CSF Gram Stain and Culture

Examination of cerebrospinal fluid by Gram stain and bacterial culture is the cornerstone of diagnosing bacterial meningitis and ventriculitis.

A cytospin or centrifuged deposit of CSF is Gram stained to detect organisms and cells. The fluid is inoculated onto blood agar, chocolate agar (for Haemophilus and Neisseria in an atmosphere with added carbon dioxide) and into enrichment broth, and incubated for 48 to 72 hours (up to 5 to 7 days when fastidious organisms or shunt infection are suspected).

Positive cultures are identified and tested for antimicrobial susceptibility. Results are interpreted with CSF cell count, protein and glucose, blood cultures, molecular panels and clinical findings.

Bacterial meningitis occurs in 2.5 to 3 cases in every 100 000 adults each year in high income countries and 10 to over 100 in every 100 000 in the African meningitis belt during epidemics.

The commonest causes are:

• Streptococcus pneumoniae and Neisseria meningitidis in children and adults.
• Streptococcus agalactiae (group B streptococcus), Escherichia coli and Listeria monocytogenes in neonates.
• Listeria in older and immunocompromised adults.
• Haemophilus influenzae type b (now rare where vaccinated).
• Staphylococcus aureus, coagulase negative staphylococci and Gram negative bacilli after neurosurgery or with shunts.

Mortality is 10 to 30% and neurological sequelae affect up to 30% of survivors, and each hour of delay in antibiotics increases adverse outcome.

Indications. Fever, headache, neck stiffness, photophobia, altered consciousness, seizures or petechial rash (suspected meningitis). Neonates and infants with fever, irritability, poor feeding or bulging fontanelle. Fever after neurosurgery, external ventricular drain or shunt placement. Head injury with CSF leak and fever. Fever of unknown origin with neurological signs. Subacute or chronic meningitis (with additional tests for mycobacteria and fungi).

Specimen. CSF 1 to 2 mL in each of three sterile tubes (tube 1 for chemistry and serology, tube 2 for microbiology, tube 3 for cell count). Obtain by lumbar puncture before antibiotics when this does not delay treatment, and after neuroimaging if there are contraindications (focal signs, papilloedema, reduced consciousness, seizures, immunosuppression).

The sample is delivered to the laboratory immediately at room temperature (not refrigerated, as meningococci and Haemophilus die in the cold). For shunt and ventricular drain infection: ventricular or shunt fluid. Blood cultures are taken at the same time.

Reference values (adult CSF).

• Gram stain: no organisms, few or no cells.
• Culture: no growth.
• White cell count: 0 to 5 cells/microlitre (neonates up to 20 to 30).
• Protein: 0.15 to 0.45 g/L. Glucose: 2.5 to 4.4 mmol/L (60% or more of the paired plasma glucose).
• Opening pressure: 10 to 20 cmH2O in adults.

Interpretation.

• Gram stain positive: sensitivity is 60 to 90% in untreated bacterial meningitis (Streptococcus pneumoniae 90%, Neisseria meningitidis 70 to 80%, Haemophilus influenzae 70 to 85%, Listeria 30 to 35%), with specificity of nearly 100%.
• Gram positive diplococci suggest pneumococcus, Gram negative diplococci meningococcus, small pleomorphic Gram negative coccobacilli Haemophilus, Gram positive rods Listeria, and Gram negative bacilli E. coli or others.
• Culture positive: sensitivity 70 to 85% when taken before antibiotics, falling to 40 to 60% after antibiotic pre-treatment. The organism and its susceptibility define the final antibiotic.
• Gram stain negative with culture positive: occurs in low bacterial loads and after antibiotics.
• Cell count and chemistry help in culture negative cases: CSF neutrophils over 1000/microlitre, glucose under 1.9 mmol/L or ratio under 0.4, protein over 2.2 g/L, and lactate over 3.5 mmol/L favour bacterial meningitis (bacterial meningitis score and similar rules help in children).
• Shunt or external ventricular drain infection: lower cell counts, and coagulase negative staphylococci and Cutibacterium acnes (grows slowly, needs prolonged anaerobic culture for 10 to 14 days) are common. A single positive culture with few cells may be contamination.
• Partially treated meningitis: lymphocytic pleocytosis with low glucose and negative culture, where PCR and latex or antigen tests may identify the organism.

Critical values and findings needing immediate action.

• Any organisms on Gram stain, or turbid CSF, or clinical suspicion of bacterial meningitis: call the clinician with the result at once.
• Start empirical intravenous antibiotics without waiting for results: third generation cephalosporin, with ampicillin for Listeria risk in neonates, older adults and immunosuppression, and vancomycin where penicillin resistant pneumococci are present.
• Give dexamethasone before or with the first dose in adults and children.
• Positive culture from CSF: critical result, urgent telephone notification, and susceptibility testing.
• Meningococcal disease: isolation for 24 hours of effective therapy, notification and chemoprophylaxis for close contacts (ciprofloxacin, rifampicin or ceftriaxone).
• Suspected raised intracranial pressure before lumbar puncture: do not delay antibiotics and take blood cultures first.
• Positive Gram stain with Gram positive diplococci in a patient with recent head injury or skull base fracture: pneumococcal meningitis with CSF leak, neurosurgical review.

Interferences and pitfalls.

• Antibiotics before lumbar puncture reduce culture yield (sterilisation of CSF may occur within 2 to 4 hours for meningococcus and 4 to 10 hours for pneumococcus) and Gram stain sensitivity, but antibiotics must not be delayed for lumbar puncture. Blood cultures, PCR and latex agglutination are alternatives.
• Contamination by skin flora (coagulase negative staphylococci, Cutibacterium) causes false positives, especially in single positive cultures of low count, and is judged by CSF cell count, repeat cultures and clinical features.
• Gram stain artefacts (stain precipitate, debris, Gram variability in older cultures and in pneumococci that appear Gram negative) may mislead, and examination by an experienced observer is important.
• Delay in transport and refrigeration kill fastidious organisms, so samples go to the laboratory immediately.
• CSF Gram stain and culture do not detect viruses, mycobacteria, fungi or parasites, and requests are added for PCR, acid fast stain and culture, cryptococcal antigen, India ink, syphilis serology and others as the clinical picture requires.
• Bacterial antigen tests (latex agglutination) have poor sensitivity and are not used routinely, and multiplex PCR panels are fast and sensitive but can give false positives (for example, Streptococcus pneumoniae and Streptococcus agalactiae), and a negative PCR does not exclude bacterial meningitis.
• A bloody tap raises the white cell count (about 1 white cell for every 500 to 1000 red cells) and protein, and the correction is applied.
• Lumbar puncture is delayed when there are focal neurological signs, papilloedema, Glasgow Coma Scale under 10 to 12, new onset seizures, immunocompromise, coagulopathy or local skin infection.

Special groups.

• Neonates: group B streptococcus, E. coli and Listeria. CSF cell counts are higher in healthy neonates, and a positive blood culture with normal CSF does not exclude meningitis.
• Children: pneumococcus, meningococcus and Haemophilus (in unvaccinated), with dexamethasone for Haemophilus and pneumococcal disease.
• Older adults and immunocompromised: Listeria, pneumococcus and Gram negative bacilli.
• Post-neurosurgical and shunt: staphylococci, Gram negative bacilli and Cutibacterium, treated with drain removal and prolonged antibiotics.
• Regions: epidemic meningococcal meningitis in the African meningitis belt (Neisseria meningitidis serogroups A, C, W and X), where CSF is often unavailable and treatment is empirical with ceftriaxone, and low resource settings rely on microscopy and rapid tests.

Follow up tests.

• CSF cell count and differential, protein, glucose and lactate, paired blood glucose.
• Blood cultures, multiplex meningitis and encephalitis PCR, pneumococcal and meningococcal PCR.
• Serum C-reactive protein and procalcitonin, full blood count, coagulation screen, HIV test.
• Cryptococcal antigen, acid fast smear and culture, Xpert MTB/RIF on CSF, syphilis serology.
• CT or MRI head, and audiology for survivors.