteur Institute in 1891. Yet more than 100 years later, there still was not any type of (AV) in Vietnam. Since 1990, responding to urgent clinical requirements, Dr. Trinh Xuan Kiem and his colleagues from the venomous snake research Unit of Cho Ray Hospital have successfully created Naja kaouthia AV.  At the same time, 16-year-old patient (Le Quang T) was bitten by Naja kaouthia and suffered acute respiratory failure and was on a ventilator until the 58th hour. He was saved his life by the Naja kaouthia AV that Dr. Kiem’s Unit had just successfully manufactured. This was the first snake bite patient to be saved by AV made by Vietnamese people right in Saigon City, VN.

Fig.27: The first snake bite victim: Le QT: had acute respiratory failure due to N.K venom, requiring mechanical ventilation for the 58th hour at Cho Ray Hospital, 1991.	Fig.28: Le QT recovered after 6 hours of treatment with specific AV,  it produced by Trinh X Kiem at Cho Ray, 1991.

4.2. Image illustrating the treatment effectiveness of AVs in Vietnam

Fig.29: PQN patient envenomed by N. kaouthia venom, recovered with specific AV treatment at Cho Ray Hospital, 1998.

Fig.30: Patient NV.Ph envenomed by Caloselasma rhodostoma (C.R) venom admitted to Cho Ray Hospital, 1998.

Fig.31: Patient NV.Ph completely recovered from C.R bite, after treatment with specific AV at Cho Ray Hospital, 1998.

The highest award for the Vietnam Society of Toxicology & Vietnam Institute of Toxicology is the research and production antivenom program for clinical application, which has directly contributed to saving the lives of over three thousand snake bite patients in Vietnam.

The next goal is to focus all resources on manufacturing AVs, providing free of charge to save the lives of venomous snake victims in our country and then to support Laos and Cambodia.

Fig.34: The VN Government granted the Vietnam Science and Technology Innovation Award for AV manufacturing & clinical applications successfully.

Trinh Xuan Kiem. Associate Professor, PhD.BSCC. President of the Vietnam Toxicology Association. Director of Vietnam Institute of Toxicology Phone: (84) 88 617 69 97 Email:  trinhkiem0211@gmail.com Trinh Xuan Long. MSc MD. Phone: (84) 91 837 99 66 Email: longxtrinh@gmail.com dragontxl@yahoo.com

       Fig.35:  The International Society of Toxicology recognized that Dr. Trinh Xuan Kiem: Life Long Achievement and Contribution to Toxicology in Viet Nam.

4.3. Results of research and manufacturing of Antivenoms combined with training

1. Naja.kaouthia AV patented, 2003.
2. Calloselasma rhodostoma AV patented, 2003.
3. Result of CR.AV Multi-center clinical trial research, combined with successful training of  Master Chu Thi My, 2019.

Fig.36: Master Chu Thi My with her thesis on multi-center clinical trial of C.rhodostoma AV, 2019.

4. Research and manufacture N. siamensis AV, combined with successful training of PhD Le Khac Quyen. 2019.

Fig.37: Dr. Le Khac Quyen with his thesis on successful manufacturing N. siamensis AV, 2019.

5. Research and manufacture of North + South Bungarus candidus AV combined with successful training of Ph.D. Thai Danh Tuyen, 2014.
6. Manufactured bivalent kaouthia & N.atra AV, combined with successful training of Master Vu Cong Thanh, 2010.
7. Research and manufacture of King cobra AV, combined with successful training Master Truong Bich Thuy, 2010.
8. Supporting the successful training of Ph.D. Le Van Dong at Singapore University (NUS). Title: Biochemical and Immunological studies on snake venoms of South Vietnam. 2005.
9. Supporting the successful training of Ph.D Ha Tran Hung at Karolinska Institute, Stockholm, Sweden. Title: Clinical research on the effectiveness and safety of the N. multicinctus AV, 2010.
10. Successfully trained level 2rd specialist Doctor Ngo Van Hong with the topic of researching eye damage caused by snake venoms, 2000.
11. Results of Science-Technology Cooperation (Vietnam-USA): Research on manufacturing an anti-cancer biological product (Disintegrin) from snake venom in Viet Nam, 2018.

1. Vietnam Institute on Toxicology (VIT), Email: trinhkiem0211@gmail.com

2. Taiwan National Health Research Institute, Email: sung23@nhri.edu.tw

I. INTRODUCTION

1) The ecological characteristics of Vietnam, Laos and Cambodia are very favorable for the development of venomous snakes.

Vietnam (VN) is an agricultural and tropical country with a population of 97.261.435 people (United Nations data, April, 2019), located on the S-shaped strip of land in the Indochina peninsula. Which is determined from the 102o8’ to 109o27’ Meridian East, and from the 8o27’ to 23o23’ Latitude North. VN has a land area of 331.699 km2 and the East Sea is more than 1 million km2, surrounded by 3.260 km long coastline. The length of VN from Nam Quan to Ca Mau cape is 1.650 km. The width of the narrowest place is Quang Binh, only 50 km. There is Bachma mountain 1.444 m high with Haivan pass which is part of Truong Son mountain range that cuts directly to the East Sea. This topography has created 2 different climate zones: The North has 4 distinct seasons: Spring, Summer, Autumn and Winter. The Hoang Lien Son mountain range has many bows along with the valley of the Red, Lo, Da and Thai Binh rivers creating the Red river Delta with an area of 15.000 km2, where is the largest population in VN.

The South is determined from Hai Van pass to cape Ca Mau with the Central highlands and two geographical regions (East and West) with 2 distinct seasons: Dry season (from December to April) and Rainy season (from May to November). Average temperature for the whole year is (21-28oC). Temperature fluctuates all year round (5oC). Average annual rainfall is (from 1.500 to 2.000 mm) with moisture (80-82%). This is a very favorable environment for poisonous snakes to develop.

The East is a highland area (Average: 200 m compared to the West), with rubber forests, pepper and cashew trees. It is here that the Viper species are concentrated, the most dangerous is Calloselasma rhodostoma.

In the West, there is the CuuLong river with nine branches that make up the Mekong Delta as wide as 40.000 km2. This is the granary of the world. However, it is the low-lying, boggy land along the wilderness with interlaced canals. Which is a favorable place for the development of cobra species (Fig.1).

Two neighboring countries: Laos and Cambodia forming three Indochina countries with similar ecological characteristics: Laos has an area of 236.800 km2 with a population of 7.040.844 (2019), full access to the North of VN. Cambodia has an area of 181.035 km2 with a population of 16.428.267 (2019), full access to the South of VN. Therefore, venomous snakes in Laos are identical to those found in Northern VN and similarly venomous snakes in Cambodia are identical to those in Southern VN.

2) The accidents caused by snake bites and AV production in Vietnam.

WHO (2019). Dr Tedros Adhanom Ghebreyesus Director-General wrote: “Snakebites are a significant risk to health and well-being for 5.8 billion people around the world, and for those affected carry a high financial burden that often cannot be met.” [28]

WHO (2023): “A high-quality antivenom provides the best available treatment for approximately 5.4 million people who are bitten by snakes each year. Safe, effective antivenoms could prevent many of the 83.000-138.000 deaths caused by snakebites and reduce the severity of serious disabilities that impact many thousands more victims”. WHO has listed snakebite envenoming as a highest priority neglected tropical disease and launched the snakebite envenoming roadmap with the goal to halve the global burden of snakebite by 2030 [27, 29].

According to the results of an epidemiological investigation under the Ministry of Health of VN, there are about 30.000 (Thirty thousand) snakebite victims each year in VN. Most of these victims were the main workers of each family (Fig.2). With reference to research method of Hung DZ. (2004): Taiwan’s venomous snakebite and Blessmann J’s research, also have similar results as VN [3, 9].

The mortality rate in the severe envenoming snakebite group was very high (19.5%) because there was no AV. Although it’s more than 100 years after the invention of AV by Dr A. Calmette right at Pasture Institute in Saigon, VN [1, 2]. Meeting urgent clinical requirements, Dr. Kiem Trinh and his colleagues of the Venomous Snake Research Unit, have opened a new history of AV manufacturing and successful clinical application in VN [13, 14, 18, 22].

As the result, more than 3,000 (three thousand) victims of severe snake venom poisoning were saved by AVs made by Vietnamese people. The mortality rate of victims of severe snake venom poisoning has decreased to 1.5% [16].

However, the scale of AV manufacturing is still limited to the laboratory. The shortage of AV in VN is very serious. It is in great need of the attention of the State, the help of domestic, international organizations and individuals to develop AV production on an industrial scale, promptly meeting the requirements of saving the lives of thousands of snakebite victims in Vietnam. This is the goal of the AV production research program that the (VIT) has been focusing all our energy and wisdom on for many years.

II. METHODS

2.1. Method of identification and classification of venomous snakes in Vietnam.

According to the method for family and species identification of venomous snakes, Prof. Volfgang Wuster &. A. Malhotra, Ecology, Evolutionary biology University of Aberdeen, UK and Prof. D.Warrell [5, 30]. We had a good opportunity to collaborate on a survey of venomous snakes throughout VN. There are 12 basic criteria that need to be defined. Example of identifying Calloselasma rhodostoma according to the following standards below:

1) Kingdom: Animalia.

2) Phylum: Chordata.

3) Class: Reptilalia.

4) Order: Squamata, Scab.

5) Suborder: Serpentes.

6) Family: Viperidae.

7) Subfamily: Crotalinae.

8) Genus: Calloselasma.

9) Species: Rhodostoma.

10) Common name (English name): Malayan Pit Viper).

11) Scientific name: (Calloselasma rhodostoma).

12) Vietnamese name: Choam Quạp (The name was given by Kiem Trinh, the Snake Research Unit, Cho Ray hospital, 1990). [2].

2.2. Antivenom manufacturing method in Vietnam

According to the Guidance of WHO and experts in AV manufacturing, we carried out the fabrication at AV according to the following the technical process (Diagram.1) [5, 10, 30].

Diag.1: Summary the technical process of AV manufacturing in Vietnam.

a) Make snake venom antigen

After identifying and classifying typical snakes from each geographical area, it is necessary to select healthy snakes of different ages. Milking the venom, centrifuge, freeze dried and then store in (2-8 oC). Making snake venom Antigen (Ag) according to D.Theakston’s technique by using glutaraldehyde to detoxify the venom [23]. Ag was divided into doses suitable for the purpose to stimulate the horse to produce the highest antibody (Ab), but the horse remains healthy.

b) Equine immunity produces specific antibody with the highest titer.

Select at least 03 healthy horses for each type of AV. Administered subcutaneously a dose of snake venom Ag according to a definite schedule at monthly intervals (Fig.3). Monitor horses for antibody reproductive response by using Ouchterlony technique. [29].

c) Extraction of horse plasma by plasmapheresis technique.

d) Purification of AV by F(ab’)₂ (Fig.4) [5,10, 29].

e) AV Quality control at the manufacture factory and at the National Institute for Quality Control of Vaccine and Bioproduct, VN Ministry of Health.

According to the main standards prescribed by the Vietnam Pharmacopoeia V of Antivenom, Ministry of Health, 2018 [26]. The AV produced by Vietnam Institute on Toxicology (VIT) must be quality tested at the manufacturing facility to achieve good results. Next, the AV must be checked by the National Institute for Quality Control of Vaccine and Bioproduct, VN Ministry of Health, then issue a certificate for each batch. There are 7 standards as follows:

1. Sterility: Sterility criteria were determined by the AV culture technique in 02 media: Thioglycollate and Sabouraud. After 14 days of following-up, no bacteria or mold was detected, the result is satisfactory.
2. No pyrogens: This standard was defined by the following technique: Select 03 healthy rabbits, each one weighs (2-2.5 kg). Raising rabbits in a standard room for 24 hr. Measure the rabbit’s rectal temperature for 03 times, each time 01 hr apart. Required temperature difference between 03 measurements less than 1oC.

AV injection into rabbit ear vein at a dose of (0,5 ml/kg). After 03 hours of AV injection, measure the rabbit’s rectal temperature. The rabbit’s temperature requirement was increased less than 0.6 oC. Total temperature rise of all 03 rabbits must be less than 1.3 oC. The result of the pyrogen-free test was satisfactory.

3. General safety test: This test was done as follows: choose 03 guinea pigs each with a weight (250-350g). Intraperitoneal AV injection in each guinea pig at a dose of 05 ml. After 07 days of follow-up, guinea pigs did not lose weight and showed no clinical sigs. General safety test result was satisfactory.
4. 4. AV’s pH requirement in the range: 6,0 to 7,0.
5. AV’s total protein requirement was (170 g/l). Albumin was only a trace on the electrophoresis tape.
6. Preservative: Merthiolate (1/10.000), less than (0,02 %). Phenol: less than (0,025 %).
7. Potency: The goal of AV manufacturing is to neutralize the snake venom in the victim’s body, so it has been strictly regulated by international units (IU). Vietnam’s pharmacopoeia stipulates:

– Determine the 50% Lethal Dose (LD₅₀).

– Determine the Effective Dose (ED₅₀) of each manufactured AV.

f) Cold storage AV at (2-8 oC) and distribution to medical facilities in time to treat the snakebite victims.

III. Results

3.1. The successfully identified families and species of venomous snakes in Vietnam

A. Elapidae:

1. Naja naja or Naja atra (Chinese cobra): They are only available in the North of Vietnam and Laos.
2. Naja kaouthia (Monocellate cobra): They are only available in the Southwest region of Vietnam and Cambodia.
3. Naja siamensis (Thai spitting cobra): They are found in the Southeast, Central south of VN and Cambodia.
4. Ophiophagus hannah (Yellow king cobra). They are found in the Southwest of VN and Cambodia.
5. Ophiophagus hannah (Black king cobra): They are found in the Central and Northern VN, Laos and Cambodia.
6. Bungarus fasciatus (Banded krait: They are available in all 3 regions of VN, Laos and Cambodia.
7. Bungarus candidus (Malayan krait)): They are only available in the South of Vietnam, Laos and Cambodia.
8. Bungarus multicinctus (Chinese or many banded krait): They are only available in the North of VN and Laos.
9. Bungarus flaviceps (Red River Krait), (Bungarus slowinskii): They are discovered in the North of VN. However, the consequence of this snake is not clear.

B. Viperidae:

1. Trimeresurus albolabris (White-lipped green pit viper): They are everywhere in VN, Laos and Cambodia.
2. Agkistrodon rhodostoma or Calloselasma rhodostoma (Malayan pit viper): They live mainly in the Southeast region of VN and Cambodia.
3. T. mucrosquamatus (Chinese habu, Taiwanese pit viper): Rarely see them in the border provinces of Vietnam and China.

C. Hydrophiidae (Sea snakes):

There are 17 species of sea snakes. They concentrated on the central coast of VN. The most popular is Enhydrina schistosa.

Images of venomous snakes mainly in Vietnam.  

Fig.5: The main families and species of venomous snakes are circulating in VN

Note:

1. Naja atra (A)
2. Naja kaouthia (K)
3. Naja (N.S)
4. fasciatus (B.F)
5. Yellow Ophiophagus hannah
6. Black Ophiophagus hannah
7. candidus (B.C)
8. Bungarus multicinctus(B.M)
9. Trimeresurus albolabris (T.A)
10. Viperidae with a pit as a thermosensitive organ.
11. Calloselasma rhodostoma (CR)
12. Trimeresurus mucrosquamatus (T.m)
13. Enhydrina schistose (E.S)
14. Hydrophis cyanocinctus (H.C)

3.2. Research results on Antivenom manufacturing and clinical application in Vietnam.

In 1991, Naja kaouthia AV was successfully manufactured by Kiem Trinh and et al, directly saving the life of Le Q T victim, who had been seriously poisoned by NK bite, to be on a ventilator for up to 58^th hour at Cho Ray hospital. This was the first venomous snake victim saved by an AV made by Vietnamese people (Fig.6,7,8) [1,18].

Following that, the technical process of 05 specific AV types were studied and fully established in the laboratory. Which are:

-Technical process for making Calloselasma rhodostoma. AV [2, 21].

– Technical process for making Bungarus candidus. AV [13,14,25].

-Technical process for making Bungarus multicinctus. AV [8, 25].

– Technical process for making Yellow and Black Ophiophagus hannah. AV [17, 24].

– Technical process for making Naja siamensis.AV [20].

35 AV lots of the 6 specific AV types mentioned above have been approved by the National Institute for Control of Vaccine and Biologicals, MOH,Vietnam.

The most important result was that the AV batches manufactured by Dr. Kiem Trinh and et al of (VIT) have been clinically applied for clinical treatment according to Divid Warrell’s guidelines, directly saving the lives of over three thousand victims of severe envenoming. As a result, it has contributed to reducing the mortality rate of severe envenoming group from 19.5% to 1.5% [6], (Fig.10)

Since then, the Vietnam Society on Toxicology (VST) and Vietnam Institute on Toxicology (VIT) have been established in 2015.This is where thousands of experts and philanthropists gather to contribute to the snakebite disease has been forgotten in VN.

Fig.9 & Fig.10. Patient NV.Ph envenomed by Caloselasma rhodostoma (C.R) venom admitted to Cho Ray Hospital, 1998.

Fig.11 & Fig.12: Patient NV.Ph completely recovered from C.R bite, after treatment with specific AV at Cho Ray Hospital, 1998.
Fig.13 & Fig.14. PQN patient infected with Earth Tiger snake venom recovered with Earth Tiger Treatment System at Cho Ray Hospital, 1998.

2 patents of Calloselama rhodostoma (CR) and Naja kaouthia (NK)AV manufacturing have been granted by the State of Vietnam for technical process.

During the course of AV research and manufacturing for clinical applications, 04 PhDs and 6 Masters of medicine, specializing in venomous snakes were successfully trained. [7, 8, 20, 24, 25].

They are the experts who have been making effective contributions to the snakebite management in VN.

In December 2008, the important event was that the IST-Asia-Pacific Conference has been successfully organized by Dr. Kiem Trinh and et al in VN. It brought together more than 250 scientists around the World (Fig.11). This is the proud honor of Vietnamese toxicologists, key people continue to develop international and domestic cooperation for the success of snakebite management in VN.

IV. CONCLUSION

Snake bite is a serious tropical disease that has caused up to thirty thousand victims in Vietnam every year. The mortality rate of severe envenomed victims is up to (19.5%).

Since 1990, Dr. Kiem Trinh and the colleagues of Vietnam Institute on Toxicology (VIT) have researched and identified two venomous snake families (Elapidae and Viperidae) with 14 main venomous snake species circulating in VN.

06 main types of monospecific AVs have been successfully manufactured by VIT. They have applied to clinical applications, contributed to save the lives of over three thousand severe envenomed victims. The mortality rate has dropped to (1.5%). Once again, we have proven the strategic direction of WHO: “AV is the most effective and specific drug that has been used worldwide for many years”.

However, our researched program is still limited to the Lab. AV shortage is a serious problem in VN.

We urgently call on the State, charitable organizations, individuals from country to the World: Please take care and support the AV manufacturing program on an industrial scale for promptly and ready to rescue the snakebite victims in Vietnam, even Laos and Cambodia.

Reference

1. Anh T.K, Kiem T.X et al (1997): “Research and production of Naja Kaouthia antivenom, successful clinical application in Vietnam. A project of Ministry of Health, Decision N^o: 1223 KHDT, 1997.
2. Anh T.K, Kiem T.X et al (1997): “Research and production of Calloselasma rhodostoma antivenom, successful clinical application in Vietnam. A project of Ministry of Health, Decision N^o: 1224 KHDT, 1997.
3. Blessmann J, Nguyen TPN, Bui TPA, et al (2018): Incidence of snakebites in 3 different geographic regions in Thua Thien Hue Province, Central Vietnam: green pit vipers and cobras cause the majority of bites. Toxicon.2018;156:61–65.doi:10.1016/j.toxicon.2018.11.009 [PubMed] [CrossRef] [Google Scholar].
4. 4. Chanthawat Patikorn et al, 2021: Situation of snakebite, antivenom market and access to antivenoms in ASEAN countries. BMJ Global Health. http://dx.doi.org/10.1136/bmjgh-2021-007639.
5. Chippaux JP (2010):  Guidelines for the production, control and regulation of snake antivenom immunoglobulins. Biol Aujourdhui. 2010 10.1051/jbio/2009043 [PubMed] [CrossRef] [Google Scholar]
6. David Warrell -WHO, August, 2016: Guidelines for the management of snakebites.
7. Dong L.V (2004): Biochemical and immunological studies on snake venoms of South Vietnam. Ph.D. Thesis, National University of Singapore.
8. Ha T H, Höjer J, Trinh X K, Nguyen T D (2010): “A controlled clinical trial of a novel

antivenom in patients envenomed by Bungarus multicinctus”, J Med Toxicol, Dec, 6, pp. 393-

9. Hung DZ. (2004): Taiwan’s venomous snakebite: epidemiological, evolution and geographic differences. Trans R Soc Trop Med Hyg. 2004;98(2):96–101. doi: 10.1016/S0035-9203(03)00013-0 [PubMed] [CrossRef] [Google Scholar]
10. Guillermo León, Álvaro Segura, Aarón Gómez, et al, 2014: Industrial Production and Quality Control of Snake Antivenoms. Living reference work entry. First Online: 01 January 2014.

11. Kiem T.X, Long X.T(2022): “Venomous snakes, from clinical practice to manufacturing anti-venom and anti-cancer bioproducts”. Vietnam Medical Publishing House, 2022.

12. Kiem T X, Francis Markland Jr et al (2016): “A Novel Anti-tumor Protein from Calloselasma rhodostoma Venom in Vietnam”. Anatomy & Physiology: Curent Research.
13. Kiem T.X, Quyen L.K, Long T.X, David A. Warrell (2010): “Hyponatraemia, Rhabdomyoly- sis, alterations in blood pressure and persistent mydriasis in patients envenomed by Malayan kraits (Bungarus candidus) in Southern Vietnam”, Toxicon.
14. Kiem T.X, et al (2009): “Research and manufacturing Bungarus candidus antivenom, clinical application successfully in Vietnam”. Vietnam Medical Journal, N^o:1, 2009.
15. Kiem XT; Tuyen DT; Long XT (2009): “The production of Bungarus candidus & Bungarus multicinctus antivenom from horses immunized with venom & its application for the treatment of snake bite patients in Vietnam”, The XVI^th World Congress of International Society on Toxinology, Brazil.
16. Kiem Trinh et al (2008): Venomous snakes and snakebite management in Vietnam. The 8th IST-Asia Pacific Meeting on Animal, Plant & Microbial Toxins organized in Vietnam, December, 2008
17. Kiem T.X et al (2007): “Research to develop production Ophiophagus hanna antivenom, successful multi-center clinical application in Vietnam”. Department of Science and Technology, Ho Chi Minh City, 2008.
18. Kiem XT, Quyen LK, Phuoc NB (1997), “Snake bites in Vietnam, the initial results of research and production of Naja kaouthia antivenom and its application in the treatment of victims at Cho Ray hospital, Ho Chi Minh city, since 1990”, 12th the World congress of IST on animal, plant and microbial toxins, Cuernavaca Morelos, Mexico.
19. PatikornC, et al:Global systematic review of cost of illness and economic evaluation studies associated with snakebite. J Glob Health 2020; .doi:10.7189/jogh.10.020415pmid:http://www.ncbi.nlm.nih.gov/pubmed/33312499
20. Quyen L K, (PhD student), Kiem Trinh (Instructor), 2022: “Research to perfect the technical process of manufacturing Naja siamensis Antivenom in Vietnam”. Ph.D. Thesis. Vietnam Military Medical Academy, 2022.
21. Quyen L.K, Kiem T.X (2008): A case with brain hemorrhage caused by Calloselasma rhodostoma successful treated with C.R antivenom. The 8th IST-Asia Pacific Meeting on Animal, Plant & Microbial Toxins.
22. Quyen L.K (2003): Clinical Evaluation of Snakebites in Vietnam: A Study from Cho Ray hospital. A thesis for the Degree of Master of Science. National University of Singapore.
23. R.D.G.Theakston*, D.A.Warrell, E.Griffiths (2003) “Erratumto Report of WHO workshop on the standardization and control on antivenoms” [Toxicon41(5)(2003)541–557]
24. Thuy T.B (Master student), Kiem T.X (2008): “Research to perfect the technical process of manufacturing Ophiophagus hannah Antivenom in Vietnam”. A thesis for the Degree of Master of Science. Vietnam Military Medical Academy, 2008.

25.Tuyen T.D (PhD student), Kiem T.X (Instructor) (2014): “Research to perfect the technical process of manufacturing Bungarus candidus Antivenom in Vietnam”. Ph.D. Thesis. Hanoi Medical University, VN, 2014.

26. Vietnam Pharmacopoeia 5. Ministry of Health. Medical Publisher, 2018.
27. WHO (2023): Guidelines for the management of snake-bites World Health Organization.

28.WHO (2019): Snakebite envenoming: a strategy for prevention and control. Available: https://apps.who.int/iris/bitstream/handle/10665/324838/9789241515641-eng.pdf

29. 29. WHO (2010): Guidelines for the production, control and regulation of snake antivenom immunoglobulins. 134. Geneva: WHO, 2010.
30. Wolfgang Wuster (1994): Ecology and evolutionary biology of venomous snakes. University of Aberdeen, UK.

By 1990, determined to save lives, Dr. Kiem and his colleagues researched and prepared HTKN on their own under conditions of lack of equipment and finances. Research was so expensive that he had to sell his house. Another major obstacle to the work is that there has not been any complete research on venomous snakes in Vietnam. Therefore, the research team had to travel all over the country to survey poisonous snakes in each region. Survey results show that in Vietnam there are two families of venomous snakes: the cobra family (earth tigers, cat tigers, cobras, king tigers, kraits, kraits…) and the viper family (the most common is the snakehead snake). crow and green). The venom of the cobra family often causes neuromuscular paralysis while the viper venom causes hemostatic coagulation disorders (patients bleed to death).

It is estimated that in Vietnam, up to 30,000 people are bitten by snakes each year, of which 2/3 are poisoned. They are foresters, farmers, new economic workers, etc. The only method to save the lives of patients infected with snake venom is to prepare venom-neutralizing antibodies or specific serum (for snake venom). each type of poisonous snake).

Initially, Dr. Kiem’s ​​research team focused on preparing anti-venom serum for the two most common venomous snakes in the South: earth tiger (Southwest) and chom quac (Southeast). According to statistics, the rate of patients bitten by cobra is 30% while the rate of patients bitten by cobra is approximately 25%. To prepare serum, they take snake venom and process it so that the venom is no longer toxic but still retains its antigenic properties. Next, the venom is injected into the horse’s body to stimulate the horse’s immune system to produce antibodies according to the prescribed process. After a while, the horse’s blood is siphoned off to get the antibodies and finally purified into a serum that neutralizes snake venom. The job of extracting snake venom is quite dangerous and there was a time when Dr. Kiem was bitten on the hand by a crow.

Anti-venom serum… attracts investment!

The turning point occurred in 1991 when patient Le Quang Truong, 16 years old, in Dong Nai was bitten by a cobra and was in a coma for 58 hours. It is known that the Department of Hematology and Blood Transfusion is testing HTKN, so Truong’s family begged to use it, with the hope of saving lives. Based on scientific basis and firm belief in research results, doctor Trinh Kim Anh, then Director of Cho Ray Hospital, decided to allow the use of the serum under research. After two hours of receiving the HTKN infusion, Truong regained consciousness. Thus, Truong is the first snakebite patient in Vietnam to be saved by the first type of serum prepared by domestic scientists. Currently, Truong is working as a PhD student in the UK.

By 1993, the Ministry of Health allowed Cho Ray Hospital to conduct research and produce HTKN. In 1995, HTKN was approved for clinical use there. In fact, southern provinces, including Bach Mai hospital, also ask for medicine from Cho Ray to treat patients. In July 2003, Dr. Kiem’s ​​research team was granted a patent by the Intellectual Property Office on the technological process of anti-venom serum for cobras and snakes. Currently, Dr. Kiem is applying for a project to perfect the technology for producing cobra and cobra snakeskin medicine to be able to produce pharmaceutical products on a larger scale to serve the community in 2007.

Not stopping at the above successes, Dr. Kiem’s ​​research team has continued to research the scorpion snake skin system since 1999 and has used it on patients. The rate of patients bitten by scorpions accounts for 3-5%. After that, seeing that the king cobra was also very dangerous, Dr. Kiem asked to carry out a research project on preparing HTKN for the king cobra. King cobra HTKN was born in 2002 and currently Dr. Kiem continues to perfect production technology at the National Poison Control Center (Bach Mai Hospital-Hanoi), where he is working. His intention is to prepare HTKN of all types of venomous snakes in Vietnam to reduce the number of deaths due to snake bites. It is estimated that the cost of the HTKN he produces is only about 200,000-300,000 VND/bottle, 50 times cheaper than serum from other countries.

A lot of good news came to Dr. Kiem earlier this year when Taiwan confirmed that it would sponsor more than 1 million USD in the period 2005-2010 to help Vietnam produce HTKN. Some American universities, such as the University of Southern California, also offered to cooperate with him to research snake venom to create cancer treatment drugs. He confided that his greatest wish is to cooperate with foreign countries to establish a snake venom research laboratory to treat cancer because that will help the community more effectively and in the long term. Hopefully, with his passion for research and domestic and foreign sponsorship, Dr. Trinh Xuan Kiem will realize this dream.

Source: Nhan Dan Newspaper

According to the conclusion of the World Health Organization, Vietnam’s HTKNR meets international standards and is successfully applied clinically. In particular, the price is much cheaper, only 1/20 compared to HTKNR produced in other countries.

“No matter when a poisonous snake bites, a patient is hospitalized as long as their heart has not stopped beating, we can still save their life.” That is the assertion of Doctor Trinh Xuan Kiem, Poison Control Center of Mai Hospital when I came to see him after a working day that ended very late.

The patient will immediately be infused with a powerful anti-venom serum extracted from the snake venom itself. Doctor Kiem recounted that on November 24, 1991, patient Le Quang Truong (Dong Nai) was admitted to Cho Ray hospital in a state of unconsciousness due to being bitten by a cobra. Endotracheal intubation is the “best” method that the hospital has so far applied to cases of venomous snake bites. However, with this method the possibility of the patient being cured is very small.

At that time, Truong’s family received information about Dr. Trinh Xuan Kiem (then Head of the Department of Hematology and Blood Transfusion, Cho Ray Hospital) who was researching and manufacturing anti-snake venom serum (HTKNR). The family begged for a serum transfusion even though they knew this serum had not yet been officially approved for treatment and it was unclear how effective the treatment would be. Only when he had the decision of Cho Ray Hospital Director Trinh Kim Anh, “agreeing to use the serum made by Dr. Kiem”, did Mr. Kiem boldly mix the medicine and give it intravenously to Truong. . The medicine was infused from 12:00 a.m. to 3:00 a.m., and at 5:00 a.m. the same day, Truong was able to open his eyes. By 6:00 a.m., Truong was awake and healthy and could self-extubate. The knee wound has completely subsided. The danger was over, Truong was saved.

From suggested information

In the 80s, while working at the Department of Hematology and Blood Transfusion, Cho Ray Hospital, Mr. Kiem witnessed and directly interacted with many cases of poisonous snake bites, but could not do anything to save him. Surname. Patients often have blood clotting disorders, are hospitalized with bleeding in multiple forms, multiple organs on the skin, oral mucosa, eyes, vomiting and urinating blood… Sometimes patients also suffer from neurotoxicity. nerves, causing nerve-muscle paralysis and respiratory arrest. At this time, Cho Ray Hospital has not yet found the most effective treatment and only applies endotracheal intubation. However, with this method, the possibility of the patient being cured is very low. Many patients died after a few days or were left with serious complications such as local necrosis, limbs having to be amputated due to deep and widespread necrosis…

In extremely difficult economic conditions combined with Vietnam’s climatic and geographical characteristics, the number of people in rural, low-lying and mountainous areas bitten by poisonous snakes each year is estimated to be up to tens of thousands. Mr. Kiem felt heartbroken when he witnessed patients bitten by poisonous snakes facing death every hour while the country’s medical system has not yet found a specific medicine. He believes that this is a “neglected medical problem” in Vietnam. Because NR systems are specific to each geographical area and each country, each country needs to produce their own NR HT systems for their own country.

This troubled him for a long time. Until one time, on his assignment as an interpreter for a group of World Health Organization professors visiting a snake farm in Binh Quoi, he was prompted by Professor David Warrell with some information that could help him. Treat venomous snake bites with antivenom serum. Later, he called Professor David “The person who paved the way for treatment with specific HTKNR in Vietnam“. Since then, Doctor Kiem has researched documents, books and newspapers day and night to find information related to the treatment of poisonous snakebites.

Successful clinical application

It is necessary to prepare antibodies capable of neutralizing venom in the body based on the types of snakes living in Vietnam. Forgetting all dangers, Mr. Kiem personally caught and identified each type of snake and personally took the snake venom. Based on scientific research, the necessary steps to make HTKNR are: taking snake venom, creating an antigen, then injecting that antigen into the animal’s body (preferably a horse), stimulating stimulates the horse’s immune system to produce horse antibodies against snake venom. After about 7 to 8 months, the horse’s blood is taken to purify it into anti-snake venom serum.

The good news spread far and wide. After Truong was cured, the number of patients bitten by poisonous snakes coming to Cho Ray Hospital for treatment increased day by day. Due to seeing the effectiveness and necessity in treating poisonous snake bites of HTKNR, in 1993 the Ministry of Health licensed Cho Ray Hospital, in the name of doctor Trinh Xuan Kiem, to conduct research and treatment. create HTKNR. In 1995, HTKNR was allowed for clinical use at Cho Ray Hospital.

In July 2003, the group of doctors Kiem was granted a patent by the Intellectual Property Office on the HTKNR technological process. Currently, HTKNR is undergoing multicenter clinical trials (used at hospitals nationwide). Up to now, Dr. Kiem and his colleagues have successfully prepared anti-venom serum for the 7 most venomous snakes in Vietnam: king cobra, earth tiger, cat tiger, cobra, scorpion, green snake, and krait.

In particular, the doctor’s group is the first group in the world to successfully manufacture scorpion HTKNR. At international toxicology conferences, many leading experts have confirmed that the quality, safety and effectiveness of HTKNR manufactured in Vietnam meet international standards and are successfully applied in clinical practice. In particular, the price is much cheaper, only 1/20 compared to HTKNR produced in other countries. Normally, after 12 to 24 hours of treatment with specific HTKNR, the patient recovers completely.

Source: Consumer Consulting – No.8, April 20, 200

The Institute of Vaccines and Medical Biologicals (IVAC) in Nha Trang invests in a horse farm to prepare anti-snake venom serum. Over the past 10 years, this place has produced and marketed serum against the venom of cobras and bamboo vipers – common snakes and many people who are bitten by them have to be hospitalized. These are also two types of serum that Vietnam actively produces, so they meet market demand. Dr. Duong Huu Thai, Director of IVAC, said that the Institute is researching and developing two types of serum against northern krait and indigo snake venom. These two types have completed the product creation stage and are being tested and evaluated on animals. According to the international standard vaccine and serum research process, these two types must then complete human studies, evaluate, complete documents and proceed to apply for a license. It is expected that in the next 1-2 years these two serums will be introduced to the market.

“If calculating economically, the investment and development of these 4 types of antisera is a loss, but it is meaningful to save lives and serve patients so The Institute still focuses its time and effort on research and production,” Mr. Thai shared with VnExpress.

According to him, the antiserum production process is quite expensive, while investing in research to produce a product is difficult, and maintaining it is even more difficult. There are many types of Vietnamese snakes that are only We encounter a few cases each year, requiring the maintenance of a large technological line just to produce a few vials of serum. Meanwhile, if the quantity used is small, the re-licensing procedures will be affected because there is not enough quantity needed to evaluate effectiveness, side effects… Serum is an antidote to snake venom, a preparation contains globulins capable of specifically neutralizing corresponding snake venom, taken from the serum of healthy cattle (horses, sheep, mules, donkeys) that are immune to snake venom. If a victim is bitten by a snake, if they use anti-venom serum promptly, it will immediately neutralize the snake venom’s toxins, thereby preventing the organs in the body from being damaged.

Normally, the sooner a patient receives a detoxifying serum infusion, the more effective the treatment will be, and can recover in about 2-3 days. There are two types of serum: monovalent serum (used to detoxify one snake species) and polyvalent serum (used to detoxify multiple snake species). Except for the two monovalent serums produced by IVC as above, the remaining serums being used by hospitals are mainly polyvalent and imported. Therefore, domestic supplies depend on foreign countries, which are often scarce, lacking or non-existent. Like krait antivenom serum, according to Dr. Le Quoc Hung, Head of the Department of Tropical Diseases, Cho Ray Hospital, polyvalent antivenom serum for krait, krait, king tiger, and earth tiger is commonly used. imported from Thailand.

However, since the outbreak of Covid-19 last year, this supply has not been available again, so the market is short of serum. An  A 4-year-old girl in Phu Yen just died 5 days after being bitten by a garter snake, because hospitals do not have this anti-snake antidote serum. Doctor Dinh Tan Phuong, Head of the Emergency Department , Children’s Hospital 1, said that some snake species such as fire tigers (also known as red-necked flower snakes or school snakes), tiger cats… currently do not have anti-poison serum on the domestic and foreign markets. Therefore, people bitten by these snakes are very likely to be in danger, even die. Last year, two children baby died at Children’s Hospital 1 due to being bitten by a school snake without antidote serum. At that time, doctors contacted many countries around the world to search for anti-blood. Payment for the two grandchildren but still nothing. Currently, only a research institute in Japan is testing anti-serum against snake venom, but the product is still in the research stage and does not have enough three-phase testing to bring the finished product to market.

Talking about the reason why the serum production industry has not yet received investment attention, Dr. Hung agreed with IVAC’s difficulties and said that “producing anti-snake venom serum is not simple, it must be went through many stages of research and testing before becoming a commercial product.” Meanwhile, the number of snakebite patients is less than other common diseases, making investors afraid to spend large amounts of money to research and produce serum.

Sharing this opinion, Dr. Phuong said that producing antiserum requires a team of skilled human resources as well as great financial capacity. For example, student snakes have too little need for use, while any medicine only has a certain shelf life, if not used it must be thrown away. Patients who are bitten by kraits and kraits and do not receive serum injections can be replaced with respiratory support and blood filtration methods, which have a high survival rate, so the manufacturer is not interested in researching. The Center for Cultivation, Research and Processing of Medicinal Materials, Logistics Department of Military Region 9 (Dong Tam Snake Farm) in Tien Giang, which receives 2,000 cases of snake bites each year, has not yet prepared its own anti-snake venom serum. This place only raises snakes for venom and supplies IVAC to produce HTKN.

Another reason why anti-snake venom serum has not been researched in many places around the world is that the effectiveness of the serum depends on the species of snake and the geographical area in which they reside. According to Dr. Hung, drugs to treat other diseases can be used globally. Anti-snake venom serum is different, it can be effective against snake venom in this country but it may not be effective against the same snake species in another country. The reason is that the poison of the same snake species can be different depending on the food and characteristics of each locality. Without antivenom serum, nowadays, thanks to the development of medicine, people bitten by snakes will receive symptomatic treatment from doctors with many measures.

Doctor Nguyen Minh Tien, Deputy Director of the City Children’s Hospital, said that if the patient has muscle paralysis or difficulty breathing, they will receive early respiratory support, use medication to strengthen muscles, and wait about 1-2 weeks for the venom to subside. Snakes are semi-destructive depending on the amount of venom in the body. Patients with thrombocytopenia will receive platelet transfusions, and patients with coagulation disorders will receive blood transfusions and blood products. In cases of severe progression, doctors may prescribe advanced techniques such as dialysis, plasma exchange… “When there is no serum, the average treatment time is 7-14 days or longer. The cost is higher, there may be more complications,” Dr. Hung said, saying that such treatment is usually only effective for patients who come to the hospital early, without much disorder, and the amount of poison entering the patient’s body. medium. Neutralization of the poison cannot be as proactive as with antivenom serums.