Meningitis - Laboratory diagnosis, Culture, Microscopy, Biochemical Examination
Laboratory diagnosis of Meningitis
The laboratory diagnosis of meningitis begins with the collection of samples:
Specimen
CSF
Specimen processing
If the volume of CSF is greater than 1 ml, they should be centrifuged at 1500g for 15 mins. If CSF is less than 1ml, the specimen should be gram stained and plated directly to BA and CA plates.
After centrifugation, the supernatant (0.5ml) is removed to a sterile tube. The remaining fluid (0.5ml) is used to suspend the sediment.
Sediment is mixed (vortexed) with remaining fluid by forcefully aspirating the sediment up and down into a sterile pipette several times so that microorganisms are adequately dispersed and have not adhered to the tube.
A drop of sediment is Gram-stained (smear is never spread) to observe microorganisms, RBC, and inflammatory cells. The supernatant can be used to test for the presence of Ag, rapid diagnostic tests, etc.
Microscopy
It is done by gram staining a drop of sediment on a clean grease-free slide is done to observe the presence of bacteria, RBC, and inflammatory cells at 100x. Smears are not spread to avoid the difficulty of finding a small number of microorganisms.
Fluorochrome staining using acridine orange can be done at 400x.
Cell count in CSF
The lymphocytes are counted in CSF in order to classify meningitis into one of 3 clinical types:
Hazy, cloudy, turbid CSF indicates tumors and bacterial meningitis
Haemorrhagic CSF fluid may be indicative of anthrax meningitis
Pellicle formation in CSF might indicate excess protein
Biochemical examination of CSF
The glucose and protein concentration of CSF is examined
normal glucose concentration is 45-72 mg/dl
normal protein concentration is 15-40 mg/dl
CSF glucose concentration is less than 45 mg/dl is indicative of bacterial meningitis while CSF protein concentrations of more than 55 mg/dl are diagnostic of bacterial, fungal, and tuberculosis meningitis.
Direct Ag detection
Rapid Ag detection from CSF for diagnosis of meningitis has been accomplished by latex agglutination tests.
Molecular methods
PCR is one of the most important molecular methods used for the diagnosis of meningitis.
Culture
The culture media used for diagnosis of meningitis includes:
Chocolate Agar (CA)
5% sheep blood agar (BA)
MacConkey agar (MA)
New York City (NYC) agar
Lowenstein-Jensen media
Enrichment broth-Thayer Martin Agar
After vortexing sediment and remaining fluid, the specimen is inoculated into the appropriate medium. Plates should be incubated at 37°C in 5-10% CO2 for at least 72 hours while the broth should be incubated aerobically at 37°C for 5-10 days.
Anaerobic blood agar plate must be incubated if suspected microorganisms are anaerobic.