Meningitis and Encephalitis PCR Panel

The meningitis and encephalitis multiplex PCR panel is a syndromic nucleic acid amplification test performed on 200 microlitres of CSF, detecting up to 14 pathogens in about 1 hour: bacteria (Escherichia coli K1, Haemophilus influenzae, Listeria monocytogenes, Neisseria meningitidis, Streptococcus agalactiae, Streptococcus pneumoniae), viruses (cytomegalovirus, enterovirus, herpes simplex virus 1 and 2, human herpesvirus 6, human parechovirus, varicella zoster virus) and yeast (Cryptococcus neoformans and gattii).

Pooled sensitivity is about 90 to 95% and specificity 97 to 99% against culture, with lower sensitivity for Streptococcus pneumoniae and herpes simplex virus 1 and 2 in some evaluations, and for Cryptococcus compared with antigen testing (sensitivity 85 to 95%).

Acute bacterial meningitis has mortality of 10 to 30% and neurological sequelae in up to 30%, while viral meningitis is benign and self-limiting in most cases, and herpes simplex encephalitis has mortality over 70% untreated.

The panel provides a rapid identification at a time when Gram stain (sensitivity 60 to 80%) and culture (up to 2 days) are slow, and allows stopping unnecessary antibiotics and hospital stay in enteroviral meningitis.

Positive results in the first hours support early targeted therapy, but a negative result does not exclude infection by organisms not on the panel (Mycobacterium tuberculosis, Treponema pallidum, Borrelia, Toxoplasma, Streptococcus suis, Taenia, arboviruses, West Nile virus, Japanese encephalitis, rabies and others).

Indications. Suspected acute meningitis or encephalitis with abnormal CSF (raised white cell count, raised protein, low glucose) or when CSF is abnormal in the context of fever, headache, neck stiffness, confusion, seizures or focal signs. Neonates and infants with sepsis and suspected CNS infection (parechovirus, enterovirus, herpes simplex virus, Group B streptococcus).

Patients who received antibiotics before lumbar puncture, and so have negative cultures. Immunocompromised patients. Culture negative meningitis.

Not used for routine testing of every CSF specimen, or where the pre-test probability is low (normal white cell count), because false positives (particularly Streptococcus pneumoniae, Staphylococcus, cytomegalovirus and human herpesvirus 6) reduce the positive predictive value.

Specimen. CSF 0.2 to 1 mL from lumbar puncture in a sterile tube, with cell count, protein, glucose, Gram stain and culture, lactate, and extra volume stored for further testing. Transport at room temperature and test within 1 hour, or refrigerate for 24 hours. Plasma or blood is not suitable. Contamination from skin flora or the environment during lumbar puncture, and cross-contamination in the laboratory, are controlled by strict aseptic technique.

Reference values. All targets not detected. Normal CSF: white cells under 5 cells/microlitre (under 20 in neonates), protein 0.15 to 0.45 g/L (up to 1.0 g/L in neonates), glucose over 60% of paired plasma glucose, lactate under 3.5 mmol/L.

Interpretation.

• Bacterial target detected (Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Listeria, Escherichia coli K1, Streptococcus agalactiae) with neutrophilic CSF pleocytosis, high protein and low glucose: bacterial meningitis, confirmed by culture whenever possible (culture is needed for susceptibility testing).
• Streptococcus pneumoniae detected with a normal white cell count, in a well patient: probable false positive from contamination, and repeat testing or culture is needed.
• Enterovirus or human parechovirus detected: viral meningitis or encephalitis (neonatal sepsis like illness with parechovirus), supportive care, and antibiotics stopped when bacterial cultures remain negative and the clinical picture agrees.
• Herpes simplex virus 1 or 2 detected: herpes simplex encephalitis (type 1) or meningitis (type 2, Mollaret recurrent meningitis), treated with intravenous aciclovir. Negative PCR early (first 72 hours) with a high clinical suspicion needs repeat PCR and continued aciclovir.
• Varicella zoster virus detected: zoster meningitis or encephalitis and vasculopathy, with or without rash.
• Cytomegalovirus detected: immunocompromised hosts (HIV, transplant), with encephalitis, ventriculitis or polyradiculitis. Positive human herpesvirus 6 in an immunocompetent child or adult may reflect chromosomally integrated virus or latent reactivation and is interpreted with caution.
• Cryptococcus detected: cryptococcal meningitis, with CSF antigen and India ink for confirmation, and a negative PCR does not exclude it (use antigen).
• Multiple targets detected: contamination, co-infection, or reactivation, and clinical correlation is needed.
• No targets detected: does not exclude infection, especially if pre-test probability is high. Continue to look for tuberculosis, syphilis, fungi, parasites, autoimmune encephalitis (anti-NMDA receptor), malignancy and non-infectious causes.

Critical values and findings needing immediate action.

• Bacterial pathogen detected: immediate intravenous antibiotics with dexamethasone for pneumococcal and Haemophilus meningitis, ICU review as needed, close contact chemoprophylaxis for meningococcal disease, and notification of public health.
• Herpes simplex virus or varicella zoster virus detected: intravenous aciclovir without delay and neuroimaging.
• Listeria detected: ampicillin plus gentamicin (cephalosporins are inactive).
• Cryptococcus detected: amphotericin B-based induction therapy and management of raised intracranial pressure by lumbar drainage.
• Neonate with enterovirus or parechovirus and sepsis like illness: admission, close monitoring and exclusion of bacterial and herpes infection.
• Treatment should never be delayed or withheld in suspected bacterial meningitis while awaiting PCR.

Interferences and pitfalls.

• False negatives for Streptococcus pneumoniae, Cryptococcus gattii, herpes simplex virus (low viral load in early disease), and organisms with sequence variants not covered by primers.
• False positives from contamination (Streptococcus pneumoniae and Staphylococcus aureus from skin, reagents or the environment), traumatic taps and carry over, so results must be interpreted with CSF cell count, protein and glucose.
• A detected virus (for example, human herpesvirus 6, cytomegalovirus, Epstein-Barr virus) in an immunocompetent patient may represent latent or integrated virus and not the cause.
• The panel does not give antimicrobial susceptibility, and culture remains necessary. Positive PCR with negative culture is common after antibiotic treatment.
• Cost is high, and a restricted testing policy (abnormal CSF only, or defined clinical criteria) improves value.
• The panel does not detect tuberculous meningitis, which needs Xpert MTB/RIF Ultra or culture, neurosyphilis (VDRL, treponemal tests), Lyme disease, arboviruses or autoimmune encephalitis.
• Repeat lumbar puncture may be needed when the patient is not improving or when the first sample was taken early.
• Corticosteroid or antibiotic timing, opening pressure, imaging before lumbar puncture (focal signs, papilloedema, immunosuppression, reduced consciousness) and coagulation status are checked before the procedure.

Special groups.

• Neonates and young infants: Group B streptococcus, Escherichia coli, Listeria, herpes simplex virus, enterovirus and parechovirus (parechovirus causes severe sepsis like illness and white matter damage, and CSF may have no cells).
• Children: meningococcus, pneumococcus, enterovirus.
• Older adults and pregnant women: Listeria, pneumococcus, and herpes zoster.
• Immunocompromised: cytomegalovirus, varicella zoster virus, Cryptococcus, Listeria and Toxoplasma.
• Regions: meningitis belt of sub-Saharan Africa (Neisseria meningitidis serogroups A, C, W, X), tuberculous meningitis in high tuberculosis burden countries, cryptococcal meningitis with HIV, and arboviral encephalitis in Asia and the tropics.

Follow up tests. CSF cell count, protein, glucose, lactate, Gram stain and culture, blood cultures (before antibiotics), CSF cryptococcal antigen and India ink, Xpert MTB/RIF Ultra and TB culture, VDRL and treponemal tests, arbovirus serology and PCR, autoimmune encephalitis antibodies, HIV test and CD4 count, procalcitonin and C-reactive protein, CT or MRI brain,

electroencephalogram, repeat herpes simplex virus PCR in 3 to 7 days when initially negative, and antimicrobial susceptibility on culture isolates.