Methods

Study population and inclusion criteria

The study population will be all the children between age of >1 month to <18 years who present one of the following criteria.

Sudden onset of fever (> 38.5 °C rectal or 38.0 °C axillary) and one of the following signs:
• Neck stiffness and photophobia
• Bulging fontanels (in children < 2 years)
• Altered consciousness with no other alternative diagnosis
• Characteristic purpura that does not blanch on pressure
• Seizures in infant 1-6 months old
• A combination of at least three of the following signs/symptoms:
•Leg pain
•Arthralgia
•Abdominal pain
•Myalgia
•Lethargy
•Irritability
•Abdominal pain
•Toxic appearance
•Cold extremities
•Delay in capillary refill time
•Purpuric/haemorrhagic skin lesion

Clinical samples and collections:

The samples to be taken should be according to the clinical presentation of IBI and may include cerebrospinal fluid (CSF), joint fluid, blood and skin lesion biopsy. The choice of clinical sample is therefore an important issue and must follow the procedures recommended by the hospital. Samples should be transported to the laboratory at ambient temperature and as early as possible (preferably within 30 minutes). At arrival in the laboratory, for each body fluid, 200 μl should be transferred in a nuclease-free tube. For blood, 1ml of EDTA blood is required. For skin lesions, the biopsy should be transferred into a sterile tube without any additive/fixative. Tubes should be stored at 4°C for daily processing or at -20°C for longer periods. The final storage of samples will be at (-80°C) at the central laboratory.

The main part of the samples should be processed according to the standard procedures of the bacteriological laboratory, including macroscopic assessment, gram staining, protein and glucose measurement and culture. If culture is successful, isolates should be stored frozen at -80°C in appropriate media at the central laboratory for further molecular subtyping and sequencing.

The clinical and epidemiological information should be sent with the samples according to a standardized Case Report Form.

Biosafety

Laboratory staff are at risk for exposure to aerosolized bacteria and particularly Nm. It is therefore mandatory to ensure qualification and training of the staff and their protective vaccination status. Manipulations should be performed under biological safety cabinet using appropriate personal protective equipment. New laboratory staff should be trained and habilitated by expert staff using standardized protocols. Training should be documented in written.
Vaccination of laboratory staff should be up to date (against meningococci A, B, C, Y, and W and Hepatitis B). A protocol to deal with accidental exposure and accidental contamination of laboratory should be established.

Case definition

The project aims at improving biological confirmation of IBI and reporting the findings to the central laboratory of each country. The basis of detection is culture and/or PCR of one of the three agents (Nm, Sp and Hi) in a sterile site beyond the portal of entry of these three agents (underlying the invasiveness). Data will be linked to the reporting system of each country through an annual data reporting to the Department of Health.

PCR methods

Real-time PCR relies on the use of primers/probes where probes are conjugated to fluorophores and the detection of fluorescence, which increases during DNA amplification. Appropriate software is required to view and analyze the results. High specificity and sensitivity are achieved by the TaqMan method. Data are listed according to values where each sample crosses the cycle threshold (Ct) that is the fluorescence level higher than the negative controls and is within the start of the exponential phase for positive control. The target gene, the primers/probes and cut-off values are used according to the WHO recommendation for the diagnosis of meningitis.

Quality assurance

PCR facilities need to be separated from other activities in the laboratory. The PCR laboratory should be compartmentalized with ‘one way’ workflow and a gradient of atmospheric pressure. The DNA extraction module should be separated from the place where PCR reagents are prepared. Protocols and equipment should be validated.

The performance of the PCR and the staff should be regularly monitored using appropriate tools such as the comparison of the distribution (mean, variance, and SD) of Ct values of the positive controls. It is paramount that the laboratory participates in regular quality assays. An inter laboratory external quality assurance (EQA) will be organized by the WHO collaborating center at AUB for the 3 participating CPHL.