Laboratory diagnosis of Meningitis-Key points

Meningitis is an inflammation of the membranes covering the brain and spinal cord known as the meninges. The most common etiologic agents of acute meningitis are enteroviruses (primarily echoviruses and coxsackieviruses) and bacteria (Streptococcus pneumoniae,  Neisseria meningitidis, Haemophilus influenzae) etc. Organisms expected to cause chronic meningitis (symptoms ≥4 weeks) include Mycobacterium tuberculosis, fungi, and spirochetes.

Viral infections usually get better without treatment but bacterial infection of meninges are very serious and is a major cause of deaths and disability world-wide. Patient age and other factors (i.e, immune status, post neurosurgery, trauma) are associated with specific bacterial pathogens.

Common causes of bacterial and fungal meningitis

Common causes of bacterial meningitis vary by age group:

CSF Collection

1. Whenever possible, collect CSF (1 mL minimum, 3-4 mL if possible) prior to initiating antimicrobial therapy.
   CSF is generally obtained by lumbar spinal puncture (at the L4-L5 level) and is collected usually in three separate tubes:
   Tube 1: For cell count and differential stains
   Tube 2: For Gram’s stain and culture (A minimum of 0.5–1 mL of CSF should be sent to the microbiology laboratory in a sterile container for bacterial testing. )
   Tube 3: For protein and glucose or for special studies such as VDRL, Cryptococcal antigen or cytology depending on the clinical situation.
   When the specimen volume is less than required for multiple test requests, prioritization of testing must be provided to the laboratory.
2. Two to four blood cultures should also be obtained if bacterial meningitis is suspected.
3. Inform the microbiology laboratory if unusual organisms are possible (such as Nocardia, Fungi, Mycobacteria, etc.), for which special procedures are necessary.
4. Do not refrigerate cerebrospinal fluid.
5. Attempt to collect as much sample as possible (approximately 5–10 ml of CSF should be collected) for multiple studies (minimum recommended is 1 ml); prioritize multiple test requests on small volume samples.

If only one tube of CSF is available, it should be sent to the microbiology laboratory. If CSF sample is collected in multiple tubes, the first tube collected (which could contain contaminating blood from the lumbar puncture) should not be the tube sent to the microbiology laboratory.

CSF volume & Production in Human

CSF should never be refrigerated prior to culturing because fastidious organisms may not survive lowered temperatures. If bacterial culture cannot be set up immediately, store the CSF in a 35°C, to maintain body temperature or leave at room temperature.

Processing of CSF Sample

N. meningitidis, S. pneumoniae, and H. influenzae are fastidious and fragile bacteria. For successful isolation of these bacteria, CSF  must be cultured within 1-hour of collection or inoculated into Trans-Isolate (T-I) medium for transport to the laboratory if processing within 1 hour is not feasible.

A delay in examining CSF:

• Reduces the chances of isolating pathogens
• Lower cell count due to WBCs being lysed
• Falsely low glucose value due to glycolysis

Tests:

Microscopy and Staining: CSF Gram stains should be prepared after cytocentrifugation and positive results reported immediately to clinicians. If Cryptococcal meningitis is suspected, India ink preparation should be done.

1. N. meningitidis may occur intracellularly or extracellularly in PMN leukocytes and will appear as gram-negative, coffee-bean shaped diplococci.
2. S. pneumoniae may occur intracellularly or extracellularly and will appear as gram-positive, lanceolate diplococci, sometimes occurring in short chains.
3. H. influenzae are small, pleomorphic gram-negative rods or coccobacilli with random arrangements

Culture and Sensitivity: Identification and susceptibility testing of bacteria recovered from cultures are routinely performed by growing the organisms in their specific culture media unless contamination during collection or processing is suspected. Following media are routinely used in the diagnostic microbiology laboratory for the isolation of common bacterial agents;

1. Chocolate Agar
   1. On chocolate agar plate, H. influenzae appear as large colorless to grey, opaque
      colonies with no discoloration of the surrounding medium.
2. Blood Agar

1. Overnight growth of N. meningitidis on blood agar plate appears as round, moist,
   glistening and convex colonies.
2. S. pneumoniae appear as small greyish mucoid (watery) colonies with a greenish zone of
   alpha-hemolysis surrounding them on the blood agar plate.

3. MacConkey Agar

Summary of identification scheme (Gram staining and culture)

Antigen-Antibody Reactions 

1. Antigen Testing: Cryptococcal antigen latex agglutination test is the preferred method in the suspected cases of Cryptococcal meningitis. Bacterial antigen testing on CSF is not recommended.
2. Serology: Serologic diagnosis is based on CSF to serum antibody index, 4- fold rise in acute to convalescent immunoglobulin G (IgG) titer, or a single positive immunoglobulin M (IgM). Submission of acute (3–10 days after onset of symptoms) and convalescent (2–3 weeks after acute) serum samples is recommended.

Molecular Diagnosis: Nucleic acid amplification tests (NAAT) are now available for most pathogens in developed countries but such facility may not be available in developing  countries or resource-poor settings. Molecular testing has replaced viral culture for the diagnosis of enteroviral meningitis.

Reference and further reading

1. CDC meningitis home page
2. Laboratory Methods for the Diagnosis of Meningitis, WHO Manual, 2nd Edition