Snakebite Envenoming a Challenging Diagnosis for the Forensic Pathologist: A Systematic Review

Snakebite envenoming (SBE) is a public health issue in developing countries. The estimated annual global incidence of snakebites is about 5.4 million snakebites per year, resulting from 1.8 to 2.7 million cases of SBE and from 81,000 to 138,000 deaths with 400,000 survivors suffering permanent physical and psychological disabilities. There are more than 3000 species of snakes around the world: 600 are venomous and over 200 are considered to be medically important because of their clinical effects. The severity of SBE depends on several factors among which bite localization, snake’s size, condition of glands and teeth, bite angle and bite duration, the microflora of the snake’s mouth and victim’s skin, age of the victim, weight, health status, and victim’s activity after a bite. Snake venoms are mixtures of protein families, and each of these families contains many different toxins or toxin isoforms. Based on their effects, snake venoms can be classified as hemotoxic, neurotoxic, or cytotoxic and they can all act together involving multiple tissues and organs. When the bite is fatal, the mechanism of death is primarily related to the paralysis of respiratory muscles, which causes asphyxia and hypoxic-ischemic encephalopathy, but also anaphylactic shock, hemorrhagic shock, cardiomyopathy, acute tubular necrosis (ATN). The purpose of this literature review is to evaluate epidemiological and post-mortem examination findings in fatal SBEs in order to better understand the pathophysiological mechanisms, thus helping pathologists in defining the correct diagnosis.


Introduction
SBE is a potentially life-threatening disease resulting from the injection of a mixture of different toxins (venom) that particularly afflicts the poorest people living in developing countries with the worst quality of life [1]. In 2017, the World Health Organization (WHO) listed SBE as a priority neglected tropical disease (NTD). WHO reports 5.4 million snakebites per year, resulting from 1.8 to 2.7 million cases of SBE and from 81,000 to 138,000 deaths with 400,000 survivors suffering permanent physical and psychological disabilities [1,2]. The promptness of the therapy is a key element for a good outcome after SBE. However, given the socio-cultural and economic context of the territories, effective treatment is often delayed due to the lack of hospital infrastructure, the non-availability of a species-specific antivenom or delay in its injection caused by the not prompt identification of the offending snake, and the use of traditional healers or popular remedies [3,4]. Pathogenesis induced by snake venoms is complex and characterized by local and systemic alterations represented mostly by bleeding, dermo/myonecrosis, inflammation, and coagulation disorders. Mechanisms of toxicity remain poorly understood because the venoms are complex mixtures of components that induce different effects [5]. Snake venom can be detected post-mortem through many toxicological ways, such as bioassays, radioimmunoassays (RIA), fluorescence immunoassays, immunoelectrophoresis, ELISA (enzyme-linked immunosorbent assay) [6]. Cytotoxins, neurotoxins, and hemotoxins are commonly identified depending on the species of the offending snake.
There are more than 3000 species of snakes around the world: 600 are venomous and over 200 are considered to be medically important because of their clinical effects [7]. New species of snakes are still being discovered, and many species formerly recognized as being widespread have been found to comprise multiple separate species [8]. Although the majority of snake species do not have venom and kill their prey by squeezing them, snakes are classified by WHO guidelines [8,9] based on production, control, and regulation of snake antivenom immunoglobulins and according to risk assessment: in category 1 (highest medical importance) there are highly venomous snakes that are common or widespread and cause numerous snakebites, resulting in high levels of morbidity, disability or mortality; in category 2 (secondary medical importance) there are highly venomous snakes capable of causing morbidity, disability or death, for which exact epidemiological or clinical data may be lacking; and/or are less frequently implicated. The aim of this study is to evaluate retrospectively the characteristics of fatal SBE reported in the literature.

Characteristics of Eligible Studies
The search of PubMed, Scopus, Web of Science, and Google Scholar databases provided a total of 263 articles. After adjusting for duplicates and reviewing titles and abstracts, 228 studies were discarded. Finally, only 35 papers reporting, in total, 56 cases of fatal SBE satisfied the inclusion criteria. A flowchart depicting the selection of studies according to PRISMA standards [10] is reported in Figure 1. Details of the 35 papers satisfying the inclusion criteria are summarized in Table 1.

Epidemiological Findings
The most common families of snakes involved in fatal SBE were Viperidae (n = 28-50.000%) followed soon after by Elapidae (n = 23-41.071%) and Lamprophiidae (n = 1-1.786%). In four cases (7.143%) no additional information about the snake was available. Most of the fatal cases of SBE reported can be classified into category 1 (n = 28) according to the WHO Guidelines on production, control, and regulation of snake antivenom immunoglobulins [8]. Just in two cases [22,33], the snake was not included in that list and in both cases, the snake was kept in captivity far from his habitat.
Most of the SBE victims were males (n = 35 cases; 62.500%) with a male/female ratio (M/F) of 1.67. Male victims were older than females. The median age in years for males was 40.77 and for females 28.71. The overlapping mean age was 36.25 years.
The body part most frequently bitten by snakes was the lower limb in 31 cases (55.357%), the upper limbs in 15 cases (26.786%), in one case the face (1.786%), and in one other case the genitalia (1.786%). Unfortunately, in eight cases (14.286%) the site of the snakebite was not reported. The vast majority of the SBE was accidental, but in one case study involving three victims, the manner of death was assessed as homicide [31].
It is worth mentioning that 16 out of 56 cases were related to work activities. Most of the cases occurred in a rural context (countryside, paddies, etc.).
Regarding clinical history, the time interval between snakebites and death ranges from 25 min up to 35 days. Deaths occurred mostly after 24h for 22 cases (39.286%) and before 6h in 19 cases (33.929%). Most of the victims, 40 out of 56 cases (71.429%), were hospitalized and the specific antivenom was administered to 26 individuals. In a few cases, the victim was at first treated by a local healer or with traditional remedies [15,20,28,43].

Post Mortem Examination Findings
Most of the bite marks (19 out of 56 cases) are described as two puncture wounds from the animal's fangs. Just a single puncture wound has been found in three cases, sometimes scratched or lacerated by the movement of the victim and/or his clothing deviating the fangs [44]. Additional wounds caused by other teeth or additional bites have been also described [29,31,43]. In two cases there were three fang marks [24,33]. Wankhede (2004) [44] described an unusual case in which four puncture wounds due to a single snakebite were found. In this strange case, the presence of the fang marks was consistent with a snakehead bigger in size than usual where the old fangs were still present, along with two new fangs.
In this regard, it is worth mentioning that for the identification of the offending snake the number of puncture wounds must be related to the number of teeth, which is unfortunately not the same in all kinds of snakes. Different species of snakes can have different anatomical conformation of the teeth and fangs. Fangs are sharp, long, hollow, or grooved teeth that are connected to a small sack full of venom in the snake's head behind its eyes but not all snakes have fangs. Most snakes have six rows of teeth in total: one row on each lower jaw and two rows on each maxillary and palatine or pterygoid bones of the upper jaw [46]. It is also important to highlight that the victim's clothes or gloves can represent an obstacle to the penetration of a fang [41].
With regard to the morphology of the fangs, sometimes the bite injuries are also described as linear or curvilinear stripes [15] fresh linear or multiple punctate abrasion [29]. The morphology of this kind of injury is quite similar to those produced by sharp force [47]. Snakebites in the multiple punctate abrasion pattern are often the result of the typical chewing activity of the coral snake. Instead, in most of the pit viper bites one or more fang marks are usually found [48] due to the stabbing motion of this snake specie's bite [29]. Although important for the identification of the offending snake, only in very few cases (n = 8) [3,12,20,22,24,37,44,45], the distance between the fang marks is reported. At the external examination, the area surrounding the bite mark shows dermal alterations represented mostly by signs of flogosis such as edema, cellulitis, and erythema. Bruises related to the snake bites are also reported due to the bleeding into the skin. It is strongly recommended to check the signs of the vital reaction of the bite mark and to take samples from bitten tissue. Subcutaneous hemorrhages are pretty common at the puncture skin site along with skin necrosis caused by the snake venom components. Only very few cases report additional dermal effects of the venom such as multiple blisters [32], a rash [41], and a purpuric area due to microthrombosis of the skin [42]. The main histological findings are subcutaneous hemorrhage, rhabdomyolysis, and necrosis of the dermis. Autopsy findings are mostly non-specific general pathologic changes and consistent with a rapid death such as multi-visceral congestion or edema and petechial hemorrhages. In the brain, other than congestion and edema, petechial hemorrhages from pin-point size to large hematoma in the white matter have been reported in 13 cases out of 56 in total (23.214%) [14][15][16]18,20,[23][24][25]27,39,44]. Microscopically, signs of ischemic and hemorrhagic encephalopathy have been found in six cases out of 56 [14,24,26,28,34,36] and extensive perivascular demyelination in a single case [21] Myocardial hemorrhages have been reported in seven cases out of 56 (12.500%) [12,14,16,27,35,39] and alterations consistent with myocardial infarction in a single case [14]. Signs of rapid failure of the left ventricle were mostly represented by pulmonary edema and congestion. Additional histological findings are hyaline membranes due to diffuse alveolar damage (DAD) in three cases [16,24,25] and widespread microthrombosis consistent with a disseminated intravascular coagulation (DIC) in four cases (7.142%) [12,19,20,39]. Renal injuries other than vascular congestion were mainly represented by tubular and/or glomerular necrosis found in 13 cases out of 56 in total (23.214%) [11,15,19,21,24,26,37,39,42]. Cortical and glomerular necrosis has been reported in one single case [30] and widespread ischemic necrosis in a different one [43]. Microthrombosis of the small inter-lobular vessels was found in a single case. Additional renal alterations are represented by acute interstitial nephritis (AIN) in three cases [3]. Intestinal injuries are reported in only three cases, but no better described [20,30,42]. Pituitary hemorrhage/necrosis and adrenal gland hemorrhage were present in another three cases [39].

Discussion
SBE is an acute medical emergency and a major public health problem that kills thousands of people each year, particularly in rural territories [49]. Most of the case studies considered in the present retrospective study were coming from tropical countries such as Brazil, Congo, India, Martinique, Myanmar, Nigeria, and Sri Lanka. The majority of fatal SBE involved snakes belonging to the Elapidae family (e.g., kraits-Bungarus spp., cobras-Naja spp., taipans-Oxyuranus spp., coral snakes-Micrurus spp., death adders-Acanthophis spp., and tiger snakes-Notechis spp.) and to the Viperidae family (e.g., rattlesnakes-Crotalus spp., Russell's viper-Daboia russelii). According to the geographical latitudes, the species of snakes causing the largest numbers of bites and fatalities are Echis sp. (saw-scaled vipers) in Northern Africa, Bothrops asper and B. atrox (lance-headed pit vipers) in Central and South America, and Naja sp. (cobras) and Bungarus sp. (kraits) in Asia [50]. SBE is both a method of hunting and a means of protection [51]. Snake venom is produced by specialized venom glands located in the upper jaw and associated with fangs, which are specialized long teeth with a superficial groove, along which the venom runs. Sometimes the fangs can also have an enclosed canal, down which the venom flows inside the tooth [52]. The severity of SBE depends on several factors which include bite localization, snake's size, condition of glands and teeth, bite angle and bite duration, microflora of the snake's mouth and victim's skin, age and weight of the victim, health status, victim's activity after a bite [53][54][55].
However, the main deadly factor is represented by the components of the venom. Snake venoms are mixtures of protein families, and each of these families contains many different toxins or toxin isoforms [56]. A very recent proteomic study on the venom of the viperid genus indicates the presence of proteins belonging to at least 12 families, with a predominance of phospholipases A2 (PLA2s) (54%) and metalloproteinases (21.5%) [57]. In order to perform rapid detection and quantification of venom antigens, commonly found in higher concentrations from wound swabs or wound aspirates (within 15-30 min after bite) compared to body fluids, commercial highly sensitive venom detection kits are available [58] but they cannot distinguish between venoms of closely related species. Moreover, the detection of venom in a wound swab is not enough evidence that the patient has been envenomed, then also serum and urine samples should be stored for enzyme immunoassays [59][60][61][62], liquid chromatography-mass spectrometry with time-of-fight (LC-TOF/MS), DNA fingerprinting, and antibody microarrays [63]. Highly specific methods such as the detection of venom gland mRNA by reverse-transcription PCR [64,65] or snake-derived DNA in bite-wound swabs [66] are being developed.
Based on their effects, snake venoms can be classified as hemotoxic, neurotoxic, or cytotoxic and they can act all together involving multiple tissues and organs [51]. Neurotoxicity is a well-known effect of SBE by snakes of the Elapidae family (e.g., cobras, kraits, mambas, Australasian species, and sea snakes) but also of the Viperidae family (e.g., rattlesnakes, lance-headed pit vipers, and true vipers) [67]. Neurotoxic envenomation can cause a broad spectrum of early clinical manifestations varying from ptosis and ophthalmoplegia to paralysis of respiratory muscles, which causes asphyxia. Elapid venom can interfere with transmission across the motor end-plate as it can bind to the postsynaptic nicotinic receptors that would normally bind acetylcholine [14]. Delayed neurotoxic manifestations are represented by peripheral neuropathies, optic neuritis, and cortical blindness [68]. Cerebellar involvement, ataxia, and "locked-in syndrome" are very uncommon but they have been also described [68]. Outcomes are pretty good when prompt treatment has been provided.
Usually, snakes from the Viperidae family are considered hemotoxic [51]. Viperidae snakes were involved in most of the fatal SBE reviewed (28 cases out of 56 in total) followed soon after by Elapidae snakes (23 cases out of 56). Therefore, the 56 victims reviewed in this retrospective study suffered mostly hemotoxic and neurotoxic effects. Hemotoxic effects act on the heart and cardiovascular system and they can promote or inhibit hemostatic mechanisms including coagulation in different ways [53]. Hemotoxins can be pro-coagulant proteases such as prothrombin activators, thrombin-like enzymes, factor X, and factor V activators but also anticoagulant proteases like the factor IX and X inhibitors, protein C activator, anticoagulant PLA2s [69]. The venom components acting on fibrinolysis are fibrinolytic enzymes and plasminogen activators [69]. Bleeding disorders can occur everywhere in the body from the bite site to the internal organs. Major complications related to hemotoxic effects include life-threatening diseases like acute kidney injury (AKI), ARDS, or its less serious variant acute lung injury (ALI), capillary leak syndrome (CLS), and DIC [70]. Victims suffering from cytotoxic envenoming are characterized by painful and progressive swelling at the bite site, developing further into blistering and bruising, which is sometimes coupled with systemic effects including hypovolemic shock [51]. Tissue necrosis is one of the major problems in patients with SBE [71]. Anaphylactic shock secondary to SBE has been reported in very few cases [72][73][74]. In suspected anaphylactic or anaphylactoid episodes related to drugs or venoms, a careful and complete review of the clinical history and the results of allergy testing might be useful in making the diagnosis or distinguishing them from other causes [75].
In the cases included in our review, the gross findings were non-specific. Autopsy findings reported are mostly non-specific pathologic changes consistent with a rapid death such as multi-visceral congestion, edema, and petechial hemorrhages. Signs of ischemic and hemorrhagic encephalopathy can be found mostly represented by intracranial or subarachnoid hemorrhages as well as myocardial hemorrhages. Widespread microthrombosis has been also described as the result of the coagulation disorders caused by the hemotoxic effects of SBE [76]. A relationship between SBE and myocardial infarction (MI) can also occur [77]. In particular, after SBE different factors such as coronary thrombosis due to the procoagulant factors in venom, direct toxic effect on cardiomyocytes, decreased oxygen-carrying capacity of blood due to hemolysis, coronary vasoconstriction brought about by endothelins and safatoxins in venom, and myocardial hemorrhage and microvascular thrombin deposition can determine an acute myocardial injury due to an imbalance between myocardial oxygen supply and demand [77,78].
Other than pulmonary edema and congestion related to acute cardiac failure, pulmonary injuries by SBE are mostly represented by DAD and widespread microthrombosis in the DIC scenario. In particular, the effects of Crotalus durissus terrificus venom (CdtV) on the pulmonary mechanic events have been studied in male swiss mice [78,79]. The histological analysis has reported perivascular/interstitial pulmonary edema 6 h after venom injection and pulmonary hemorrhages 12 h after. The microscopic quantitative analysis has also reported a significant increase of pulmonary inflammation 3 and 6 h after venom injection, returning to control values 24 h after [79]. The systemic inflammatory response and the mechanisms of pulmonary injuries by Crotalus durissus cascavella venom have been also investigated in mice by Azavedo et al. (2020) [80]. They reported a significant increase of peribronchial inflammatory infiltrates, emphysema, and focal atelectasis indicative of severe pulmonary inflammation 3 h after venom injection followed by a decrease in the activity of inflammatory response, despite an increase in vascular congestion, alveolar septal thickening, and emphysematous areas at 6 h. Between 12 and 48 h, there were perceptible inflammatory infiltrates in the pulmonary parenchyma, hemorrhagic focuses, vascular congestion, and bronchial muscle distention [80].
Acute nephrotoxicity is a relevant complication of SBE mostly represented by AKI that is a major cause of morbidity and mortality. AKI following SBE is mostly related to ATN or ACN and has multifactorial pathogenesis including, hypotension, coagulopathy, intravascular hemolysis, thrombotic microangiopathy (TMA), and DIC, capillary leak syndrome, rhabdomyolysis, complement activation, and sepsis [81]. Once developed ATN, AKI, and death can follow quickly [81,82]. A total of 22 cases out of a series of 121 snake bite-induced AKI have been studied histologically [83], 91% of the study sample mostly after SBE by Viperidae family showed signs of ATN classified as severe in 55% of the cases. Tubulointerstitial lesions represented, in particular, by ATN, were observed in 70% to 80% of patients with AKI [83]. However, kidney injuries have been also found in three cases of SBE by Bungarus Fasciatus, Elapidae family [3]. Among the intestinal injuries, bowel ischemia due to thrombosis of the mesenteric vessels has been reported in very few cases. [84,85]. Pituitary hemorrhage/necrosis and adrenal gland hemorrhage are additional autopsy findings reported [55].

Conclusions
SBE is a public health issue in tropical countries. The identification of snake species can be crucial for appropriate clinical management and can improve the prognosis. Although snake venom can be easily detected by toxicological analysis, diagnosis of SBE related death is challenging for the forensic pathologist. The cause of death from SBE is primarily related to the neurotoxic, cytotoxic, and hemotoxic effects of the venom components including the paralysis of respiratory muscles, which causes asphyxia and hypoxic-ischemic encephalopathy, hemorrhagic shock, cardiomyopathy, ATN, and death from anaphylactic shock [15]. Key elements are still represented by investigation of the bite mark and circumstantial data. Although rare in the European context, [86][87][88] it is important for the forensic pathologist to be aware of the details of the bite experience, local and systemic symptoms, and to check the bite marks by taking samples from bitten tissue, mostly located in the lower limbs.

Materials and Methods
The review of the literature was conducted independently by two examiners and carried out according to the PRISMA statement [10].

Literature Search
This retrospective study was performed using the following electronic databases: PubMed, Scopus, and Web of Science. The following keywords were searched in all fields: "snake bite" AND "autopsy" OR "snakebite" AND "autopsy" to recognize relevant research available until 18 April 2020. No time limit was set. The systematic search was further extended by snowball search and hand searching by using Google Scholar.

Inclusion and Exclusion Criteria
The following inclusion criterion was adopted: (1) papers reporting autopsy cases of fatal SBE with a description of the autopsy and/or histological findings. Exclusion criteria comprised the following: (1) animal studies; (2) scientific article not published in English, and (3) full text not available. For duplicate studies, only the article with more detailed information was included.

Data Extraction
Two examiners independently provided the initial selection of the articles to determine whether they might potentially fit the inclusion criteria. The title, abstract, and full text of each potentially pertinent study were reviewed. Disagreements on the eligibility of the studies were solved between the two examiners; if no agreement could be reached, it was resolved using a third co-author as a reviewer. A data extraction sheet was developed and for each selected study the following items were recorded: snake species; details of the victims (sex; age); location (country, working place); clinical history (time interval between snakebite and death; admission to hospital; administration of antivenom); autopsy features (morphology of the bite; macroscopic and microscopic features of the bite site; macroscopic and microscopic findings at autopsy).
Funding: This research received no external funding.