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A proposal essay on the Development of Rapid Diagnostic Test Kit (RDT) for Snake Envenoming in the Republic of Korea

1. INTRODUCTION (173+197+258+206)

More than 3,000 species of snakes are in the world, among them 600 species seem to be venomous, and only 250 are enlisted in the ‘medically important snakes’ by the World Health Organization (WHO) (WHO, 2010). Snakebites are a serious public health concern in many rural areas all over the world, in particular, where is poorly accessible to the health facility. (Fig. 1. a). The WHO estimates around 5 million snakebite cases occur a year resulting in 2.7 million people envenomed (WHO, 2017). Another report says that 400,000 amputation cases are known to be occurred and between 20,000 and 125,000 people killed (Kasturiratne et al., 2008). Although the 10^th meeting of the Strategic and Technical Advisory Group for Neglected Tropical Diseases added the snakebite envenoming to WHOs’ neglected tropical diseases (NTDs) list (WHO, 2017b), it continuously needs to call for a solidary action globally because the mortality rate per year is still enormously higher than other NTDs, including dengue fever, cholera, and yellow fever (Tab. 1) (Williams et al., 2010).

The major barrier to treat the envenomed is that health professionals often encounter the difficulties to determine the snake responsible for envenoming by looking at wound and symptoms, thus making treatment with the correct antivenom much tougher, especially in regions where only monospecific antivenoms are available (Theakston and Laing, 2014). Therefore, it is necessary to create a rapid diagnostic test (RDT) kit not only for decreasing the mortality rate but also for betterment in prognosis (Maduwage, O’Leary and Isbister, 2014). Unfortunately, there is no available the RDT kit for snakebites in the world except Australia to date. By all means, three critical factors led to the success of the RDT kit development are the ecological factor, academic factor, and economic factor. In this aspects, Australia meets all of these requirements. Firstly, there are only five snake species such as Notechis scutatus; Pseudonaja textilis; Pseudechis australis; Acanthophis antarcticus; Oxyuranus scutellatus accounted for most snake envenoming (CSL Antivenom Handbook, 2018). This is the driving-factor of the control that all rapidly detectable venom types are neurotoxin (Fig. 1). Also, Australia has well-prepared and organised research infrastructures that are capable of studying the snake venom with sustainable financial support from a variety of funders.

Species	PLA2	SVMP	LAO	3FTx	KUN	CRISP	NP	3FTx + PLA2
Notechis scutatus	74.5	–	–	5.6	6.9	0.3	2	80.1
Oxyuranus scutellatus	68–80	5–9	–	0–9	<10	<1	1	68–89
*Pseudechis papuanus	90.2	2.8	1.6	3.1	–	2.3	–	93.3
Figure 2. The SDS-PAGE gel image of the medically important snakes in Australia and the proportion of each of the seven major protein families in venom (Jackson et al., 2016; Tan, Tan and Tan, 2016; Tasoulis and Isbister, 2017; Arbuckle et al., 2018)            *Some data not found or substituted.

When it comes to requirements mentioned above, Korea is also capable to control of snakebites. The medically important snakes in Korea are as simple as Australia, it has only four; Gloydius brevicaudus, G. intermedius, G. ussuriensis, Rhabdophis tigrinus (Fig 2.). And Korea has the capability to study the snakebite and many funding sources as well. However, snakebites are entirely neglected. For example, ‘WHO guidelines for the management of snakebites’ says the current situation in snakebite occurrence in the Republic of Korea with one sentence that “Published data are restricted to the Republic of Korea” (Warrell, 2010a). In fact, there is no consensus data, no baseline survey, and no consistent care guidelines and regimens. Hence, nobody knows how many and often snakebite victims occurred a year (Lim, Kang and Kim, 2013). Only a few clinical data written in either Korean or English are available. A report from one hospital published in the Journal of Korean Ophthalmology Society, 6 cases were treated by the Pyridostigmine, acetylcholinesterase inhibitor, due to Ptosis as a result of snakebites during 2006 to 2008 (Lee, Ahn and Jung, 2009). Besides, a venomous snakebite occurred in a water park located in Daegu in June 2016. Yonsei University Wonju College of Medicine Hospital dealt with approximately 20 cases of snakebites in the emergency room during the research period, indicating that it is not a rare injury (Rha et al., 2015). It is roughly reckoned that at least 200 snake envenoming cases annually occurred in Korea and around 5-10 people killed on average (Lim, Kang and Kim, 2013).
Primarily, G. ussuriensis, G. brevicaudus and G. intermedius accounted for approximately 60~70% of snakebite in Korea (Shim et al., 1998). Patients clinically undergo a variety of symptoms from local pains, oedema to systemic complications like rhabdomyolysis, renal failure, and neurologic abnormalities (Kim et al., 2008). But, it is impossible to identify the venom by looking at the wound and symptom when the snakebite victim transferred to a hospital. Even though the overall health indices are relatively high, research infrastructure and relevant information about snakebites are as badly insufficient as other endemic countries. In this background, a game changer not only for improving the survival rate but also public awareness is to develop a rapid, and reliable test to determine the appropriate regimens. When it comes to the environmental condition, developing the RDT kit for the indigenous snakes in Korea seems to be needed as well as desirable. The ultimate aims of this essay are to suggest the possibility of the development of RDT kit for four indigenous snake species in Korea to increase the diagnostic accuracy. Secondly, it is to reveal the venom profiling in comparison with the practical results and other literature. And lastly, it will justify the snakebite research in Korea through evidence-based research.

Glyoidius brevicaudus (Short-tail Mamushi)	Glyoidius intermedius (Rock Mamushi)
Glyoidius ussuriensis (Ussuri Mamushi)	Rhabdophis tigrinus (Japanese keelback)
Figure 2. A map of the geographical distribution of the indigenous snake species in Korea.  The geographical distribution of each species is generated in accordance with the Taxonomic Databases Working Group (TDWG) (The Reptile Database, 2018)

2. VENOM & ANTIVENOM

2.1. Venom (98+109)

Snake venom is a product as a result of a modification of the salivary gland which extraordinarily containing various toxins using for immobilisation and digestion its prey or carrying out as a defence mechanism (Hayashi, 2005). The venom-producing gland is homologous and, it is mechanically delivered by an injection system via front fangs that enable the venom to penetrate into the targets. Snake venoms are mixtures of diverse proteins, and each peptide contains a lot of toxins or isoforms (Warrell, 2010b). It includes more than 20 compounds, approximately 200 proteins and polypeptides (Tab. 2) (Gutiérrez et al., 2017).

2.2. Antivenom (136+177)

To date, intravenous immunoglobulin injection is the primary method to neutralise the venom for more than 100 years since Dr. Albert Calmette introduced the serum antivenom for the treatment of envenoming in 1895. (WHO, 2017a). Antivenom is a biological product created by venom from the target snake. The venom is then diluted and injected into an antivenom host such as horse, sheep, goat, or camel. Accordingly, the host animal will undergo immune response in which immunoglobulin produce against the venom molecules. Through this series of procedures, the antidote can then be harvested from the blood and used to treat envenomation. Antivenoms can broadly be classified either monovalent which are effective against a specific snake venom or polyvalent which are effective against a wide range of species, or several different species at the same time (Tab. 3).

3. FINDINGS

3.1. MCD-P Assay reveals Crotalinae snake venoms make a plasma clotting. (106+138+82)

The venoms were collected from taxonomic, pathologic and geographical representative Viperinae, Crotalinae, and Elapinae snakes from Africa, Asia, Australasia, and Americas. The compositional studies were identified for 16 snake species: 5 from 239 Crotalinae, 5 from 101 Viperinae, 5 from 360 Elapidae, and the only 1 from Colubridae snake. To determine the pathological feature of each venom to blood, we carried out the MCD-P assay. The MCD-P assay is to define the least amount of venom that clots standard citrated solution of human plasma in 60 seconds at 37 °C (Theakston and Reid, 1983). And then, the clotting time of each venom was recorded (Tab. 4).

Interestingly, all Elapidae snake venoms did not make plasma clotting, even their total concentration was much higher than other snake venoms. Whereas, all Crotalinae snake venoms successfully made a plasma coagulation. Only 2 out of 5 Viperinae venoms were able to produce a plasma clotting. Although the venom is much less concentrated than other samples, coagulation relatively occurred in short time in comparison with the most Crotalinae samples. Lastly, the only one Colubridae snake, Dispholidus typus, venom made clotting in a minute.

3.2. The composition of proteins of Crotalinae snake venoms is complex. (80)

The 1-dimensional SDS-PAGE gel electrophoresis carried out to determine what kind of proteins included in each venom (Fig. 3). This technique is commonly used for separation of the macromolecules based on its size by the electric charge in vitro. Venom profiling unveiled that venoms are composed of a variety of proteins. But, Snake Venom Metalloprotein (SVMP) is strongly expressed in haemotoxin venoms such as Echis ocellatus, Bothrops jararaca, Lachesis muta, and Dispholidus typus, most of them are haemotoxin snake species.

Figure 3. Characterisation of venom proteins through 1-dimensional SDS-PAGE gel electrophoresis. Viperinae, Crotalinae and Colubridae snake venoms made a plasma coagulation by the MCD-P assay.

3.3. Enzyme-linked immunosorbent assay (ELISA) results give a clue the development of the RDT. (72+38)

The ELISA performed to confirm the neutralisation of the six antivenoms such as SAIMR Echis carinatus (AV1), Beafrique-10 Pan African (AV2), SAIMR polyvalent (AV3), Malayan pit viper (AV4), Anti-Bothropico (AV5), CSL-Polyvalent (AV6) against four haemotoxin venoms. AV5 is manufactured to treat the snakebite patients bitten by Bothrops spp., however, the plots as a result of the ELISA are shown that it has universally neutralised in haemotoxin venoms beyond the families (Fig. 4).

Figure 4. Anti-Bothropico (Green plot) resulted in neutralisation in the haemotoxin venoms collected from Echis ocellatus, Bothrops jararaca, Lachesis muta, and Dispholidus typus.

Whereas, AV5 did not work as the antidote to most neurotoxic venoms collected from Elapidae (Fig. 8). Therefore, it is presumably deduced that an antigen detected by AV5 is likely to be a venomic marker of haemotoxic snakes.

Figure 5. Anti-Bothropico (Green plot) was not able to neutralise the neurotoxin venoms collected from Bungarus candidus, Naja nivea, N. nigricollis, and Oxyuranus scutellatus.

3.4. Venoms of Gloydius spp. consist of many protein families but SVMPs are predominant. (183+184)

G. brevicaudus venom has many different types of proteins such as Disintegrin, PLA₂, SP, CRISP, CTL, LAO and SVMP. Among them, SVMP is the predominant protein (64.4%), followed by PLA₂ (25.0%) (Gao et al., 2014). On the other hand, the proportion of the protein families of G. intermedius venom is slightly different, even though the whole composition has high similarity with G. brevicaudus venom. The four most abundant families are SVSP (36.2%), NP (25.3%), LAO (13.1%), and PLA₂ (9.9%) (Fig. 8) (Yang et al., 2015). Although the clade of R. tigrinus shows no phylogenetic relationship with Crotalinae, its bite resulted in venom-induced consumptive coagulopathy as similar as many pit vipers’ patterns (Silva et al., 2014). Also, one clinical report published by Japanese researchers demonstrated that R. tigrinus venom shows strong plasma coagulant activity, with prothrombin activating effects and weak thrombin-like effects; therefore, main symptoms of the patients are disseminated intravascular coagulation, fibrinogen degradation (Hifumi et al., 2014). Based on the literature published in China and Japan, the main venomous proteins of Gloydius is the metalloproteinase which is likely to be seen from haemotoxin snake.

The proportion of the protein families of G. intermedius and G. intermedius venom are slightly different even though the whole composition has high similarity, for instance, the cytotoxic and haemotoxin proteins (SVMP, SVSP) are predominant. However, G. ussuriensis venom is no data.
Species	PLA2	SVMP	LAO	SVSP	DIS	CRISP	CTL	NP
Gloydius brevicandus	25	64.4	0.9	3.7	*4.6	1.1	0.2	–
Gloydius intermedius	9.9	2.6	13.1	36.2	–	6.2	0.8	*25.3
SVMP PII and PIII: snake venom metalloproteinase of classes PII and PIII, PLA2: phospholipase A2, SP: serine proteinases, LAAO: L-amino acid oxidase, CTL: C-type lectin-like protein, CRISP: Cysteine-rich protein, NT: nucleotidase, BPP-CNP: bradykinin-potentiating and C-type natriuretic peptide, EST: expressed sequence tag, HAase: hyaluronidase, NGF: nerve growth factor, PDE: phosphodiesterase, PLB: Phospholipase B, NP: natriuretic peptides.  *Red letters are likely to be a candidate for Gloydius spp. as a venomic marker
Figure 6. Comparison of the total protein composition of venoms from G. brevicaudus & G. intermedius and 1-dimensional SDS-PAGE gel of Rhabdophis tigrinus (Gao et al., 2014; Silva et al., 2014; Yang et al., 2015; Komori et al., 2017).                           *Some data not found.

4. DISCUSSION (47+139+101+100)

In Korea, developing the RDT kit is much more feasible than other parts of the world due to various reasons. This essay has been focusing on the possibility and technical challenge in comparison with other literature and practical results. Accordingly, there are three things could be suggested.

First of all, the MCD-P results represent that all haemotoxin snake venoms were able to make the plasma coagulation, although the time varied. All Crotalinae snake venoms made clotting in 120 seconds on average (± 67 seconds, 95% CI). Its median was 94 seconds, and the venom collected from Lachesis muta resulted in the shortest time (38 seconds). But, recording time cannot be significant since the deliberate involvement of the venom concentration to deal with the technical problem during the practical session. Ainsworth and his colleagues published the procoagulant potency of snake venoms to normal citrated human plasma and their capability to clot plasma (Tab. 5) (Ainsworth et al., 2018). When it comes to the development of RDT kit for Korean snakes, the MCD-P practical results will give a tip whether each venom has a coagulant potency or not.

Figure 7. Snake taxonomy and the relative taxonomical positions of its venom type (Li, Fry and Kini, 2005). According to the clades of each snake group, Viperinae and Crotalinae families are evolutionary closed relationship amongst all groups. Whereas, the Elapidae family is phylogenetically far from Viperinae, Crotalinae, and Colubridae.

Secondly, venoms which were able to coagulate share the common things such as high proportion of metalloproteinases as a result of the 1-dimensional SDS-PAGE gel electrophoresis. Also, these venoms were well-neutralised by Anti-Bothropico antivenom based on the ELISA; therefore, the antibody neutralised by Anti-Bothropico antivenom in these venoms might have been able to be a general venomic marker. Although full profiling information is scarce, ‘disintegrin’ is remarkably expressed in G. brevicandus venom, as well as ‘nucleotidase’ is detected in G. intermedius only. In this background, specific venomic markers to be used for developing the RDT kit are likely to be achievable.

Lastly, although the composition and feature of snake venom are highly relying on the environment, evolutionary and taxonomical relationships cannot be negligible as a critical factor (Davidson and Dennis, 1990). The dominant species of Korean venomous snake is Crotalinae with one minor from Colubridae. In regarding of composition of venom, all snakes are likely to have the similar venoms, hence, the snakebites in Korea have shown the similar symptoms based on the literature. It is not only because of the ecological similarity but also the phylogenetic relationship. But, this aspect should be more studied whether it is correct or not.

5. CONCLUSION (173+189+130)

When the victim of snakebite transferred to a medical facility, accurate diagnosis of the venom type is the urgent treatment. Therefore, the RDT kit plays a critical role in reducing mortality of snakebites. However, the venomous proteins of snakes vary relying on many factors such as species, regions, prey, and ecological environment. Furthermore, the toxin development has various genetic mechanisms such as genetic synteny, alternative splicing, domain loss/duplication and these are evolutionary well-conserved (Cousin et al., 1998; Casewell et al., 2011; Vonk et al., 2013). Technically, Chinese researchers tried to design an RDT kit for identification of three venomous snakes in China, but it failed because the cross-reacting fractions and avoiding the false positive could not be eliminated (Gao et al., 2013). The effort to develop the trustworthy RDT kit has been more than 40 years since the 1970s, but only one RDT kit is successfully released to the market so far (Currie, 2004). Due to this reason, developing the snakebite RDT kit is time-consuming, labour intensive and it needs lots of budgets.

Most venoms from Gloydius spp. and R. tigrinus have the similar components (metalloproteinases), even though its proportion might be disparate. This is a critical factor in the reflection to the history of the successful development of RDT kit in Australia. Due to this reason, Snake Venom Detection Kit manufactured by CSL Antivenom in Australia is the most trustworthy reference. General Principles of this RDT will be based on the sandwich ELISA technique. It is estimated that most Korean snake venoms have their distinctive venomic marker and generic markers. Based on this information, the proposed draft of the RDT is as similar to CLS Snake Venom Detection test (Fig. 8). According to the sample drawing, its size, physical feature will be relatively much bigger than malarial RDT kit. It is likely to be used in the medical facilities such as primary public health centres and secondary hospitals rather than using for non-professionals in the field as a Point-Of-Care testing. Indeed, this concept would be more acceptable because it aims to increase and support the regimen for the snake envenomed as a diagnostic tool at the hospital when the patient transferred.

Figure 8. The Preliminary version of the RDT for Korean Snakes. The general principle of this RDT is the sandwich ELISA, therefore all wells are coated with the primary Antibody. The venom sample will be collected by swabbing the envenomed part and it will be diluted with the buffer. This diluted sample will be placed in each well. After incubating for a while and then, wash out all wells with saline/D.W. And the 2’ antibody and the reagent will be successively aliquoted.

To sum up, there are more than 3,000 primary public health centres in Korea and which are responsible for securing the public health in the remote area (MOHW-Korea, 2017). Most snakebites may occur in this remote area. In the economic point of view, this market seems to have purchasing power because the appliance will be consumable to the public area via ‘Business to Government strategy’, if it developed. There might be high demands and needs in the clinical environment, so it is commercially competitive as well. Above all, in terms of economy, ecology and scientific background, developing the RDT kit for indigenous snakes in Korea is relatively suitable based on other data and literature. Also, classical approaches to toxinology of snakebites can tremendously contribute to this understudied field in Korea.

6. PROSPECT & LIMITATION (152)

This research may give a number of advantages. It is likely to pay attention the current research trends in snakebite as a neglected tropical disease when it makes relevant research outcome. Also, it would commercially have market competitiveness in East Asia. However, there are more critical data added in order to be significant in the academic field. At first, the venom profiling research of Korean snakes has not been carried out yet. That is why logic associated with the results of the practical session was weak. Secondly, the literature data were insufficient. All cited data for G. brevicaudus, G. intermedius, G. ussuriensis, R. tigrinus came from either Japan or China. Furthermore, G. ussuriensis is ecologically distributed sole in the Korean peninsula, so that there was no research data for this species. Lastly, biochemical experiments such as HPLC, 2-dimensional SDS-PAGE gel electrophoresis et cetera should be conducted in collaboration with the statistic approaches.

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