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PLOS Neglected Tropical Diseases logoLink to PLOS Neglected Tropical Diseases
. 2021 Aug 10;15(8):e0009657. doi: 10.1371/journal.pntd.0009657

The 20-minute whole blood clotting test (20WBCT) for snakebite coagulopathy—A systematic review and meta-analysis of diagnostic test accuracy

Thomas Lamb 1,2,*, Michael Abouyannis 3,4, Sâmella Silva de Oliveira 5,6, Rachana Shenoy K 7, Tulasi Geevar 7, Anand Zachariah 7, Sanjib Kumar Sharma 8, Navin Bhatt 9, Mavuto Mukaka 2,10, Eli Harriss 11, David G Lalloo 3, Elizabeth A Ashley 1,2,12, Wuelton Marcelo Monteiro 5,6, Frank Smithuis 1,2, Michael Eddleston 13,14
Editor: Jean-Philippe Chippaux15
PMCID: PMC8405032  PMID: 34375338

Abstract

Background

The 20-minute whole blood clotting test (20WBCT) has been used to detect coagulopathy following snakebite for almost 50 years. A systematic review and meta-analysis of the 20WBCT was conducted to evaluate the accuracy of the 20WBCT to detect coagulopathy, indicative of systemic envenoming.

Methods and findings

Databases were searched from inception up to 09/12/2020 to identify studies that compared the 20WBCT and INR/fibrinogen on five or more subjects. Data was extracted from full-text articles by two reviewers using a predetermined form. Authors of 29 studies that lacked sufficient details in the manuscript were contacted and included if data meeting the inclusion criteria were provided. Included studies were evaluated for bias using a tailored QUADAS-2 checklist. The study protocol was prospectively registered on PROSPERO database (CRD42020168953).

The searches identified 3,599 studies, 15 met the inclusion criteria and 12 were included in the meta-analysis. Data was reported from 6 countries and included a total of 2,270 patients. The aggregate weighted sensitivity of the 20WBCT at detecting INR >1.4 was 0.84 (CI 0.61 to 0.94), the specificity was 0.91 (0.76 to 0.97) and the SROC AUC was 0.94 (CI 0.91 to 0.96). The aggregate weighted sensitivity of the 20WBCT at detecting fibrinogen <100 mg/dL was 0.72 (CI 0.58 to 0.83), the specificity was 0.94 (CI 0.88 to 0.98) and the SROC AUC was 0.93 (0.91 to 0.95). Both analyses that used INR and fibrinogen as the reference test displayed considerable heterogeneity.

Conclusions

In the absence of laboratory clotting assays, the 20WBCT remains a highly specific and fairly sensitive bedside test at detecting coagulopathy following snakebite. However, clinicians should be aware of the importance of operator training, standardized equipment and the lower sensitivity of the 20WBCT at detecting mild coagulopathy and resolution of coagulopathy following antivenom.

Author summary

Snakebite is a neglected tropical disease and responsible for an estimated 130,000 deaths per year. Coagulopathy is the commonest clinical manifestation of systemic snakebite envenoming and is indicative of a need for antivenom. The 20WBCT is a simple, cheap and quick bedside clotting test devised to detect coagulopathy after snakebite. The test is used widely and is incorporated in multiple national and WHO guidelines. However, following a publication questioning its diagnostic accuracy, there is a lack of consensus regarding its diagnostic accuracy and utility. In this review, we conclude that the 20WBCT is capable of detecting coagulopathy following snakebite with high specificity and acceptable sensitivity. Diagnostic test accuracy of the 20WBCT is improved when assessing for the presence of severe coagulopathy. Inclusion of data in this analysis from a variety of countries, make the findings widely applicable. However, the heterogeneity in study findings indicates the need for local assessment of external validity. The results of this study support the WHO guidelines that recommend its continued use in the absence of conventional laboratory clotting assays. Urgent efforts are required to identify an optimal method of detecting systemic snakebite envenoming and the response to treatment through pharmacokinetic and pharmacodynamic studies of venom and antivenom.

Introduction

Snakebite is a neglected disease of the rural tropics with an estimated 1.8 to 2.7 million envenoming cases and 81,000 to 138,000 deaths per year [1]. Coagulopathy is a common clinical manifestation of systemic snakebite envenoming, arising from a huge geographical and taxonomic array of snake genera across the world. Examples include old world viper genera such as Echis, Daboia and Bitis, pit viper genera such as Calloselasma, Trimeresurus, and Bothrops, and Australasian elapid genera such as Pseudonaja, Notechis and Oxyuranus.

Clinical manifestations

Despite the common descriptive term of ‘haemotoxic’ envenoming, systemic envenoming can cause a wide spectrum of haematological effects. Bleeding diathesis can manifest as spontaneous bleeding from the bite site, ecchymosis and bleeding from mucous membranes [2,3]. Occasionally patients develop devastating gastro-intestinal, retroperitoneal or intracranial haemorrhage [3,4]. However, many patients with coagulopathy have no obvious clinical features of systemic envenoming at presentation [5,6]. Distinguishing these patients, who may benefit from antivenom, from those without systemic envenoming e.g., patients with a dry or non-venomous bite, is vital to ensure early and judicious use of antivenom. This is particularly pertinent given the importance of early administration of antivenom [7,8], high cost of antivenom [9], high frequency of serious adverse reactions with low quality antivenoms [10] and the global shortage of antivenom [9].

Clotting assays

In the absence of clinical manifestations of systemic envenoming, for certain species, clinicians rely upon the detection of clotting abnormalities to determine whether systemic envenoming has occurred and antivenom is warranted. Despite the wide spectrum of snakebite pathophysiology resulting in disorders of haemostasis (Box 1), the standardized laboratory assays of prothrombin time and fibrinogen have been found to be sensitive at detecting coagulopathy from a variety of different snake genera [5,11,12]. Unfortunately, as most snakebites occur in remote geographical locations, conventional clotting assays are not commonly available. As a result, a number of bedside tests are used to detect clotting abnormalities—these include the 20-minute whole blood clotting test (20WBCT) [4], 30-minute whole blood clotting test (30WBCT) [13], capillary blood clotting time [14], Lee-White clotting test [15] and Vellore manual activated clotting time (VeMac) [16]. The 20WBCT is the most widely used bedside clotting test in snakebite envenoming and is recommended by two WHO snakebite management guidelines [17,18]. Despite this, there has been no systematic validation of this method compared to conventional laboratory clotting assays such as INR or fibrinogen concentration or consensus on the utility of the 20WBCT in snakebite management.

Box 1. Haemostasis and snake venoms

Haemostasis is a dynamic process in which multiple clotting factors function to control blood clotting by regulating coagulation, fibrinolysis and vessel integrity. Venom-induced disturbances of the clotting cascade and fibrinolytic proteins, platelets and blood vessels may be associated with failure to form a normal clot or abnormal bleeding. Venom is a complex mixture of proteins and peptides that can possess both inter and intraspecies variation. Convergent and functional evolution has resulted in the formation of venoms from just a handful of protein families [19]. Haemotoxic snake venoms consist of pro-coagulant and anti-coagulant proteins and peptides with a variety of different targets [20]. Snake venom metallo-proteinases (SVMP) and snake venom serine proteinases (SVSP) affect the clotting cascade through activation of factor V, factor X and prothrombin [20,21]. Thrombin-like enzymes, typical of pit-vipers, can disrupt the conversion of fibrinogen to fibrin without interference with the clotting cascade [20]. Less commonly, some snake C-Type lectin-like proteins (snaclecs) and phospholipases A2 (PLA2) possess anti-coagulant effects such as preventing the conversion of factor IX to IXa and inhibition of extrinsic tenase complex [21]. Further disruption to haemostasis is achieved through SVMP mediated microvascular damage and impaired platelet activation and aggregation [22].

20-minute whole blood clotting test

The 20WBCT was first described in 1977 by Warrell and colleagues and involves placing ‘a few millilitres of freshly collected blood into a clean dry glass test tube that is then left un-disturbed for 20-minutes and then tipped to discover whether the blood has clotted’ [4]. Blood that fails to clot after 20-minutes is considered positive and indicative of coagulopathy and thus systemic envenoming, whilst blood that has clotted is considered negative and not indicative of coagulopathy. The 20WBCT was specifically designed with consideration of cost, speed, limited laboratory resources and reproducibility in locations with limited antivenom supply that needed to be reserved for patients at highest risk of complications. In the original paper, the test was described as an ‘all or nothing’ test that is an ‘easy and sensitive sign of systemic poisoning in which spontaneous haemorrhage poses the greatest threat to life’ [4]. Although subtle variations in methodology have been used, the most common method, as described by the WHO, has been implemented in national snakebite guidelines, such as Myanmar [23].

Over time, the use of the 20WBCT has evolved from its initial design to identify patients most at risk of severe bleeding [4]. Increasingly, guidelines have incorporated the 20WBCT as a test of detecting systemic haemotoxic envenoming [23]. In light of the 2019 WHO strategy for snakebite prevention and control that highlighted the importance of making safe and effective treatments available, accessible and affordable to all [24], and the importance of early administration of antivenom to prevent complications of envenoming [7,8], we systematically reviewed the evidence that compared the 20WBCT against lower thresholds of detecting coagulopathy. The primary objective of the review was to identify the sensitivity and specificity of the 20WBCT at detecting coagulopathy, defined by a laboratory clotting assay, that is indicative of systemic envenoming following snakebite.

Methods

The systematic review was conducted in accordance with Preferred Reporting Items for a Systematic Review and Meta-analysis of Diagnostic Test Accuracy Studies (PRISMA-DTA) statement [25]. The study protocol is registered on the PROSPERO database for systematic reviews (CRD42020168953).

The literature review was performed in collaboration with a medical librarian (EH). The following databases were searched from inception up to 09/12/2020: Ovid Medline; Ovid Embase; Ovid Global Health; Scopus; Web of Science Core Collection; and WHO Global Index Medicus. The search strategies applied the SIGN diagnostics search filter to text words and relevant index terms to retrieve studies about diagnostic tests which detect a coagulopathy following snakebite [26]. No limits or language restrictions were applied to the search results. The full search strategies are shown in the supplementary material (S1 Text).

Studies that reported five or more human subjects that had undergone 20WBCT and an INR or fibrinogen concentration clotting assay at the same time were included. Studies that did not collect the 20WBCT and INR or fibrinogen samples at the same time-point were excluded. In all studies, the 20WBCT was considered the index test, and the paired clotting assay, the reference test. Additional information was collected from each study including year of publication, country of origin, biting snake species, method of snake identification, time from bite to index test, comparator test threshold, number of paired clotting tests, source of funding and conflict of interest.

Titles and abstracts were screened by one reviewer (TL). Studies that were selected for full text review were independently reviewed in duplicate by two reviewers (TL and MA) using a pre-designed data collection table. Discrepancies in findings were reviewed by a separate reviewer (ME) who adjudicated using consensus to reach a decision. Studies most commonly used an INR threshold of >1.4 and a fibrinogen threshold of <100 mg/dL to define coagulopathy. For this reason, these thresholds were used in the meta-analysis to define coagulopathy. For studies that assessed the accuracy of 20WBCT using a different threshold of INR or fibrinogen, or where incomplete data were reported, study authors were contacted and asked to provide true positive (TP), false negative (FN), true negative (TN) and false positive (FP) data for the 20WBCT against an INR threshold of >1.4 and fibrinogen of <100 mg/dL. The same eligibility criteria were applied for data not presented in publication. Sub-analyses were conducted to identify the sensitivity and specificity of the 20WBCT at detecting severe coagulopathy (defined as INR >5.0 or fibrinogen <100 mg/dL) and the sensitivity and specificity of the 20WBCT at detecting resolution of coagulopathy after antivenom.

Each primary reviewer conducted an independent assessment of bias using a Quality Assessment of Diagnostic Accuracy Studies (QUADAS2) checklist [27]. For published studies, the QUADAS2 assessment was completed using the methodology described in the published manuscripts. If unspecified, the assessment was completed by contacting the primary author.

A random effects meta-analysis was used to calculate aggregate sensitivity, specificity and confidence intervals using Stata-IC version 14. Simple descriptive statistics were used to describe individual patient data and a Mann-Whitney U test was used to compare distributions of skewed continuous data between two groups using Graphpad Prism version 9 and R version 4.0.3.

Results

The search identified 3,599 studies, of which 1,580 were duplicates. Two further studies [28,29] were identified through screening references and two datasets were considered following recommendation from experts (Fig 1). One thousand, eight hundred and eighty-seven studies were excluded based on review of the title and abstract (Fig 1). Of the 132 studies included for full-text review, 11 met the inclusion criteria. Authors from a further 29 studies that reported methodology suggesting synchronous 20WBCT and clotting sample collection were contacted, and a further four studies (from three papers) were included [5,30,31]. Thus, 15 studies were included in the systematic review (Fig 1).

Fig 1. PRISMA-DTA flow chart.

Fig 1

The data from the 15 studies, with 2,270 patients, included in the systematic review are summarized in Table 1. These studies report data from Thailand (n = 4), India (3), Sri Lanka (3), Brazil (2), Nepal (2) and Papua New Guinea (1), on a variety of different snake genera: Daboia (n = 7), Trimeresurus (5), Hypnale (3), Echis (2), Bothrops (2), Calloselasma (1), Ovophis (1), Bungarus (1) and Oxyuranus (1). In several studies, snake genus and species was unknown.

Test of Bias–QUADAS2 assessment

The results of the QUADAS2 assessment of bias are summarized in Table 2. Study methodology lacked details concerning patient recruitment in 6/15 studies. Four studies did not specify whether patients with pre-existing coagulopathy were excluded. In the absence of a published standard operating procedure for conducting the 20WBCT, 9/15 studies either referenced or adopted the methodology by Warrell et al or WHO guidelines [4,18], three studies put 1 mL blood into a 5 mL glass tube, but otherwise followed the methodology specified by Warrell et al and three studies did not specify the 20WBCT methodology. All participants in each study received the same reference test. The methodology of the reference test was deemed to be suitable in the 13 studies in which it was described, and one paper reported laboratory accreditation [35]. One paper provided no methodological detail relating to the index and reference tests [28]. Three studies performed the reference test on frozen samples and in two studies it was unclear how the final reported statistics were derived (Table 2).

Table 2. QUADAS-2 Assessment of bias.

Studies designed with objective to assess the validity of the 20WBCT
First author, year of Publication Was a consecutive or random sample of patients enrolled? Was a case control design avoided? Did the study use appropriate exclusion criteria? Was the 20WBCT interpreted without knowledge of reference standard? Was the 20WBCT conducted using standardized technique? Was the reference standard, its conduct, or its interpretation performed in a manner to avoid bias? Did all patients receive the same reference standard? Was the reference standard performed on site and not require freezing? Were all patients accounted for in the analysis?
Thongtonyong, 2020 Unknown Yes Yes Yes Yes Yes Yes Yes Yes
Tongpoo, 2020 Yes Yes Yes Yes Yes Yes Yes Yes Yes
DSilva, 2019 Yes Yes Yes Yes Yes Yes Yes Yes Yes
Saengnoi, 2019 Unknown Yes Yes Yes Yes Yes Yes Unknown No
Silva, 2018 Yes Yes Yes Yes Yes Yes Yes No Yes
Ratnayake, 2017
Yes Yes Unknown Yes Yes Yes Yes Yes Yes
Paiva, 2015 Unknown Unknown Unknown Yes Yes Unknown Yes Yes Unknown
Biradar,
2015
Yes Yes Yes Unknown Unknown Unknown Yes Unknown Yes
Pongpit, 2012 Unknown Yes Yes Yes Yes Yes Yes No Yes
Sano-martins, 1994 Unknown Yes Unknown Yes Yes Yes Yes Yes Yes
Studies that were not specifically designed with the objective of assessing the validity of 20WBCT
Bhatt, 2020 Yes Yes Yes Yes Yes Yes Yes Yes Yes
Wijewickrama, 2020 Unknown Yes No Yes Unknown Yes Yes Yes Yes
Sharma, 2020 Yes Yes Yes No Yes Yes Yes Yes Yes
Shenoy, 2020 Yes Yes Yes Yes Yes Yes Yes Yes Yes
Oliveira, 2019 Yes Yes Yes Yes Yes Yes Yes No Yes

Diagnostic test accuracy

Three studies were not included in the meta-analysis due to use of different reference test thresholds for defining coagulopathy with further data not being available from the study authors [14,28,29]. These studies (n = 264) were performed in Sri Lanka, Papua New Guinea and India. The sensitivity of the 20WBCT at detecting coagulopathy in these three studies varied from 0.50 (Confidence interval (CI) 0.37 to 0.63) to 0.93 (CI not calculable), whilst the specificity of the 20WBCT at detecting coagulopathy varied between 0.89 (0.76 to 0.96) to 0.94 (0.85 to 0.98) (Table 1).

Table 1. Studies included in the systematic review.

First author, year Country Funding source/ Declaration Snake species Method of snake identification Median (IQR) time to hospital Reference test Number of 20WBCT True positive False negative True negative False positive Sensitivity (Confidence interval) Specificity (Confidence interval)
Thongtonyong [32], 2020 Thailand NR / No conflict of interest Calloselasma rhodostoma Dead snake and photographs 1.2 h (IQR NR fibrinogen <100 mg/dL 296 98 23 158 17 0·81 (0·73–0·88) 0·90 (0·85–0·94)
Wijewickrama [31], * 2020 Sri Lanka NMHRC/ No conflict of interest Hypnale spp and Daboia russelli Enzyme immunoassay (EIA) 1.0 h (0.5–2.5) INR > 1·4 157 5 11 130 11 0·31 (0·11–0·59) 0·92 (0·86–0·96)
Tongpoo [33], 2020 Thailand RHMU/ No conflicts of interest Trimeresurus spp Dead snake and syndrome 1.3 h (IQR NR) fibrinogen < 100 mg/dL 38 4 8 26 0 0·33 (0·10–0·65) 1·00 (0·87–1·00)
Bhatt, * 2020 Nepal NR/NR Ovophis monticola and Trimeresurus spp Dead snakes and photographs NR INR >1·4 97 20 1 55 21 0·95 (0·76–1·00) 0.72 (0·61–0·82)
Sharma, * 2020 Nepal NR/NR Daboia russelli and Trimeresurus spp Dead snakes and photographs NR INR >1·4 33 8 1 24 0 0·89 (0·52–1·00) 1·00 (0·86–1·00)
Shenoy [16], 2020 India CMC Vellore/ No conflicts of interest Daboia russelli and Echis carinatus Not specified NR INR >1·4 66 24 1 32 9 0·96 (0·80–1·00) 0·78 (0·62–0·89)
Dsilva [34], 2019 India NR/ No conflicts of interest Daboia russelli and Echis carinatus Not specified NR INR >1·4 60 8 3 35 14 0·73 (0·39–0·94) 0·71 (0·57–0·83)
Saengnoi [35], 2019 Thailand RHMU/ No conflicts of interest Trimeresurus spp and Daboia siamensis Dead snakes and photographs NR fibrinogen <100 mg/dL 48 0 1 43 4 0·00 (0·00–0·98) 0.91 (0·80–0·98)
Oliveira [5], * 2019 Brazil CAPES, CNPQ and FAPEAM/ No conflicts of interest Bothrops spp Dead snakes and EIA NR fibrinogen <100 mg/dL 100 54 26 20 0 0·68 (0·56–0·78) 1·00 (0·83–1·00)
Silva [14], 2018 Sri Lanka NR/NR Daboia russelli, Hypnale hypnale, and Bungarus caeruleus Dead snakes NR INR >1·5 92 14 12 62 4 0·54 (0·33–0·73) 0.94 (0·85–0·98)
Ratnayake [36], 2017 Sri Lanka NMHRC/ No conflicts of interest Daboia russelli and Hypnale spp Dead snakes and EIA NR INR >1·4 987 65 14 895 13 0·82 (0·72–0·90) 0·99 (0·98–0·99)
Biradar [28], 2015 India None/ No conflicts of interest Not specified Dead snakes, photographs or toxidrome NR INR >1·5 112 33 33 41 5 0·50 (0·37–0·63) 0·89 (0·76–0·96)
Paiva [29], 2015 Papua New Guinea NR/NR Oxyuranus scuttelatus Not specified NR fibrinogen <50 mg/dL 60 NR NR NR NR 0·93 0·91
Pongpit [12], 2012 Thailand TRF and CHEMET/ No conflicts of interest Trimeresus albolabris and T. macrops Dead snake or visualisation of green snake NR fibrinogen <100mg/dL 55 6 1 46 2 0·86 (0·42–1·00) 0·96 (0·86–0·99)
Sano-martins [37], 1994 Brazil STDEPC/NR Bothrops spp Dead snake and EIA NR fibrinogen <100mg/dL 69 37 7 23 3 0·84 (0·70–0·93) 0.88 (0·70–0·98)

Key: * Data included from study that did not assess the validity of 20WBCT as primary objective

† Conference abstract

‡ Published MD thesis in repository. NR = Not reported, NHMRC = Australian National Health and Medical Research Council, RHMU = Ramanthibodi Hospital Mahidol Univeristy, CMC = Christian Medical College, CAPES = Coordenção de Aperfeiçoamento de Pessoal de Vível Superior, CNPQ = Conselho Nacional de Desenvolvimento Científico e Tecnológico, FAPEAM = Fundação de Amparo à Pesquisa do Estado do Amozonas, TRF = Thailand Research Fund, CHEMET = Commission on Higher Education, Ministry of Education, Thailand and STDEPC = Science and Technology for Development Programme of the European Community.

The remaining 12 studies (n = 2006) were included in a meta-analysis. Six studies (n = 1,400) used INR > 1.4 as the reference test. The aggregate weighted sensitivity and specificity of the 20WBCT at detecting INR >1.4 was 0.84 (95% Confidence interval (CI) 0.61 to 0.94) and 0.91 (0.76 to 0.97) respectively, with a positive likelihood ratio of 9.1 (CI 3.3 to 25.7) and negative likelihood ratio 0.18 (0.07 to 0.47) (Fig 2). The summary receiver operating characteristic (SROC) curve has an area under the curve (AUC) of 0.93 (CI 0.91 to 0.95) (Fig 3).

Fig 2. Forrest plot of 20WBCT sensitivity and specificity at detecting coagulopathy, Forrest plot of 20WBCT sensitivity and specificity at detecting coagulopathy defined as INR>1·4.

Fig 2

Studies displayed individually and pooled. Circle size is proportional to sample size, whiskers represent 95% confidence interval.

Fig 3. SROC curve for 20WBCT at detecting coagulopathy.

Fig 3

SROC curves for 20WBCT at detecting mild coagulopathy defined as INR>1·4.

Six studies (n = 606) used fibrinogen concentration as the reference test. The aggregate weighted sensitivity and specificity of the 20WBCT at detecting fibrinogen <100 mg/dL was 0.72 (0.58 to 0.83) and 0.94 (0.88 to 0.98) respectively with a positive likelihood ratio of 12.9 (6.1 to 27.5) and negative likelihood ratio of 0.30 (0.19 to 0.46) (Fig 4). The SROC AUC is 0.93 (0.91 to 0.95) (Fig 5). There was considerable heterogeneity in data for studies that used INR as the reference test (I2 = 83.4 (CI 71.0 to 95.7) and 96.2 (CI 94.3 to 98.0) for sensitivity and specificity, respectively) and fibrinogen as the reference test (I2 = 76.5 (57.5 to 94.0) and 21.0 (0.0 to 86.5) for sensitivity and specificity, respectively).

Fig 4. Forrest plot of 20WBCT sensitivity and specificity at detecting coagulopathy defined as fibrinogen < 100mg/dL.

Fig 4

Studies displayed individually and pooled. Circle size is proportional to sample size, whiskers represent 95% confidence interval.

Fig 5. SROC curve for 20WBCT at detecting coagulopathy.

Fig 5

SROC curves for 20WBCT at detecting mild coagulopathy defined as fibrinogen <100 mg/dL.

A comparison of true positive and false negative 20WBCT

Individual patient data (n = 1,865, 82.2%), were either reported, displayed in figures or provided upon request in nine studies [5,16,3032,34,36,37]. In these studies, the median INR for patients with a false negative 20WBCT (n = 33) was 1.9 (IQR 1.6 to 12.0, skewness of 1.06 and kurtosis of -0.83). This was lower than for patients with a true positive 20WBCT (n = 130) (median 12.0 (12.0 to 12.0, skewness -0.34 and kurtosis -1.78) p value <0.001. The median fibrinogen (n = 56) for patients with false negative 20WBCT was 51 mg/dL (IQR 35 to 75, skewness 0.49 and kurtosis of -1.29) which was greater than for patients with a true positive 20WBCT (n = 189) (median 31 mg/dL (31 to 35, skewness 3.3, kurtosis 10.7) p value <0.001 (Figs 6 and 7).

Fig 6. A density plot displaying the distribution of false negative and true positive 20WBCT results using a reference test of INR.

Fig 6

Fig 7. A density plot displaying the distribution of false negative and true positive 20WBCT results using a reference test of fibrinogen.

Fig 7

Sub-analyses–Accuracy of the 20WBCT to detect severe coagulopathy

Using the individual patient data values described above (n = 1,865), [5,16,3032,34,36,37] a sub-analysis was performed to assess the ability of the 20WBCT to detect severe coagulopathy, defined as INR >5.0 or fibrinogen <50 mg/dL. The aggregate weighted sensitivity and specificity of the 20WBCT at detecting severe coagulopathy were 0.91 (0.64 to 0.98) and 0.90 (0.74 to 0.96), respectively. The 20WBCT SROC AUC for detecting severe coagulopathy is 0.96 (0.94 to 0.97) (Fig 8).

Fig 8. SROC curve for 20WBCT at detecting coagulopathy.

Fig 8

SROC curves for 20WBCT at detecting severe coagulopathy defined as INR>5 or fibrinogen <50 mg/dL.

20WBCT after antivenom, a test of coagulopathy resolution

Only four studies, with a total of 238 patients, provided paired data for the 20WBCT and reference test at a time interval of 6 hours [16,37] or 12 hours [5,34] after antivenom administration. The sensitivity of the 20WBCT at identifying persistent coagulopathy (defined as INR > 1.4 or fibrinogen < 100 mg/dL) at 6 or 12 h ranged from 0.05 (CI 0.01 to 0.17) to 0.67 (0.09 to 0.99) (Table 3). The specificity of the repeat 20WBCT at 6 or 12 h ranged from 0.80 (0.68 to 0.90) to 1.00 (0.89 to 1.00).

Table 3. Summary of studies performing 20WBCT after antivenom.

First author, year Reference clotting assay Time of repeat 20WBCT Number of 20WBCT True positive False negative True negative False positive Sensitivity (Confidence interval) Specificity (Confidence interval)
Shenoy, 2020 INR >1·4 6 h 37 5 7 21 4 0·42 (0·15–0·72) 0·84 (0·64–0·95)
DSilva, 2019 INR >1·4 12 h 59 2 1 45 11 0·67 (0·02–0·45) 0·80 (0·68–0·90)
Oliveira, 2019 fibrinogen <100 mg/dL 12 h 73 2 39 32 0 0·05 (0·01–0·17) 1·00 (0·89–1·00)
Sano-martins, 1994 fibrinogen <100 mg/dL 6 h 69 8 34 27 0 0·19 (0·09–0·34) 1·00 (0·87–1·00)

Discussion

For almost 50 years, the 20WBCT has been integral to the assessment and management of snakebite in locations across the world lacking access to more sophisticated laboratory facilities [2,3,17,18]. Contrary to its initial intended use, the studies reported here were all set up to determine whether the 20WBCT could detect any coagulopathy defined as INR >1.4 of fibrinogen < 100 mg/dL. In snakebite patients, the sensitivity of 0.84 and 0.72 (using INR and fibrinogen as reference test respectively), specificity of 0.91 and 0.94 (using INR and fibrinogen as reference test respectively) and SROC AUC of 0.94 and 0.93 (using INR and fibrinogen as reference test respectively) of the 20WBCT at detecting coagulopathy, support its continued use in the absence of any alternative, but highlight the need to improve snakebite diagnostics.

The sensitivity of the 20WBCT improved when used to detect severe coagulopathy which is associated with greater risk of complications [38]. This is an important consideration for the many countries that lack sufficient antivenom supply to treat systemically envenomed patients [9]. However, the 10% of patients with severe coagulopathy that were not identified by 20WBCT remain a concern. It is important to stress that the 20WBCT should be used in addition to clinical assessment. For example, some patients with a false negative 20WBCT following Daboia russelli envenoming may be identified as having systemic envenoming through careful clinical assessment for evidence of neurotoxicity [34,36].

The comparison of admission clotting assays for patients with correctly identified coagulopathy by 20WBCT (true positive) and coagulopathy missed by 20WBCT (false negative) (Figs 6 and 7) demonstrates that the 20WBCT is less effective at identifying less severe coagulopathy. This concern has been raised by authors in Sri Lanka that have reported that the mild coagulopathy, common to systemic envenoming by the Hump-nosed pit viper (Hypnale hypnale), is usually not detected by 20WBCT [31,39]. Figs 6 and 7 support this finding, demonstrating that the 20WBCT performs better at detecting severe coagulopathy, or so-called ‘complete consumption coagulopathy’ [20]. The clinical implications of missing systemic envenoming with mild coagulopathy are uncertain but it has been reported to precede severe coagulopathy in some circumstances [40] and other systemic manifestations [31,41].

Despite the lack of sophisticated equipment and comparative ease with which the 20WBCT can be performed, the importance of using a clean, dry, glass test tube cannot be overstated. There is no clinical evidence indicating the validity of plastic containers for 20WBCT. In an assessment of commercially available plastic containers in Australia, only polyethylene terephthalate plus a clot activator ‘Vacuette Z/serum separator’ was able to identify true negatives (the identification of negative (clotted) 20WBCT in normal controls) [42]. A further study of plastic syringes for 20WBCT in Benin confirmed their inability to detect true negatives [13]. In recent years, commercially available imported glass test tubes in Myanmar were found to be coated with silicon which detrimentally impacts upon clot activation producing false positive results (positive (unclotted) 20WBCT in normal controls) (Dr. Myat Thet Nwe, unpublished communication 2020). In a small pre-clinical study by Paiva et al, soda lime glass test tubes compared favourably at detecting true negative (normal clotting) in comparison to borosilicate glass test tubes and BD vacutainer glass tubes over a range of ambient air temperatures [29].

The need for a standardised technique, equipment and operator training was illustrated by a pair of studies from Sri Lanka [36,43]. The authors identified markedly improved sensitivity of the 20WBCT at detecting coagulopathy (from 40% to 82%) following the introduction of training, a standard operating procedure and implementation of single-use glass test tubes. Whilst the discrepancy in results underlines the importance of external validation, considerable differences may be expected if the index test (20WBCT) and reference test (INR) are collected non-synchronously, particularly given the dynamic process of haemostasis. The former study [43], which raised concerns that the 20WBCT lacks sensitivity [15,44], analysed unpaired 20WBCT and INR samples collected at different time-points and showed a lower sensitivity to the studies in this systematic review, that used paired samples.

Although repeat paired sampling of the 20WBCT and clotting assays at 6 and 12 h after antivenom was conducted in far fewer patients, the comparatively poor sensitivity of the 20WBCT at detecting snakebite coagulopathy at these time points is concerning. Given the widespread use and recommendation of the 20WBCT to assess response to antivenom, (18) it is surprising that few studies have sought to determine the diagnostic accuracy of the 20WBCT at detecting coagulopathy following antivenom. Work is required to understand the pharmacodynamic response to antivenom and how this may be used to assess antivenom effectiveness. A number of studies have looked at the time taken for clotting assays to normalize after antivenom in patients envenomed from snakes that predominantly cause a venom induced consumption coagulopathy (VICC). The median time in these studies ranged from 10–24 h for INR [11,45,46] and 6–24 h for fibrinogen [5,11,47]. In a study of 18 patients administered supranormal antivenom dosing for systemic Daboia siamensis envenoming, the median time to negative (clotted) 20WBCT was 4 h [3]. In contrast to the more commonly encountered VICC, anticoagulant coagulopathy following snakebite (such as coagulopathy arising from Pseudechis australis envenoming) may be expected to rapidly reverse following adequate dosing of antivenom [41].

Alternative strategies for detecting systemic snakebite envenoming and response to antivenom therapy are limited and face additional challenges. Snake venom detection kits (SVDK) seek to identify the responsible snake species rather than the presence of systemic envenoming. The only commercially available SVDK (Seqirus SVDK, Australia) uses a point-of-care, rapid, lyophilized, enzyme immunoassay on bite site swabs or urine samples. The sensitivity and specificity of the Seqirus SVDK at identifying the correct snake species following snakebite in Australia is 83.1% and 63.5% respectively [48]. The considerable cost associated with SVDK’s have contributed to their limited uptake in other regions. SVDK’s using immunochromatographic technology are currently in development for Daboia and Naja genera [49] and require comprehensive validation before commercial role out. Due to the need for sophisticated laboratory equipment, the detection of venom in serum samples both pre and post antivenom have not been used to guide clinical practice. In research studies, the detection of venom after antivenom therapy without recrudescence of clinical features has been described and is possibly explained by the misidentification of bound venom-antivenom complexes as free venom [5,44]. Further work is required to understand the optimal method of detecting systemic envenoming in the absence of clinical features and the response to treatment through pharmacokinetic and pharmacodynamic studies of venom and antivenom. New diagnostic tests are required to be as cheap, quick, widely available and as easy to perform as the 20WBCT whilst improving upon the diagnostic accuracy. This will ensure that antivenoms and potentially novel therapies for snakebite are used judiciously and delivered in a timely manner.

Limitations

Through efforts to include as much available data in the review as possible, the inclusion of five data sets that were not previously fully published renders the conclusions of this review more susceptible to bias. Each of these studies were conducted as part of other published snakebite studies, four of which have undergone peer review (two from one paper) [5,30,31] and the last is pending submission [16]. The effect of snake species and, therefore, mechanism and extent of VICC, varies greatly [20]. It is likely that the range of snakes responsible for envenoming in this study will have significantly contributed to the heterogeneity observed in both meta-analyses. It is likely that the 20WBCT diagnostic test accuracy in snakebite envenoming varies for bites from different snake genera. Time from bite to index test is another probable confounding factor when comparing diagnostic test accuracy across studies. Unfortunately, just three studies in this meta-analysis reported the median time from bite to index test, precluding a bivariate regression analysis to assess the impact of heterogeneity. Other factors not controlled for in this systematic review, such as type of glassware, ambient air temperature, operator training, and prior antivenom treatment are potential causes of error.

Conclusion

This review provides important insights into the interpretation of the 20WBCT and its limitations for mild systemic envenoming and coagulopathy. In the absence of available laboratory clotting assays, particularly in LMIC settings, the 20WBCT remains the best available method for detecting systemic haemotoxic envenoming and a good method for detecting severe coagulopathy following snakebite. However, urgent efforts must be made to identify a quick, cheap and more reliable method of detecting envenoming that causes lesser degrees of clotting disturbance. Further research is also needed to better define the relationship between changes in clotting abnormalities following antivenom and clinical outcome.

Supporting information

S1 Text. Systematic review search criteria.

A full list of search strategies and results broken down by database.

(DOCX)

Acknowledgments

The authors would like to thank all corresponding authors that provided data for inclusion in this review.

Data Availability

All individual patient data provided by authors of original studies has been deposited at Mendeley data repository (https://data.mendeley.com/datasets/3mdzkr5b4k/2).

Funding Statement

TL is supported by a Hamish Ogston Foundation Fellowship (https://www.hamishogstonfoundation.org/). MA is supported by a Wellcome Trust fellowship (grant number:203919/Z16/Z). This research was funded in whole, or in part, by the Wellcome Trust [grant number:220211]. For the purpose of Open Access, the author has applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission

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PLoS Negl Trop Dis. doi: 10.1371/journal.pntd.0009657.r001

Decision Letter 0

Jean-Philippe Chippaux

17 Jun 2021

Dear Dr Lamb,

Thank you very much for submitting your manuscript "The 20-minute whole blood clotting test (20WBCT) for snakebite coagulopathy - a systematic review and meta-analysis of diagnostic test accuracy." for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Jean-Philippe Chippaux, M.D., Ph.D.

Deputy Editor

PLOS Neglected Tropical Diseases

Jean-Philippe Chippaux

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: -Are the objectives of the study clearly articulated with a clear testable hypothesis stated? - yes

-Is the study design appropriate to address the stated objectives? yes

-Is the population clearly described and appropriate for the hypothesis being tested? yes

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested? yes

-Were correct statistical analysis used to support conclusions? yes

-Are there concerns about ethical or regulatory requirements being met? N/A

Reviewer #2: (No Response)

Reviewer #3: It is a meta-analysis to meet a clearly defined objective. the methodology is appropriate.

the statistical analysis is properly conducted to meet the objectives.

Compliance with ethical or regulatory requirements is correct.

Reviewer #4: The objectives of the study are clearly stated and study design is quite appropriate to address the stated objectives. Selection criteria of the studies are well described and SROC curves for sensitivity/specificity analysis strongly support the conclusions. There are no concerns about ethical or regulatory issues.

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Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: -Does the analysis presented match the analysis plan? yes

-Are the results clearly and completely presented? yes - but can be improved - see the detailed comments

-Are the figures (Tables, Images) of sufficient quality for clarity? yes - but can be improved - see the detailed comments

Reviewer #2: (No Response)

Reviewer #3: The analysis does presented correspond to the protocol. The results are presented in a clear and appropriate manner.

Figures and tables are of sufficient quality to be understood.

Reviewer #4: Systematic review was said to include 15 studies. However, there was only one study from Nepal [Ref. 30]. In fact, data presented in table is not in accordance with the study published. Navin Bhatt is the first and corresponding author, while Samir Kumar Sharma the last one. Please, review reference in which Sharma was the first author in 2020.

Line 252: paragraph is incomplete.

Correct typing for Thongtonyong, 2020 (ref. 32) in Table 1 and 2.

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Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: -Are the conclusions supported by the data presented? yes

-Are the limitations of analysis clearly described? yes

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study? yes

-Is public health relevance addressed? yes - but can be improved - see detailed comments

Reviewer #2: (No Response)

Reviewer #3: The limits of the study are well specified. The conclusions are adapted to the study conducted. The discussion allows a good understanding of the problem and raises important points for the use of the 20WBCT (glass tube, user training). This study confirms the role of the 20WBCT in the management of ophidian envenomations as well as its limitations.

Reviewer #4: Coagulation tests are important in indicating the snakebite patients who should receive antivenom, and in monitoring the recovery of the coagulopathy. Oddly, this review showed that 20WBCT has been performed after antivenom treatment in the minority of the studies, although most of the studies were specifically designed to assess the validity of the test. Please, discuss this apparent inconsistence.

Furthermore, the four studies in which clotting assays were performed hours after antivenom treatment revealed low sensitivity of the 20WBCT, suggesting the inefficiency of test to assess antivenom effectiveness. Would 20WBCT be more effective to detect a “all or nothing” coagulation disturbance, as originally described by Warrell, and less efficient to detect partial coagulation abnormality, as expected few hours after antivenom administration?

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Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: Line 173: “Although subtle variations in methodology have been used, the most common method, as described by the WHO, has been implemented in national snakebite guidelines” : is this a general statement or a statement specific to Myanmar?

Line 252: incomplete

Page 15: Dsilva – please correct the spelling of Daboia

Fig 4b: The unit of fibrinogen value must be indicated on the x-axis label

Line 368: g/dL or mg/dL?

Reviewer #2: (No Response)

Reviewer #3: no suggestion

Reviewer #4: (No Response)

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Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: The manuscript titled “The 20-minute whole blood clotting test (20WBCT) for snakebite coagulopathy – a systematic review and meta-analysis of diagnostic test accuracy.” by Lamb et. al. investigates the accuracy of the 20WBCT to detect coagulopathy (VICC) through a systematic review and meta-analysis. This study addresses a highly important topic related to snake envenoming, particularly relevant to the low-resource settings where an early diagnosis of snake envenoming is a challenge.

I general, the study has been well-designed, follows standard guidelines for methodology, the selection of the cut-off points of comparison tests are justified, analyses the results in adequate depth, presents the results clearly and the conclusions drawn out are supported by the results. Therefore, I have only minor comments.

Although the authors have compared the outcome of WBCT20 with the INR and fibrinogen measurements taken at the same time point, across different studies, the time from the snake bite to the test has not adequately appreciated in analysis. The utility of WBCT20 depends not only on the performance of the test in effectively detecting various severities of VICC. The diagnostic utility of WBCT20 may also depend on how early it detects VICC. This aspect is cannot-be adequately addressed by sub-group analysis of on-admission (admission times could variry greatly across settings) and post-antivenom. WBCT20 This may be very important in envenomings by certain snake species that often leads to the gradual transformation of mild VICC to severe VICC. For example, if a patient bitten by Russell’s viper presents to a hospital two hours after the bite and the WBCT20 becomes negative at an INR of 2.0 (so that the antivenom is not indicated) but six hours later WBCT20 becomes positive at an INR of >10, the diagnostic utility of the test is quite low. Therefore, I suggest the authors address the above issue and include a column to table 1 showing the median and IQR/ range of the time from the bite to test in individual studies, if they have presented the data. In addition, if it is possible, a subgroup analysis for <4hr and >4hr from the bite or even <6hr and >6hr would be quite important.

Reviewer #2: The authors systematically reviewed diagnostic accuracy of the 20-min whole blood clotting test (20WBCT) in snakebite-induced coagulopathy. The pooled specificity was high, but the sensitivity was variable among studies. Limitations and caveats of the tests were also discussed.

1. There are different mechanisms of coagulopathy among snake families. For example, pit vipers mainly consume fibrinogen, while true vipers also affect other clotting factors. I wonder if there are differences in 20WBCT performances for pit vipers vs. other snakes.

2. Line 171: ‘poisoning’ suggests ingestion of toxins. For snakebite, envenoming may be more appropriate.

Reviewer #3: (No Response)

Reviewer #4: This is a comprehensive review of the predicting value of 20WBCT demonstrating its accuracy to the prompt diagnose of venom-induced consumption coagulopathy and antivenom prescription.

As pointed out by the authors, some results do not allow to validate the bedside coagulation test because of the methodological limitations, but truthfully reflect the clinical and laboratory practices in most healthcare services where snakebite patients usually seek medical assistance.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

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PLoS Negl Trop Dis. doi: 10.1371/journal.pntd.0009657.r003

Decision Letter 1

Jean-Philippe Chippaux

16 Jul 2021

Dear Dr Lamb,

We are pleased to inform you that your manuscript 'The 20-minute whole blood clotting test (20WBCT) for snakebite coagulopathy - a systematic review and meta-analysis of diagnostic test accuracy.' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Jean-Philippe Chippaux, M.D., Ph.D.

Deputy Editor

PLOS Neglected Tropical Diseases

Jean-Philippe Chippaux

Deputy Editor

PLOS Neglected Tropical Diseases

***********************************************************

PLoS Negl Trop Dis. doi: 10.1371/journal.pntd.0009657.r004

Acceptance letter

Jean-Philippe Chippaux

5 Aug 2021

Dear Dr Lamb,

We are delighted to inform you that your manuscript, "The 20-minute whole blood clotting test (20WBCT) for snakebite coagulopathy - a systematic review and meta-analysis of diagnostic test accuracy.," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

We have now passed your article onto the PLOS Production Department who will complete the rest of the publication process. All authors will receive a confirmation email upon publication.

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Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Text. Systematic review search criteria.

    A full list of search strategies and results broken down by database.

    (DOCX)

    Attachment

    Submitted filename: 20WBCTSR_Response to reviewers.docx

    Data Availability Statement

    All individual patient data provided by authors of original studies has been deposited at Mendeley data repository (https://data.mendeley.com/datasets/3mdzkr5b4k/2).


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