Value of multimodal ultrasound in the assessment of snakebite

Abstract

Background

This study aimed to analyze the ultrasound features of snakebite-affected limbs and explore the diagnostic utility of multimodal ultrasound.

Methods

An analysis was conducted on 70 patients with snakebites admitted to The Affiliated Hospital of Southwest Medical University from July 2023 to October 2023. Two-dimensional ultrasound was used to observe subcutaneous tissue edema, color Doppler flow imaging to observe hemodynamic changes, and shear-wave elastography to measure subcutaneous tissue elasticity. Patient demographics and multimodal ultrasound findings were recorded, comparing affected versus unaffected limbs.

Results

In all patients, the thickness of the subcutaneous fat layer, ultrasound grayscale median value, elasticity, and limb circumferences were significantly higher on the affected side than those on the unaffected side (P < 0.05). Continuous testing was conducted on 39 patients hospitalized for ≥ 3 days, and a gradual decrease in the thickness and elasticity of the subcutaneous fat layer was observed with treatment progression.

Conclusion

Multimodal ultrasound can assess limb edema and provides useful indicators for assessing the risk of compartment syndrome. This provides clinicians with a decision-making reference for treatment after snakebite.

Introduction

Snakebites are a common medical emergency worldwide. Statistics estimate that up to 2.7 million people are bitten by snakes annually, resulting in approximately 400,000 permanent disabilities and between 81,000 and 138,000 deaths [1]. Clinical manifestations depend on the type of snake. If a snake attacks without releasing venom, resulting only in a simple fang bite and no obvious symptoms or signs of envenomation, this is classified as a “dry bite,” which has relatively minor effects [2]. However, if symptoms of envenomation occur, immediate medical treatment is required. Studies indicated that patients treated within 12 h of a venomous snake bite have better outcomes than those who seek treatment after 12 h [3], highlighting the importance of early diagnosis and treatment.

Currently, clinicians mainly rely on patient symptoms and laboratory tests to assess their conditions, with some treatment methods based on clinical experience, which introduces subjectivity. However, with advancements in ultrasound technology, its utility in snakebite management has grown. Modern ultrasound can clearly visualize the structures of subcutaneous tissues, including skin, fat, and muscle, and can also identify subcutaneous tissue damage, such as the site of fluid collection and the degree of muscle damage, while non-invasively monitoring hemodynamic changes and assessing blood flow in affected limbs in real-time [4]. This visualization diagnostic technology significantly improves the objectivity and timeliness of clinical decision-making.

Snakebite-induced compartment syndrome is a rare yet potentially life-threatening complication. Without prompt surgical intervention, it can result in irreversible ischemic damage to nerves and muscles, potentially leading to amputation of the affected limb or even death [5]. Although measuring compartment pressure is the most objective method to diagnose compartment syndrome, there is a lack of consensus on its use, and it is not universally available [6]. In the absence of compartmental pressure measurement, most clinicians rely on empirical fasciotomy treatment, which is subject to a degree of subjectivity and delay. As a widely utilized clinical imaging modality, conventional ultrasound combined with shear wave elastography (SWE) for quantitative tissue stiffness assessment may provide a novel noninvasive solution for evaluating compartment pressure changes. Therefore, this study aimed to investigate the role of multimodal ultrasound, including two-dimensional (2D) ultrasound, color Doppler flow imaging (CDFI), and SWE, in assessing the severity of snakebite injuries.

Materials and methods

Study population

Patients hospitalized with snakebite in the Emergency Department of the Affiliated Hospital of Southwest Medical University between July and October 2023 were selected as the study subjects. All patients received antivenom therapy immediately upon admission and underwent ultrasonography within 12 h of hospitalization. Patients with stable vital signs who could be safely transported for ultrasound examination were included in the study. Exclusion criteria were: patients with severe illness or antivenom allergy who could not be transported; patients with a history of arterial stenosis or other vascular disease; patients with severe systemic comorbidities (e.g., advanced hepatic/renal dysfunction, malignancy); and patients with poor ultrasound image quality.

This study was approved by the Ethics Committee of the Affiliated Hospital of Southwest Medical University [2023(028)], and informed consent was obtained from all participants.

Data collection

Demographic information (including sex and age) and medical histories (snake species and bite site) were collected from physician-documented records in the hospital’s electronic medical system. The circumference of the limb was measured on both the affected and unaffected sides. Wound characteristics, such as bruises and blisters, were also documented.

Ultrasound examinations

Ultrasound examinations were conducted using the Aixplorer color Doppler system (SuperSonic Imagine, France) with an SL10-2 linear transducer (2–10 MHz) and XC6-1 convex transducer (1–6 MHz). Standard imaging parameters included a gain setting of 48–50%, focal zone adjustment to the subcutaneous fat layer level, and an initial depth setting of 3 cm, which was adaptively modified based on individual patient body type and subcutaneous tissue thickness. All examinations were conducted by two ultrasound clinicians with over 5 years of experience, who were blinded to the clinical examination results and circumference measurements. Patients were positioned supine, with upper or lower limbs fully exposed to avoid compression by clothing. Specific measurement locations were identified to facilitate ultrasound examination and minimize measurement errors.

For reliability assessment, 15 patients were randomly selected. Inter-observer agreement was evaluated by two physicians (A and B) experienced in SWE, while intra-observer agreement was assessed by Physician A through repeated examinations with a 24-hour interval.

This study initially investigated the influence of region of interest (ROI) size on SWE measurements. Twenty healthy volunteers of different age groups were recruited, and ROIs of varying sizes were applied to measure the elasticity of the subcutaneous fat layer in normal limbs. The aim was to determine whether differences in ROI size would affect the measurement results.After confirming that ROI size had no significant impact on the measurements, formal SWE examinations were conducted.

Using the SL10-2 ultrasound probe, for patients with upper-limb bites, the ultrasound probe was placed on the swollen middle portion of the forearm. For lower-limb bites, the probe was positioned approximately 0.5 cm inside the anterior tibial edge, about 5 cm below the knee joint for lower-limb bites. Next the SWE mode was activated and an ROI size was set with a 4 mm diameter in the subcutaneous fat layer with minimal fascial tissue. If the patient’s subcutaneous tissue was thinner than 4 mm, the ROI size was adjusted. The thickness and elasticity of the subcutaneous fat layer were measured on both the affected and unaffected limbs.

Velocity and doppler indices measurements

In upper-limb bites, the peak systolic velocity (PSV), pulsatility index (PI), and resistance index (RI) of the radial and ulnar arteries were measured with the SL10-2 ultrasound probe, about 1 cm above the wrist joint. In lower-limb bites, the probe was placed about 5 cm below the knee joint and about 0.5 cm inside the anterior tibial edge, and the XC6-1 transducer was used to measure the PSV, PI, and RI of the posterior tibial arteries on both sides. The anterior tibial and peroneal arteries were measured about 5 cm below the knee joint and about 0.5 cm from the outer fibular edge, again using the XC6-1 transducer to assess the PSV, PI, and RI. Additionally, the probe was positioned about 1 cm below the midpoint of the imaginary line connecting the medial and lateral malleoli of the ankle joint, and the SL10-2 transducer measured the PSV, PI, and RI of the dorsalis pedis arteries on both sides.

When the arterial waveform changes from a normal triphasic pattern—characterized by antegrade systolic flow, brief early diastolic flow reversal, and late diastolic forward flow—to a monophasic pattern with continuous antegrade flow during diastole, it may indicate decreased vascular compliance or external compression [7]. Therefore, in this study, changes in the spectral Doppler waveforms of limb arteries were observed and recorded to further assess the degree of local vascular compression and its potential clinical implications.

In addition, venous vessels of the upper or lower limbs were examined to rule out the possibility of venous thrombosis.

Image analysis

Two-dimensional images were processed using Adobe Photoshop (version 25.0.0, Adobe Inc.) by converting to grayscale mode (‘Image’, ‘Mode’, ‘Grayscale’) to discard color information. Measurements were performed by selecting a rectangular ROI in areas demonstrating thickest subcutaneous fat with homogeneous echotexture, while deliberately avoiding hypoechoic/anechoic regions. Grayscale values (0–255 scale) were quantified using the histogram analysis tool.

Statistical analyses

Statistical analysis was performed using SPSS 29.0 software. Normally distributed data were presented as mean ± standard deviation (X ± s), while non-normally distributed data were presented as median [first quartile (Q1), third quartile (Q3)]. Group comparisons for normally distributed data were made using an independent sample t-test, and a one-way analysis of variance was used for comparisons among multiple groups. For non-normally distributed data, the rank sum test was used for comparison between groups. Pearson’s or Spearman’s correlation analysis was used to evaluate the relationship between subcutaneous fat layer thickness, elasticity, grayscale median values, and limb circumferences. The strength of correlation was interpreted according to the following criteria: |r| ∈ [0.00, 0.30) indicates a weak or negligible correlation, |r| ∈ [0.30, 0.50) represents a low to moderate correlation, |r| ∈ [0.50, 0.70) suggests a moderate to high correlation, |r| ∈ [0.70, 0.90) denotes a high correlation, and |r| ∈ [0.90, 1.00] reflects a very high correlation [8]. Statistical significance was set at P < 0.05.

Results

Patient characteristics

A total of 70 patients who met the inclusion and exclusion criteria were enrolled in the study between July 2023 and October 2023. Among the enrolled patients, the number of males was slightly higher than that of females. The majority of injuries were located on the right limbs, with a higher incidence in the lower extremities. Unfortunately, the majority of patients could not precisely identify the snake species responsible for the injury (Table 1).

Table 1.

Demographic information

		Upper limbs	Lower limbs
Sex	Female	6(12, 50%)	25(58,43.10%)
	Male	6(12, 50%)	33(58,56.90%)
Age		65.33 ± 8.18	61(52,69.25%)
Bite site	Right	8(12,66.6%)	39(58,67.24%)
	Left	4(12,33.4%)	19(58,32.76%)
Snake species	Unknown	6(12, 50%)	42(58,72.41%)
	Viperidae	6(12, 50%)	11(58,18.97%)
	Black-striped Rat Snake	0	1(58,1.72%)
	Red Large-toothed Snake	0	3(58,5.17%)
	Cobra	0	1(58,1.72%)

Comparison between the affected and unaffected sides using 2D ultrasound, CDFI, and SWE

As shown in Table 2, the intraclass correlation coefficient demonstrated excellent agreement for both inter-observer (between two operators) and intra-observer (same operator on two occasions) measurements.

Table 2.

The intraclass correlation coefficients (ICC) for the elasticity values in the intra- and intergroup consistency tests

Inter-group agreement		Intra-group agreement
ICC	P	ICC	P
0.778	<0.001	0.717	<0.001

Regardless of the bite site, the affected limbs showed greater subcutaneous fat layer thickness, grayscale median values, elasticity, and limb circumferences compared to the unaffected limbs (Figs. 1 and 2), with statistically significant differences (P < 0.05). Among patients with lower-limb bites, the PSV of blood vessels in the affected limb was higher than in the unaffected limb, while the PI was lower (Fig. 3). These differences were statistically significant (P < 0.05). However, no statistically significant difference in RI was observed, and no statistically significant differences were found in the hemodynamic parameters of upper limb vessels (Table 3).

Fig. 1.

The subcutaneous fat layer thickness, grayscale median values, elasticity and limbs circumference were compared between the affected and unaffected sides of the lower limbs.(Note: * indicates P < 0.05,** indicates P < 0.01,*** indicates P < 0.001)

Fig. 2.

The subcutaneous fat layer thickness, grayscale median values, elasticity and limbs circumference were compared between the affected and unaffected sides of the upper limbs.(Note: * indicates P < 0.05,** indicates P < 0.01,*** indicates P < 0.001)

Fig. 3.

PSV and PI were compared between the affected and unaffected sides of lower limbs vessels.(Note: * indicates P < 0.05,** indicates P < 0.01,*** indicates P < 0.001)

Table 3-2.

Comparison of related parameters between the affected and unaffected limbs in patients with upper-limb bites

Index	Affected side	Unaffected side	95%CI lower limit	95%CI upper limit	P
Subcutaneous fat layer thickness/cm	0.73 ± 0.07	0.23 ± 0.02	0.35	0.64	<0.001
Elasticity(kPa)	24.69 ± 1.24	14.74 ± 0.36	6.20	13.40	<0.001
Grayscale median values	127.26(93.45, 143.81)	81.97 ± 4.79	14.44	58.72	0.002
Radial arteries PSV/(cm/s)	75.35 ± 5.87	72.60 ± 4.63	-13.42	17.92	0.717
Radial arteries RI	0.85(0.81, 0.87)	0.80(0.74, 0.86)	-0.02	0.10	0.266
Radial arteries PI	2.37(2.08, 2.81)	2.06(1.73, 3.05)	-0.37	0.76	0.347
Ulnar arteries PSV/(cm/s)	83.19 ± 6.27	77.89 ± 5.04	-10.63	24.94	0.518
Ulnar arteries RI	0.82(0.75, 0.87)	0.82(0.75, 0.90)	-0.07	0.08	1.000
Ulnar arteries PI	2.35(1.71, 3.17)	2.27 ± 0.20	-0.54	1.07	0.630
Limb circumference/cm	23.50(22.62, 26.00)	22.59 ± 0.71	0.00	4.00	0.089

Table 3-1.

Comparison of related parameters between the affected and unaffected limbs in patients with lower-limb bites

Index	Affected side	Unaffected side	95%CI lower limit	95%CI upper limit	P
Subcutaneous fat layer thickness/cm	0.92 ± 0.05	0.27(0.63, 1.14)	0.47	0.70	<0.01
Elasticity(kPa)	24.45(20.77, 25.85)	14.14 ± 0.19	8.80	10.80	<0.01
Grayscale median values	125.14(106.83, 132.62)	71.87(60.47, 90.24)	35.67	55.02	<0.01
Dorsal pedis arteries PSV/(cm/s)	87.23 ± 3.70	73.87(60.10, 87.86)	0.99	19.80	0.025
Dorsal pedis arteries RI	0.91 ± 0.01	1.00(0.93, 1.00)	-0.10	-0.01	0.001
Dorsal pedis arteries PI	3.46(2.46, 5.26)	6.24(4.59, 9.18)	-3.62	-1.44	<0.01
Anterior tibial arteries PSV(cm/s)	84.51 ± 2.69	72.45 ± 2.44	4.85	20.14	0.001
Anterior tibial arteries RI	1.00(0.89, 1.00)	1.00(1.00, 1.06)	-0.05	0.00	0.036
Anterior tibial arteries PI	5.01(3.02, 6.45)	7.21(5.27, 10.08)	-3.42	-1.33	<0.01
Posterior tibial arteries PSV(cm/s)	87.83 ± 3.09	77.52 ± 3.15	2.51	18.67	0.021
Posterior tibial arteries RI	1.00(0.87, 1.00)	1.00(1.00, 1.09)	-0.10	0.00	0.001
Posterior tibial arteries PI	4.98(2.87, 7.62)	9.07(5.52, 11.92)	-3.24	-4.80	<0.01
Peroneal arteries PSV(cm/s)	74.10(64.55, 87.42)	63.82(52.30, 73.87)	10.34	3.49	0.002
Peroneal arteries RI	1.00(0.91, 1.00)	1.00(1.00, 1.00)	0.07	0.27	0.112
Peroneal arteries PI	4.60(3.06, 6.27)	7.37 ± 0.44	-2.26	-3.29	0.001
Limb circumference/cm	32.44 ± 0.4	30.20 ± 0.42	1.20	3.60	<0.01

Correlation analysis between 2D ultrasound and SWE

Correlation analysis revealed that on 2D ultrasound and SWE of the lower limbs, there was a positive correlation between the thickness of the subcutaneous fat layer and its grayscale median value (r = 0.268, P = 0.042), between the thickness and elasticity (r = 0.591, P < 0.001), between the grayscale median value and elasticity (r = 0.480, P = 0.000), and between subcutaneous fat thickness and the circumference of the affected limbs (r = 0.275, P = 0.037) (Fig. 4). In addition, the elasticity of the subcutaneous fat layer generally exceeded 30 kPa for the group of patients with blistering of the skin (Fig. 5).

Fig. 4.

Correlation analysis of the lower limbs between the thickness of the subcutaneous fat layer and its grayscale median value(weak correlation), between the thickness and elasticity(a moderate to high correlation), between the grayscale median value and elasticity(a low to moderate correlation), and between subcutaneous fat thickness and the circumference of the affected limbs(weak correlation)

Fig. 5.

Patients with skin blisters whose subcutaneous fat layer elasticity is greater than 30kPa

Changes in blood flow spectral analysis

Concurrently, varying degrees of edema were observed to have differential effects on the arterial blood flow spectrum. Patients without obvious edema still showed typical triphasic waveforms. As the edema progressed, the early diastolic negative waveforms disappeared. Patients with obvious edema showed diastolic retrograde arterial blood flow (Fig. 6).

Fig. 6.

Presents gross images and multimodal ultrasound findings corresponding to patients with varying degrees of edema. Figures (1 A-1D) show the spectral features of diastolic retrograde arterial blood flow in the lower limb vessels of patients with obvious edema; figure (2 A-2D) corresponds to cases with moderate edema, where the early diastolic negative waveforms disappeared; figure (3 A-3D), a patient without obvious edema is shown. Visual observation shows almost no significant difference in the bilateral limbs. However, the 2D ultrasound image reveals the thickening of the subcutaneous fat layer in the patient, accompanied by echo enhancement and high elasticity, but the blood flow spectrum shows almost no significant changes

Analysis of the results from a three-day consecutive observation of the patients

Of the 70 patients, 39 were hospitalized for ≥ 3 days, including 33 with lower-limb bites and 6 with upper-limb bites. The thickness and elasticity of the subcutaneous fat layer in the lower limbs gradually decreased as treatment progressed, with statistically significant differences observed between days 2 and 3, as well as between days 1 and 3 (P < 0.05). Hemodynamic analysis revealed significant changes only in the PI of the anterior tibial and peroneal arteries within 3 days (P < 0.05) (Table 4).

Table 4-2.

Post hoc comparisons of subcutaneous fat thickness and elasticity in affected lower limb

	(I)name	(J)name	(I)mean value	(J)mean value	Difference(I-J)	95%CI lower limit	95%CI upper limit	P
Subcutaneous fat layer thickness(cm)	D1	D2	1.112	0.988	0.123			0.142
	D1	D3	1.112	0.751	0.361	0.20	0.52	0.000**
	D2	D3	0.988	0.751	0.238	0.07	0.40	0.005**
Elasticity (kPa)	D1	D2	25.064	22.848	2.215	-0.17	4.60	0.046*
	D1	D3	25.064	20.055	5.009	2.85	7.16	0.000**
	D2	D3	22.848	20.055	2.794	0.79	4.79	0.012*

*p < 0.05 ** p < 0.01

Table 4-1.

Variance analysis of subcutaneous fat thickness and elasticity in affected lower limb

	Days (mean value ± standard deviation)			F	P
	D1(n = 33)	D2(n = 33)	D3 (n = 33)
Subcutaneous fat layer thickness(cm)	1.11 ± 0.34	0.99 ± 0.36	0.75 ± 0.31	9.722	0.000**
Elasticity(kPa)	25.06 ± 5.12	22.85 ± 4.57	20.05 ± 3.49	10.529	0.000**

*p < 0.05 ** p < 0.01

Related complications and prognosis

In this study, seven patients underwent local small-incision decompression (only skin incision with a wound length of less than 1 cm) to prevent the progressive increase in intracompartmental pressure. This decision was based on severe edema, and intense pain, and ultrasound suggested a significant increase in subcutaneous fat layer elasticity. Among these patients, five patients had subcutaneous fat layer elasticity values > 30 kPa, and two patients had diastolic retrograde arterial blood flow on Doppler ultrasound. Furthermore, these two patients’ diastolic retrograde arterial blood flow disappeared by the second postoperative day. Additionally, it is worth noting that in one patient, the diastolic retrograde blood flow appeared prior to the formation of skin blisters.

All patients were discharged without the occurrence of compartment syndrome or other complications. Follow-up was conducted for 70 patients, with the duration of edema resolution ranging from 1 week to 3 months.

No residual snake teeth or venous thrombosis was found in any patient.

Discussion

In 2017, the World Health Organization reclassified venomous snakebites as a Category A neglected tropical disease, suggesting increased global attention to this public health issue [9]. This study found that patients were mostly bitten in the evening, night, and early morning, likely related to the activity patterns of snakes and the reduced visibility of humans at these times. In terms of patient distribution, most of the 70 patients in our study were from rural areas(69/70). This may be due to frequent farming activities in rural areas, where the likelihood of encountering snakes is much higher than in other areas and where awareness of snakebite prevention is generally lower. Additionally, two-thirds of the bites occurred on the right side, consistent with the cultural preference for using the right hand or foot in China.

Venomous snakes secrete a variety of proteases that directly or indirectly affect blood vessel walls, damaging capillary endothelium cells and increasing vascular permeability, which leads to massive exudation of tissue fluid and limb edema [2, 10–15]. Through animal experiments, Sun and Mora et al. found that lymphatic dysfunction and its pathological changes may be an important factor leading to limb edema following snakebite [11, 14]. In another study, a 63-year-old patient with a lower-limb bite was evaluated by lymphoscintigraphy, which confirmed the diagnosis of lymphedema [16]. Limb edema not only causes significant pain but also increases the risk of pressure-related damage and local infections, impacting patient prognosis. Therefore, early detection and treatment of edema are crucial.

The pitting test is a common clinical method for detecting subcutaneous edema; however, significant variability can occur due to differences in the pressure applied by clinicians. Furthermore, this method is also only applicable to pitting edema. In contrast, ultrasound can clearly display subcutaneous tissue structure and fluid accumulation, and the most common findings on 2D ultrasound for patients with snakebites are subcutaneous tissue damage and edema [17]. In this study, edema of the subcutaneous fat layer was observed in all patients with snakebites. On 2D ultrasound, the subcutaneous fat layer was thickened, with enhanced echogenicity and fissure-like hypoechoic areas. The grayscale median value of the affected area was significantly higher than that of the unaffected area. In addition, a clear positive correlation was observed between subcutaneous fat layer thickness and grayscale median value. A plausible explanation is that edema causes expansion of the interlobular fat septa, leading to the appearance of visible hypoechoic clefts. In addition, the accumulation of interstitial fluid enhances backscattering, thereby increasing the overall echogenicity. Together, these factors result in a mixed echotexture composed of both hypoechoic and hyperechoic regions, which in turn elevates the mean gray scale value on ultrasound imaging [18]. Ikuta et al. used Adobe Photoshop software to analyze leg edema and found a significant positive correlation between the grading of the compression pitting test and the average pixel grayscale of images (including epidermis, dermis, and subcutaneous tissue layers), which quantitatively evaluated the level of leg edema [19]. These findings suggest that quantitative assessment of limb edema using Adobe Photoshop is a feasible approach. This method not only enhances the objectivity of ultrasound image analysis but also provides a novel technical pathway for the quantitative evaluation of subcutaneous edema following snakebite. Given its practicality, it holds promise for application in clinical practice to support diagnosis and monitor disease progression. Notably, although some patients reported good subjective feelings and did not show a significant increase in the circumference of the affected limb compared to the unaffected side, the 2D ultrasound imaging still accurately captured subtle changes in the echo enhancement of the subcutaneous fat layer, accompanied by a slight increase in thickness. These findings align with the findings of Cheng and Amr et al., which highlighted ultrasound’s ability to detect subcutaneous edema in areas that show no visible signs of edema or tenderness during physical examination [20–23]. Zhang et al. similarly reported a high degree of consistency between ultrasound and the pitting test in detecting subcutaneous edema, emphasizing that ultrasound offers a higher detection rate compared to the pitting test [24]. This suggests that even in the absence of obvious symptoms, there may be potential subcutaneous edema and inflammatory reactions, highlighting the importance of ultrasound examination in the early diagnosis of venomous snakebites.

CDFI evaluation showed that in patients with lower-limb bites, PSV on the affected blood vessels was higher than that on the unaffected side, while the PI was lower. However, there was no statistical difference in RI. Sun and Zheng et al. also observed that in the early stages of snakebite lesions (within 3 days) and in mild cases, the affected limb segment showed a significant increase in blood flow [4, 25]. This observation may be attributed to the pro-inflammatory effects of various protease components in snake venom, such as snake venom metalloproteinases, hyaluronidase, and phospholipase A2, which trigger the body’s inflammatory response, thereby promoting vasodilation and increased blood flow. These changes lead to an increase in PSV and a decrease in PI on spectral Doppler ultrasound [12, 15]. RI, which reflects both vascular resistance and compliance [26], may not demonstrate significant changes during the acute phase of snakebite-induced limb edema, as vascular compliance is unlikely to undergo substantial alterations in this early stage. However, there were no significant hemodynamic changes in upper limb blood vessels, which may be related to the small sample size in this group. Therefore, the use of hemodynamics alone cannot comprehensively and stably evaluate changes in patient conditions, and further research with a larger sample size is needed. Moreover, although no cases of thrombosis were identified in this study, previous reports have documented various thrombotic events following snakebites [27, 28]. It is possible that the absence of thrombosis in our findings may be attributed to the relatively limited sample size. Therefore, the use of color Doppler ultrasound remains clinically valuable for excluding potential thrombotic complications.

Hsu, et al. reported that when venom enters the muscle layer, fluid is released into the muscle compartment. This can lead to increased pressure in the fascial compartment and potentially trigger fascial compartment syndrome [29, 30], which can significantly affect patient prognosis. However, several studies have suggested that venom rarely penetrates the muscle layer directly. Instead, symptoms such as severe pain and edema that indicate compartment syndrome often result from subcutaneous tissue involvement rather than muscular involvement [23, 31, 32]. This is consistent with our findings where nearly all patients had thickened subcutaneous fat layers without muscle involvement. Misdiagnosing these cases as fascial compartment syndrome and performing unnecessary fasciotomies can delay wound healing and increase hospitalization time and treatment costs. The onset of compartment syndrome following a snakebite is variable and requires close monitoring of the patient’s condition for timely intervention [29]. In our study, when the patient’s limbs were severely swollen with numerous blisters, diastolic retrograde arterial blood flow was observed on spectral Doppler ultrasound. This phenomenon aligns with findings by Hou, who described diastolic retrograde arterial blood flow and multiple blisters in a patient bitten by a venomous snake on the left upper limb, indicating a higher risk of developing compartment syndrome [6]. Notably, among the seven patients who underwent localized small-incision decompression, one patient exhibited spectral changes prior to the formation of blisters. Meanwhile, Santiago observed complete diastolic retrograde arterial blood flow in two patients with compartment syndrome [33]. Therefore, the diastolic retrograde arterial blood flow is a potential early indicator, but larger prospective studies are required to confirm its predictive value. A study by Lu et al. on patients with snakebite in Taiwan identified an absence of blood flow signals on CDFI as a predictive indicator for surgical intervention [22]. However, this was not observed in the current study, possibly due to different types of venomous snakes and differences in the timing of clinical presentations.

SWE is a method that excites tissue to generate shear waves and measures the shear wave velocity (SWV) to assess tissue stiffness values quantitatively. It has been widely used in diagnosing various diseases, such as those of the breast, thyroid, and liver [34]. Preliminary data showed that the ROI size had minimal effects on SWV measurements (Table 5), consistent with the findings of other studies. Additionally, studies have found that when lesions were shallow (15–25 mm depth), SWV did not decrease significantly, indicating that the influence of shallow depth on SWE is not significant [35]. Given that the thickest subcutaneous fat layer measured in this study was 17.1 mm, the effect of depth on SWE was ignored.

Table 5.

Comparison of elasticity values for different ROI sizes in the lower extremities

ROI sizes	Elasticity Values	95%CI lower limit	95%CI upper limit	P
4 mm	15.47 ± 0.72	15.13	15.80	0.972
3 mm	15.34 ± 0.54	15.08	15.59
2 mm	15.25 ± 0.73	14.90	15.59
1 mm	15.17 ± 0.65	14.88	15.48

This study found that the degree of limb edema correlated with an increase in elasticity values. Among the 70 patients, seven had obvious blisters on the skin, and five of them had SWE elasticity values above 30 kPa, with more intense pain. Therefore, SWE can supplement diagnostic information lacking in 2D ultrasound and CDFI. Also, the elasticity value of the subcutaneous fat layer can quantitatively evaluate limb edema to predict the occurrence of compartment syndrome. However, further research is necessary to substantiate this hypothesis.

Riziyu et al. investigated the use of SWE in the follow-up of breast cancer-related lymphedema and found that changes in skin SWV may be more sensitive than volume difference measurements [36]. These results indicate the effectiveness of SWE in the dynamic monitoring of limb edema. The current study also found that the elasticity value of the subcutaneous fat layer decreased significantly on day 3 compared to day 1, further suggesting that SWE may be a useful tool in evaluating therapeutic efficacy and disease prognosis.

Snake tooth residue is an occasional occurrence and was not found in this study. However, some scholars suggest that imaging techniques should be used to eliminate the possibility of snake tooth residue. William et al. conducted simulation experiments using a gelatin model and found that ultrasound has a high negative predictive value (96%) and sensitivity (85.2%) for excluding snake tooth residue [37], making it the preferred imaging modality for this purpose.

The study has the following limitations: first, it was conducted at a single center, and all cases were collected from the same region and medical institution, which may introduce a degree of selection bias. Secondly, the time from snakebite to hospital admission varied among patients, resulting in inconsistent timing of ultrasound examinations. Given that pathological changes in tissue follow a temporal progression, variations in the timing of imaging could lead to fluctuations in ultrasound parameters, thereby affecting data comparability. However, the majority of patients were admitted within 24 h post-injury, and the time interval was relatively narrow, which may have minimized the overall impact of this variability. Third, differences in pre-hospital first aid measures (e.g., pressure bandaging, incision and drainage) may have influenced the local pathological progression to varying degrees. As this study did not standardize or systematically document these interventions, such factors could not be controlled as covariates and may have introduced potential confounding bias. Fourth, variability among different ultrasound devices makes standardization of parameters challenging, which may affect the comparability of results. Additionally, in obese patients or those with severe skin injuries on the limbs, adequate contact between the ultrasound probe and the skin can be difficult to achieve, leading to reduced image quality. Given these limitations, future studies should aim to increase sample size and conduct multicenter research to validate the reliability and generalizability of the findings.

Conclusion

Ultrasound allows timely detection of subcutaneous edema and vascular changes in patients following snakebite. Subcutaneous fat thickness was positively correlated with limb circumference, grayscale, and elasticity values. Multimodal ultrasound offers objective assessment, with SWE showing potential as a quantitative tool for clinical decision-making.

Author contributions

All authors contributed to the study’s conception and design. Shipei Xu and Yao Liu made equal contributions and co-completed the data collection and analysis. Shipei Xu and Yao Liu completed the first draft of the manuscript. Liwen Zhu commented on previous versions of the manuscript. Yingchun Hu and Jiqing Xuan gave some meaningful suggestions and theoretical support.All authors have read and approved this article.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability

The data that support the fingings of this study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

All procedures performed in studies in involving human participants were in accordance with the ethical standards of the institutional and national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Consent for publication

This study was approved by the Ethics Committee of the Affiliated Hospital of Southwest Medical University [2023(028)], and informed consent and consent for publication was obtained from all participants.

Competing interests

The authors declare no competing interests.