Describe the laboratory diagnosis of meningitis with the cerebrospinal fluid findings in different types.

Collection of Cerebrospinal Fluid

• Lumbar puncture at L3-L4 or L4-L5 under strict asepsis; the opening pressure must be measured with a manometer, which is diagnostically valuable and is therapeutic in cryptococcal meningitis
• Contraindications — signs of raised intracranial pressure with focal deficits or papilloedema, a suspected space-occupying lesion, bleeding diathesis, and infection at the puncture site. Where imaging is needed first, blood cultures are taken and antibiotics started immediately — the lumbar puncture may wait, the treatment may not
• Three or four sterile tubes, numbered in order — conventionally tube 1 for biochemistry, tube 2 for microbiology, tube 3 for cell count (least contaminated by a traumatic tap) and tube 4 for special tests
• A paired blood glucose must be taken at the same time, ideally shortly before; the cerebrospinal fluid glucose is meaningless without it, since the normal value is a ratio of about two-thirds of the blood level
• Transport immediately and DO not refrigerate — Neisseria meningitidis and Haemophilus influenzae die rapidly at 4°C. The specimen goes at room temperature or 37°C and is processed within an hour. This is among the most frequently violated rules in clinical practice
• Volume — more is better where tuberculosis or fungal infection is suspected, since organisms are scanty; 5 to 10 mL for mycobacterial culture

• Do not delay antibiotics for a lumbar puncture, and do not put the sample in the fridge. Bacterial meningitis kills in hours, and the cerebrospinal fluid remains abnormal and often PCR-positive for hours after the first dose — so nothing diagnostic is lost by treating first. What is lost is the culture if the specimen sits at 4°C, because the two commonest bacterial pathogens are killed by chilling.
• Treat early
• transport warm.

Examination of Cerebrospinal Fluid

• A low cerebrospinal fluid glucose is the single most useful discriminator between bacterial, tuberculous and fungal meningitis on the one hand and viral meningitis on the other, and it can be available within minutes
• Early bacterial meningitis may show few cells, particularly in the neutropenic and the very young, and early tuberculous meningitis may show a polymorph predominance that later shifts to lymphocytes — so the pattern must be read alongside the clinical picture rather than mechanically
• A traumatic tap is distinguished from subarachnoid haemorrhage by clearing of the blood between successive tubes and the absence of xanthochromia after centrifugation; a correction of about 1 white cell per 500 to 1,000 red cells may be applied, but heavily blood-stained fluid is best interpreted cautiously

Microbiological Tests

• Gram stain of the centrifuged deposit — positive in 60 to 90% of untreated pyogenic meningitis, and it identifies the organism within minutes: gram-negative intracellular diplococci (meningococcus), gram-positive lanceolate diplococci (pneumococcus), gram-negative coccobacilli (H. Influenzae), and gram-positive bacilli (Listeria) in neonates, the elderly and the immunosuppressed
• Culture on blood and chocolate agar in 5 to 10% CO2, with enrichment broth; the reference standard, though it is often negative after antibiotics
• ZIEHL-neelsen stain on the deposit or on the cobweb clot for tuberculosis — insensitive, so a negative result means little; CBNAAT (Xpert MTB/RIF Ultra) is far more sensitive, gives a result in hours and simultaneously reports rifampicin resistance; and mycobacterial culture on liquid media
• INDIA ink for Cryptococcus, and the cryptococcal antigen lateral flow assay, which is more than 95% sensitive and is the test of choice
• Antigen detection by latex agglutination for meningococcus, pneumococcus, H. Influenzae type b and group B streptococcus — useful chiefly in partially treated meningitis where culture has been sterilised
• Molecular tests — multiplex meningitis and encephalitis PCR panels covering the common bacteria, viruses and Cryptococcus in a single run; HSV PCR is the test of choice in encephalitis and has replaced brain biopsy
• Where the fluid is likely to be sterilised by prior treatment, blood cultures, antigen tests and PCR retain their yield and should all be used

Likely Organisms BY Age and Host

Applied Aspects

• Give the first dose of antibiotic within an hour of suspicion, taking blood cultures first; every hour of delay in bacterial meningitis worsens the outcome measurably
• Add dexamethasone before or with the first dose in suspected pneumococcal meningitis, which reduces hearing loss and mortality; giving it afterwards is of little value
• Do not refrigerate the specimen, and take it to the laboratory yourself if necessary; a sample left in a fridge overnight will often grow nothing
• Take the paired blood glucose, which is forgotten remarkably often and renders the most useful single parameter uninterpretable
• Start empirical antitubercular treatment on clinical suspicion in India where the picture fits, since confirmation is slow and insensitive and delay causes permanent damage; but send CBNAAT and culture first
• Measure and manage the opening pressure in cryptococcal meningitis; repeated therapeutic lumbar puncture saves sight and life independently of the antifungal
• Cover Listeria with ampicillin in neonates, in adults over 50 and in the immunosuppressed; cephalosporins do not treat it, and this is a common gap in empirical regimens
• A normal cell count does not exclude meningitis in the neutropenic or the very young, who cannot mount a cellular response
• Send cryptococcal antigen and CBNAAT routinely on cerebrospinal fluid in an HIV-positive patient, since both cryptococcal and tuberculous meningitis are common and present subacutely
• Distinguish a traumatic tap from subarachnoid haemorrhage by comparing successive tubes and looking for xanthochromia after centrifugation
• Antigen tests and PCR retain their yield after antibiotics, which makes them particularly valuable in the partially treated patient who arrives having already had a dose elsewhere — a very common Indian presentation
• Send enough fluid for mycobacterial work — 5 to 10 mL — since bacilli are scanty in tuberculous meningitis and a small volume guarantees a negative result
• Look for the cobweb clot on standing, which is characteristic of tuberculous meningitis and is itself a good specimen for Ziehl-Neelsen staining
• Purpura with fever is meningococcal disease until proved otherwise, and parenteral antibiotics are given immediately, before transfer and before any investigation
• Imaging before lumbar puncture delays nothing if antibiotics are given first; the rule is to take blood cultures, treat, and then image and tap
• Multiplex panels give an answer in an hour but must be read alongside the cell count, protein and glucose, since detecting nucleic acid is not the same as establishing the cause
• An early tuberculous meningitis may show polymorphs and only later become lymphocytic, so the cell type at a single time point should not exclude it
• Repeat the lumbar puncture if the picture does not fit; the evolution over 24 to 48 hours is often more informative than the first sample
• Latex antigen tests earn their place in partially treated meningitis, where culture has been sterilised but antigen remains detectable