Early Diagnosis of Native and Device-associated Meningitis

Study aim Evaluation of the analytical and clinical performance (sensitivity, specificity, positive and negative predictive value, accuracy) of a novel, pathogen-specific biomarker D-lactate in the cerebrospinal fluid (CSF) for the diagnosis and treatment monitoring of native and device-associated meningitis. The performance of CSF D-lactate will be compared with currently used conventional CSF culture and biomarkers, using standard definition criteria for infection (native and device-associated meningitis).

Specific aims

• Aim 1. Determination of the performance of D-lactate test for the diagnosis of device-associated meningitis and native meningitis in serially collected CSF samples through lumbar/ventricular puncture (pre- or intra-operatively), external ventricular drainage (EVD), lumbar drainage (ELD) and shunt aspiration.
• Aim 2. Evaluate the optimal cut-off value of CSF D-lactate test for the diagnosis of device-associated and native meningitis using the existing definition criteria.
• Aim 3. Evaluation of performance of D-lactate test for the diagnosis of device-associated and native meningitis compared to standard tests (culture, gram stain, cerebrospinal fluid leukocyte count \& differential, CSF lactate, CSF glucose, CSF-to-blood glucose ratio, CSF total protein, multiplex PCR) and novel biomarkers (CSF procalcitonin, alpha-defensin - ELISA, interleukin-6).

Study population:

Included will be consecutive patients with suspected device-associated meningitis and native meningitis treated in our institution. CSF will be aspirated following the routine diagnostic procedure. During conduction of this study no additional invasive or non-invasive examinations will be performed. The results obtained in this clinical investigation will not influence the diagnostic or treatment decisions for the patients.

Study definitions Device-associated meningitis will be defined according to modified criteria for nosocomial infections from Centers for Disease Control and Prevention (CDC).

Device-associated meningitis or ventriculitis must meet at least 1 of the following criteria:

1. Relevant growth of a pathogen in the CSF, on the device or in the wound swab of the device insertion site.
2. At least one of the following signs or symptoms without another identifiable cause: fever >38°C, headache, neck stiffness, cranial nerve signs, irritability, decrease in Glasgow coma scale (GCS) AND at least one of the following laboratory findings: increased CSF leukocytes (>5 × 10^6/l), increased CSF total protein (>0.45 g/l) or decreased CSF/blood glucose ratio (<0.5); positive CSF gram stain AND the physician initiates an antimicrobial therapy.

The following classification system for native meningitis will be applied:

Meningitis is diagnosed, if at least 1 of the following criteria is present:

1. Patient has organisms cultured from cerebrospinal fluid (CSF).
2. Patient has at least 1 of the following signs or symptoms: fever (>38°C), headache*, stiff neck*, meningeal signs*, cranial nerve signs*, or irritability*
3. And at least 1 of the following:
4. increased white cells, elevated protein, and decreased glucose in CSF
5. organisms seen on Gram's stain of CSF
6. organisms cultured from blood
7. positive laboratory test of CSF, blood, or urine
8. diagnostic single antibody titer (IgM) or 4-fold increase in paired sera (IgG) for pathogen and if diagnosis is made antemortem, physician institutes appropriate antimicrobial therapy.

• With no other recognized cause

In patients with diagnosed native or device-associated meningitis, the onset of the infection will be defined by clinical, laboratory and microbiology characteristics.

Study conduction In device-associated meningitis, CSF will be aspirated before, during or after surgery related to the device, according to the standard practice. Lumbar/ventricular punctures (pre-, intra- and/or postoperatively) and external ventricular (EVD) and lumbar drainage (ELD) aspiration will be performed by neurologist or neurosurgeon according to standardized aseptic techniques.

In patients with native meningitis, CSF will be aspirated by lumbar puncture, according the standard practice.

Laboratory procedure

• Immediately after aspiration, 0.5-1 ml of cerebrospinal fluid will be transferred into each of the following vials:
• native vial for conventional culture (agar plate \& broth)
• EDTA vial for the determination of leukocyte count and differential
• pediatric blood culture bottle (BacTec PedsPlus/F)
• native vial for biomarker detection (e.g. D-Lactate, procalcitonin interleukin-6, alpha-defensin (ELISA) and additional tests (e.g. PCR)
• The D-lactate test will be performed using spectrophotometric enzyme assay according to the manufacturer's instructions (D-Lactate Colorimetric Assay. Sigma-Aldrich, St. Louis, Missouri, USA) as follows: the reaction mixture containing 10 µl of cerebrospinal fluid samples, 2 µl of substrate mix and 2 µl of enzyme mix and blank containing only the sample and substrate mix will be analyzed for each sample. A calibration curve with solutions of D-lactate (monolithium salt) in water will be processed in each batch. The mixture will be incubated at room temperature for 30 min and absorbance at 450 nm determined by Microplate Absorbance Reader. The optical density of each sample will be recalculated into D-lactate concentration in mmol.
• The test will be performed by two trained medical laboratory technical assistants, who are available during 12 hours on working days.
• All CSF aspirates and growing microorganisms (including those growing from intraoperative samples, such as peri-implant tissue or sonication fluid) will be stored in a biobank at -70°C. This fluid and culture collection allows subsequent additional analysis to assure the correct diagnosis.

Statistical analysis:

For device-associated meningitis it is assumed that 30-40% of the CSF samples are infected and the difference in sensitivity between D-lactate and conventional tests is 10% (90% for D-lactate vs. 80% for conventional tests). For native meningitis it is assumed that 10% of the CSF samples are infected and the difference in sensitivity between D-lactate and conventional tests is 20% (95% for D-lactate vs. 75% for conventional tests). Proof of non-inferiority between the two tests is provided by a one-sided two-group test of the percentage values. The non-inferiority is determined at a difference of 10% and can be shown for foreign material-associated meningitis in 458 patients and for native meningitis in 198 patients at a significance level of 5% and a power of 80%. The power calculation was performed with nQuery Advisor 7.0. Depending on the scale-type of data, the descriptive results are expressed as mean ± SD or number of subjects (percentage). Categorical variables will be compared using x2 or Fisher's exact tests, as appropriate. Continuous variables will be compared using the Wilcoxon rank-sum test. A p value <0.05 will be considered statistically significant. Test results will be evaluated with sensitivity, specificity, positive predictive value, negative predictive value, accuracy and statistical significance will be assessed with McNemar's Chi-squared test or a binomial test depending on sample size. All p-values from secondary analyses will be considered in a non-confirmatory exploratory manner. For statistical analyses the program packages SPSS (SPSS 10.0 for Windows, SPSS, USA) and the program package R (most recent version at time of analysis) will be used.

Investigators plan to include a total of 600 patients with device-associated meningitis and 220 patients with native meningitis. The high number of cases is indicated because investigators only expect a 30-40% infection rate for the foreign device-associated meningitis and a 10% infection rate for the native meningitis as well as a 10-15% drop-out rate (e.g. lack of leukocyte count or culture).