Diagnosis of Tetanus Immunization Status: Multicenter Assessment of a Rapid Biological Test

Theoretical model of the problem.The World Health Organization acknowledges the ELISA test as the criterion standard, with a threshold of 0.1 IU/ml, to diagnose tetanus immunization status. This threshold is 10 times higher than the serum antitoxin level considered to be protective (8). As ELISA is not readily available in emergency departments, immunization status in this context is generally assessed from the immunization history reported by the patient, which is known to be inaccurate (13). This results in both the over- and underprescription of vaccine and tetanus immunoglobulin (mainly of human origin). The risk of communicable disease due to the injection of human blood products is small but should nonetheless encourage a responsible attitude to prescription, with diagnostic tests used to determine vaccine status accurately in the emergency department itself. We aim to evaluate the potential value of being able to perform laboratory tests in the emergency room that provide information about a patient's immune status. The characteristics of a test that could be incorporated into this setting should be a short turnaround time and a high availability (rural settings, nights, or weekends), technical simplicity, and ease of interpretation. However, appropriate training of doctors and nurses would be required, with supervision by a laboratory biologist. Reliability depends on tests being carried out with the same rigor applied in the laboratory, particularly in regard to quality criteria for each step.

Study design and setting.We carried out a prospective multicenter study to evaluate the feasibility and diagnostic accuracy of the Tetanos Quick Stick performed in the emergency departments of 30 French hospitals.

Eighty-two of the 550 emergency departments contacted by the French Emergency Sentinel Network agreed to participate. We selected participating centers from these 82 centers based on the following criteria: availability of a participating laboratory at or near the center and written consent of the emergency department and the laboratory for participation in the study. We selected centers so that the ratio of university to general hospitals was 1 to 3 and all types of emergency departments were represented (e.g., urban and rural).

Selection of participants.All patients over 18 years old referred to the emergency room for open wounds within 24 h of injury were prospectively included in the study after giving written informed consent and receiving appropriate treatment. Pregnant women, patients with severe injuries requiring immediate care or surgery, and patients with acute psychiatric problems requiring a special hospitalization procedure were excluded.

We wanted to recruit at least 30 centers, each including 30 patients. These numbers were considered sufficient to ensure an appropriate trade-off between representativeness of patients (largest number of patients per site) and operator multiplicity (which increases with the number of sites). Emergency physicians and biologists attended a national meeting, and they also were trained on site in the use of the TQS.

Diagnostic tests: TQS and ELISA.The Tetanos Quick Stick test was performed with blood obtained by finger prick by an attending physician or nurse from the emergency department (blood/emergency-TQS), and another blood sample was simultaneously sent to the hospital laboratory. The staffs of the emergency departments were trained in the use of the TQS.

The TQS method uses a combination of a solid phase coated with tetanus toxoid and tetanus toxoid-dye conjugate (colloidal gold). The operator takes a whole-blood sample from the patient's fingertip by means of a small pipette provided in the test kit. This sample is placed in the test sample well, to which diluent is added. The diluent flows through the absorbent pad, carrying the toxoid-dye conjugate along the chromatographic strip. A complex with antitetanus antibodies forms if such antibodies are present in the blood sample. These complexes react with the immobilized toxoid to form a pink line in the “T” (test) window. In the absence of anti-tetanus antibodies, no line appears in the test window. The excess gold conjugate binds to a control reagent immobilized in the “C” (control) window, forming a pink line, indicating that the test has been carried out correctly. The test should be read 20 min after diluent is added to the blood sample in the device.

Within 6 hours of the blood/emergency-TQS, the participating center laboratory carried out a second TQS test on a total blood sample (blood/lab-TQS) and a serum sample (serum/lab-TQS). The reference laboratory (Laboratory of Bacteriology and Virology, Lariboisière Hospital, Paris, France) performed ELISA (quantitative detection of immunoglobulin G after binding on a microplate coated with tetanus toxoid and calibration with the World Health Organization standard [Genzyme-Virotech GmbH, Russelsheim, Germany; distributed by InGen, Rungis, France]), according to the manufacturer's recommendations, for all included patients. ELISA, blood/lab-TQS, and serum/lab-TQS tests were performed blind to the results of the blood/emergency-TQS.

Data collection and processing.After inclusion, a structured clinical history was taken with (i) the main sociodemographic characteristics, (ii) a description of the wound and its risk for tetanus contamination (particularly contact with soil), and (iii) vaccination status for tetanus (items on the questionnaire are detailed in Table 1). Physicians were asked to perform the TQS test after appropriate care had been given and any prescription made, to ensure that they did not take the result of the test into account when deciding on their tetanus prevention strategy. They were encouraged to define the strategy used according to the algorithm recommended by the French Ministry of Health: (i) vaccine and immunoglobulin, in cases of no vaccination or doubts about vaccination history or severe wounds with incomplete or out-of-date (>10-year-old) vaccination; (ii) vaccine alone for minor wounds with incomplete or out-of-date (>10-year-old) vaccination or severe wounds with vaccination 5 to 10 years before; and (iii) neither vaccine nor immunoglobulin in cases of more recent vaccination (less than 5 years before for severe wounds and less than 10 years before for minor wounds) (10). The prescriptions made by each physician were recorded on the standardized form used to record the clinical history.

The feasibility of the TQS test was documented on two forms. The first concerned the procedure and result of the test (required for addition of the sample to the device, use of the pipette provided, time until reading, presence of a control line, and interpretation time) as performed at the emergency department and the laboratory. The form was filled out by the operator who performed the test for each included patient. The operator reported the TQS as positive if both the “C” and “T” lines were clearly apparent, as negative if the “C” line appeared but not the “T” line, and as inconclusive if the “C” line was not apparent or if the operator was unsure whether there was a line in either the “T” or the “C” window.

The second form (feasibility form) was filled in by each operator of the emergency department at the end of the study. Operators scored the feasibility of the test in an emergency context (with respect to time delays, simplicity, and interpretation) on a scale of 1 to 8 at the end of the study.

The results of the TQS tests were reported by the operators (a physician or nurse in the emergency department and a biologist or technician at the laboratory) on standardized forms, including details concerning the performance of the test.

Statistical analyses.The likelihood that the agreement between emergency department and laboratory TQS tests was not due to chance alone was assessed by means of the kappa test.

The accuracy of vaccination status diagnosis was assessed by determining the sensitivity, specificity, and positive likelihood ratio, with 95% confidence intervals (CI), for blood/emergency-TQS, blood/lab-TQS, and serum/lab-TQS and for status reported in the clinical history. We defined sensitivity as the proportion of subjects protected against tetanus according to the ELISA test with positive TQS results. We defined specificity as the proportion of unprotected subjects (on the basis of ELISA) with negative or inconclusive TQS tests. The positive likelihood ratio was calculated for any possible result i of the test (positive, inconclusive, or negative) as the proportion of patients with result i in protected patients divided by the proportion of result i in unprotected patients.

ELISA was considered to be the gold standard. We used a threshold of 0.1 IU/ml of serum to define positive results in ELISA and serum/lab-TQS, which implied the use of a threshold of 0.2 IU/ml of blood in the blood/emergency-TQS and blood/lab-TQS tests.

We used McNemar's tests (1 degree of freedom) to compare the sensitivities and specificities of blood/emergency-TQS and vaccine status reported in the clinical history.

Ethical aspects.The patient was required to give informed consent for inclusion. The therapeutic strategy was not modified by the result of the test, but the patient was provided with information and advice concerning his or her vaccination status based on the results of the various tests. The study was approved by the local ethics committee and was considered to correspond to research with direct individual benefit.

Two of the authors (G.C. and I.C.) were responsible for data input and statistical analysis.

Characteristics of study subjects.We recruited 37 emergency departments from 8 university hospitals and 29 general hospitals. We included 988 patients in June and July 2001: 27 centers included 30 subjects or more, and 4 centers included less than 15 patients. The main clinical characteristics of the patients and the prescriptions made are recorded in Table 1. All patients in this population underwent blood/emergency TQS tests, but ELISA was not performed for three of the patients (samples were not sent to the central laboratory). ELISA showed that 932 (94.6%) patients were protected against tetanus. A flowchart of the study is presented in Fig. 1.

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FIG. 1.

Flowchart of the study.

Main results.The blood/emergency-TQS test was inconclusive in 97 (9.8%) patients, whereas the blood/lab-TQS test was inconclusive in only 38 patients (3.8%). We compared the conditions under which the test was performed in the emergency department and the laboratory (Table 2). The main differences were the grade of the operator (physician versus nurse at the emergency department or biologist versus technician at the laboratory), use of the disposable pipette provided with the test, and time between performance and reading. The mean scores (and standard deviations) awarded by operators in the satisfaction survey (scale of 1 to 8) were 5.7 (±1.7) for ease of blood sampling from the fingertip, 4.9 (±1.8) for ease of use of the disposable pipette, 6.7 (±1.3) for the overall simplicity of the test, and 7.3 (±1.1) for the simplicity of the test in an emergency context.

Agreement between the results obtained was excellent for the blood/lab-TQS and serum/lab-TQS (0.912; 95% CI, 0.910 to 0.914) and moderate for blood/lab-TQS and blood/emergency-TQS (0.510; 95% CI, 0.508 to 0.511). Agreement did not depend on the qualification of the operator (0.513; 95% CI, 0.512 to 0.515 for physicians and 0.526; 95% CI, 0.519 to 0.533 for nurses) or the time at which the test was performed (0.497; 95% CI, 0.479 to 0.515 for nights and weekends and 0.513; 95% CI, 0.512 to 0.515, for weekdays between 8 a.m. and 6 p.m.).

Results and likelihood ratios for TQS tests performed in the emergency department and in the laboratory are presented in Table 3, together with the vaccine status reported in the patient's clinical history. If we compared positive results with pooled inconclusive and negative results, diagnostic accuracy was higher for the blood/emergency-TQS (69% [95% CI, 66 to 72] and 98% [95% CI, 90 to 99], respectively, for sensitivity and specificity) than for clinical history (62% [95% CI, 59 to 65] and 79% [95% CI, 67 to 88]). This difference was highly significant (P < 0.001). The sensitivity and specificity of the blood/lab-TQS test were 84% (95% CI, 82 to 86) and 99% (95% CI, 92 to 100), respectively. The sensitivity and specificity of the serum/lab-TQS test were 86% (95% CI, 83 to 88) and 99% (95% CI, 92 to 100), respectively. The median (interquartile range) ELISA antibody titers for ELISA-positive and blood/emergency-TQS-negative and ELISA-positive and blood/emergency-TQS-positive patients were 0.7 (1.3) and 3.0 (3.3), respectively.

Prescriptions were recorded for 969 patients, three of whom could not have the ELISA test (Fig. 1): 914 (95%) were protected according to ELISA, and 52 were not. Immunoglobulin was unnecessarily given to 120 (13.1%) protected patients. If the blood/emergency-TQS had been used for decision making, this prescription could have been avoided in 23 patients for whom positive results were obtained in this test. Eight unprotected patients (15%) received no treatment. If TQS had been used for decision making, this error would have been avoided in all these patients (all tested negative in blood/emergency-TQS).