Changes in serial laboratory test results in snakebite patients: when can we safely exclude envenoming?

This was a cohort study of patients with confirmed or suspected snakebite recruited to the Australian Snakebite Project (ASP). ASP recruitment and data collection processes have previously been described in detail.10-12 In brief, ASP is a national, prospective, multicentre study that recruits adults and children (aged > 2 years) with suspected or definite snakebite from over 100 hospitals. All patients have demographic and clinical information, laboratory test results and treatments recorded on a clinical research form, which is then entered into a purpose-built relational database. Ethics approval has been obtained from 19 human research ethics committees covering all institutions involved in the study.

Patients recruited to ASP from January 2002 to April 2009 were included in this study if at least three sets of laboratory test results were available and the patient was observed in hospital for at least 12 hours after the bite or was admitted to hospital with envenoming. We compared envenomed and non-envenomed patients, using cases classified as severe envenoming, minor envenoming or non-envenomed.

Severe envenoming was defined as any of the following:

• Venom-induced consumption coagulopathy (VICC): evidence of a complete consumption coagulopathy, indicated by either an undetectable fibrinogen level or a raised D-dimer level (at least 10 times the assay cut-off or > 2.5 mg/L), with an international normalised ratio (INR) > 3.0.

• Myotoxicity: a creatine kinase (CK) level > 1000 U/L, with myalgia and/or muscle tenderness.

• Neurotoxicity: with either two nerve groups (eg, ocular and bulbar) involved, respiratory muscle paralysis, or requirement for intubation or mechanical ventilation.

• Thrombotic microangiopathy: defined as the presence of intravascular haemolysis on the blood film, thrombocytopenia and an abnormal creatinine level with or without acute renal failure.13

Patients with minor envenoming were those with evidence of envenoming but in whom specific treatment or antivenom were rarely required. This included patients with isolated systemic symptoms (at least three of nausea, vomiting, abdominal pain, diarrhoea, diaphoresis and headache), anticoagulant coagulopathy, mild or partial VICC (incomplete consumption coagulopathy characterised by low but detectable fibrinogen level, elevated D-dimer level and a maximum INR < 3.0), mild neurotoxicity or myotoxicity not meeting the criteria for severe envenoming.

Non-envenomed patients were those who did not manifest any of these clinical or laboratory features of minor or severe envenoming during their hospital stay of at least 12 hours and did not re-present to the hospital with delayed envenoming.

Information was extracted from the ASP database on: patient demographics; clinical features of envenoming (including time of onset after bite and type of neurological effects); and laboratory test results, including a full blood count, biochemistry, coagulation studies (INR, activated partial thromboplastin time [aPTT], D-dimer and fibrinogen concentrations), and CK level.

Based on biological plausibility and visual inspection of early serial laboratory test results comparing patients with severe or minor envenoming to non-envenomed patients, only early changes in coagulation test results, CK level, white cell count (WCC) and lymphocyte count were further analysed for diagnostic utility. The cut-off values used to define abnormal results were: INR > 1.2, aPTT outside the reference range (depending on the individual laboratory), CK > 250 U/L, and WCC > 11.0 × 109/L. Based on previous concerns about cases of isolated neurotoxicity occurring in death adder envenoming,9 we included a neurological exam-ination in cases of neurotoxicity as part of the analysis.

The primary analysis was of the time from the bite to development of abnormal laboratory test result(s) or neurological examination in patients with severe or minor envenoming, with the conservative assumption that test results were normal if the test was not done (last observation carried forward). For this analysis, we plotted the proportion of envenomed patients who still had normal laboratory test results or neurological examination by time of the test after the snakebite. We then used these plots to estimate the median time until an abnormal laboratory test result or neurological examination occurred.

In addition, plots were made and the time to development of each abnormal laboratory test result (INR, aPTT, CK and WCC) or neurological examination was estimated for individual envenoming syndromes (VICC, myotoxicity and neurotoxicity). For example, the time to development of an abnormal INR was estimated for patients who developed VICC.

The final analysis was done using three of the above tests — INR, aPTT and CK — plus repeated neurological examination within 6 and 12 hours, to determine if severe envenoming could be excluded by normal laboratory test results occurring at these times. A similar analysis was done to determine if minor envenoming could be excluded with the same combination of repeat tests and neurological examination.

For descriptive statistics, median and range, interquartile range (IQR) or 95% confidence intervals were used for data that were not normally distributed. Statistical and graphical analyses were performed using GraphPad Prism version 5.03 for Windows (GraphPad Software, San Diego, Calif, USA).

VICC occurred in 206 patients, including 174 who had VICC alone and 16 who had VICC with thrombotic microangiopathy. The remainder had VICC with either neurotoxicity (6) or myotoxicity (8), or both (2).

In 178 of the 206 patients (86%), the INR was abnormal (> 1.2) on the first set of tests using blood samples taken a median of 1 hour and 36 minutes after the bite (range, 20 min – 11 h 30 min) (Box 3). The remaining 28 patients had a normal INR recorded before VICC developed (including one patient who had a second normal INR at 3 hours and 45 minutes after the bite). For these 28 patients, the median time after the bite to the normal INR was 1 hour (range, 7 min – 3 h 45 min) and the median time after the bite to the first abnormal INR was 4 hours and 17 minutes (range, 1 h 55 min – 10 h 30 min). In seven patients where the time from the bite to the first abnormal INR was greater than 6 hours, this reflected a delay of 4 or more hours between the first (normal) INR and the second (abnormal) INR. For all patients with VICC, the median time to an abnormal INR was 1.8 hours (range, 20 min – 11 h 30 min) (Box 4, A).

Of the 28 patients whose first INR was normal, 22 had other parameters measured at the same time: 12 of these had an abnormal D-dimer (> assay cut-off) or low fibrinogen (< 1.5 g/L) level. Twenty-five of the 28 patients (89%) with an initial normal INR had PBI at the time, compared with 149 of the 178 patients (84%) whose first INR was abnormal despite PBI. The relative risk for an initial normal INR with PBI was 0.65 (95% CI, 0.21–2.00; P = 0.58).

Of 31 patients with partial VICC, 26 had an abnormal INR within 12 hours. Of the remaining five, two had an abnormal aPTT within 12 hours; one had myotoxicity with CK > 250 U/L within 12 hours; and two never had an abnormal INR, and identification of their partial VICC was based on a low fibrinogen or high D-dimer level.

There were 20 patients with thrombotic microangiopathy, and all had preceding evidence of VICC, with an abnormal INR within 12 hours. Seventeen of these patients had VICC and three had partial VICC.

Myotoxicity occurred in 33 patients with severe envenoming who were bitten by mulga snakes (Pseudechis australis) (8), red-bellied black snakes (Pseudechis porphyriacus) (4), Collett’s snake (Pseudechis colletti) (3), snakes of the tiger snake group (Notechis spp, Tropidechis carinatus) (14), taipans (Oxyuranus scutellatus) (2), a death adder (Acanthophis praelongus) (1) and a sea snake (1).

The median maximum CK level was 5390 U/L (range, 1054–203 110 U/L) in patients with myotoxicity. An abnormal CK level had occurred in 10 of the 33 patients (30%) at 6 hours and in 24 patients (73%) at 12 hours. An abnormal aPTT had occurred in 20 patients (61%) at 6 hours and in 25 patients (76%) at 12 hours.

Two patients with myotoxicity did not have an abnormal CK level or aPTT within 12 hours (Box 3). One of these patients had a normal CK level but an elevated WCC at 2 hours and 15 minutes, and the next sample was not taken until 33 hours and 30 minutes after the bite, which then had a CK level of 4770 U/L. The second patient had a CK level of 220 U/L at 4 hours and no further testing until 20 hours after the bite, with a CK level in that sample of 691 U/L. This patient developed myotoxicity with localised myalgia and a CK level peaking at 12 028 U/L at 46 hours after the bite, with ptosis and diplopia but no VICC.

For patients with myotoxicity, the median time to an abnormal CK level was 9.1 hours (range, 45 min – 33 h 30 min), and median time to an abnormal aPTT was 4.2 hours (range, 45 min – 9 h 30 min) (Box 4, A).

Of 120 non-envenomed patients who had their CK level measured in the first 6 hours, 18 (15%) had an abnormal CK level. Of these 18, the CK level decreased on the next blood test for 13, and on the second subsequent blood test for three. It was not re-tested for two patients. This compared with an increase in CK level on the next blood test in all cases of myotoxicity.

There were nine cases of isolated severe neurotoxicity, and all nine were from death adder bites. The median time of onset of neurotoxicity after the bite was 4 hours (range, 35 min – 12 h) (Box 4, A). Eight patients had ptosis, and the ninth had bulbar weakness. Three patients who developed respiratory muscle paralysis and required mechanical ventilation had evidence of neurotoxicity after 35 minutes, 3 hours, and 4 hours, respectively.

An abnormal WCC developed in 192 of the 240 patients with severe envenoming (80%), and the median time to an abnormal WCC in these patients was 5.7 hours (range, 30 min – 26 h 45 min) (Box 4, A). Leukocytosis occurred in similar numbers of mildly and severely envenomed patients, and only identified one case of severe envenoming not identified by the other three tests within 12 hours in this study (Box 3). Patients with brown snake (Pseudonaja spp) bites were less likely to have leukocytosis occur at any time, compared with patients with bites from the tiger snake group (Notechis spp and T. carinatus) or red-bellied black snakes (P. porphyriacus).

Using a combination of three laboratory tests (INR, aPTT and CK) and repeated neurological examination, 238 of the 240 patients with severe envenoming (99%) had an abnormal laboratory test result or evidence of neurotoxicity by 12 hours (Box 3 and Box 4, B). Both patients who did not have abnormal test results within 12 hours had myotoxicity and did not have laboratory tests performed at 6 or 12 hours, when it was likely their CK levels would have been elevated. There were 222 patients with severe envenoming who presented within 6 hours of the bite, and 213 of these (96%) had documented abnormal laboratory test results at 6 hours. Applying the same laboratory criteria to the minor envenoming cases, 64 of the 75 patients (85%) had abnormal results within 12 hours (Box 4, B). The median time to an abnormality (laboratory test result or neurological examination) was 1.75 hours (range, 20 min – 33 h 30 min) for severe envenoming and 2.1 hours (15 min – 30 h 45 min) for minor envenoming.