7. LABORATORY INVESTIGATIONS

7.1 Blood Culture

Blood must be collected in blood culture specimen bottles using strict aseptic technique (s) from all suspected cases and sent to the laboratory as quickly as possible. Specimens should reach the laboratory within 3-4 hours and not beyond 24 hours. Ideally two sets (taken from different sites and at different times) of blood cultures should be submitted prior to antibiotic therapy but treatment should not be delayed in order to obtain specimens. Even in cases of meningitis, blood for culture must be collected. About 1-5 ml of blood is needed in children and 5-10 ml in adults. Ideal volumes may vary depending on the blood culture system in use. Laboratories can be consulted for optimal blood volumes. Specimens must be kept at room temperature (not in a refrigerator) whilst awaiting transport to the laboratory.

If the clinical picture is compatible with meningococcal septicaemia, do not delay giving penicillin or ceftriaxone if laboratory confirmation cannot be immediately obtained. Early use of antibiotics in this setting can be lifesaving.

7.2 Cerebrospinal fluid (CSF)

Cerebrospinal Fluid (CSF) Examination and Culture

A lumbar puncture should be performed for suspected meningitis where no contraindications exist. In the primary care setting a lumbar puncture does not need to be done. Emphasis should be placed on administration of lifesaving care and urgent transfer to a referral hospital.

In adults

Where lumbar puncture is not contraindicated and can be safely performed; this should be done, as it provides valuable diagnostic information on the specific cause of meningitis.

In Children

The clinical signs indicating the presence or absence of raised intracranial pressure in children are notoriously inaccurate and should never be relied upon. A lumbar puncture is not indicated in a child with clinical meningococcemia even if meningism is found. A lumbar puncture should never be done if there is any suggestion of impaired level of consciousness. 

A blood culture and urgent treatment based on clinical assessment is more appropriate.

Contraindications to lumbar puncture in adults

Lumbar puncture is contraindicated in patients with raised intracranial pressure. Classical signs of RAISED INTRACRANIAL PRESSURE such as bradycardia, papilloedema or hypertension are often absent, especially in children. Neurological imaging, e.g. CT scanning should be considered before doing a lumbar puncture in all patients who have signs of:

• Raised intracranial pressure (impending cerebral herniation) with focal neurological signs or papilloedema.
• New onset seizures and an abnormal level of consciousness should prompt a careful examination to exclude raised intracranial pressure.
• Intracranial pathology with mass effect. Signs include:

        •Deep coma (Glasgow Coma Scale (GCS) less than 13),

        •Sudden deterioration of level of consciousness,

        •Decerebrate or decorticate posturing

        •Neurogenic hyperventilation

        •Unequal dilated or poorly reactive pupils and

        •Absent doll’s eye reflex

Lumbar puncture should also be delayed in patients with haemodynamic instability (low blood pressure or uncorrected bleeding tendency).

Transport and processing of CSF

The CSF should be kept as close to body temperature as possible whilst awaiting transport. The meningococcus is highly susceptible to heat, cold and direct sunlight. So the specimen should not be refrigerated, left on the window sill or transported in a hot car boot!

Tests to be requested on the cerebrospinal fluid (CSF) include:

• Protein and glucose determination (a blood glucose should also be done)
• Direct microscopy (cell count and Gram stain)
• Culture and antibiotic susceptibility testing

If HIV infection is suspected an Indian ink stain and cryptococcal latex antigen test for cryptococcal meningitis should also be requested.

The typical findings in CSF of adults may include the following:

• White blood cell count: often above 1000 cells/mm3 with 60% polymorphonuclear cells*
• Protein level: >0.80g/l (should be <0.45 g/l in normal CSF)
• CSF glucose concentration 2/3 lower than blood glucose
• Gram stain showing Gram-negative diplococci (intra and/or extracellular).

* may be low in immunocompromised patients

7.3 Aspirates from other normally sterile sites specimens, skin rash aspirate or biopsy culture

should be taken as clinically indicated, but if culture from these sites yield meningococci, this would confirm invasive disease.

7.4 Oropharyngeal swabs

Oropharyngeal swab specimens are less likely to be affected by prior antibiotic use, and may assist in making the diagnosis when used in conjunction with other laboratory test results and the clinical characteristics of presenting disease. In the absence of isolation of meningococci or positive PCR results from normally sterile sites, a positive culture from an oropharyngeal swab does not confirm disease, and may only reflect asymptomatic carriage.

Non-culture diagnostic tests

Polysaccharide antigen testing

Rapid detection tests for bacterial antigen using latex agglutination may give false positive and false negative results and should be interpreted with caution. They should not be routinely requested on all CSF specimens but reserved for certain circumstances e.g. patients in whom antibiotic therapy has been given prior to lumbar puncture, which may result in a negative culture.

PCR (polymerase chain reaction)

PCR-based assays for detecting specific DNA sequences of N. meningitidis are available at the meningococcal reference laboratory at the National Institute for Communicable Diseases, Johannesburg. The test has been validated and performs well for CSF and blood specimens; however, specimens from other normally sterile site can also be tested. Whole blood (EDTA or other unclotted specimen) and/or CSF specimens can be sent for PCR if cultures are negative, if the diagnosis is suspected and facilities for culture are not available, or if specimens are taken after commencing antibiotics. Direct communication with the laboratory in Johannesburg (011 555 0315/ 0327/ 0316) will assist in the urgent transportation of the specimen and expedited testing.

Skin scrapings/impression smears

The practice of performing skin scrapings and impression smears for Gram stain from the petechial/purpuric site is not recommended. A negative Gram stain does not exclude the diagnosis. In addition (Neisseria) species may form part of normal skin flora and will resemble meningococci on Gram stain thus yielding false positive results.

Post-mortem specimens

Post-mortem specimens can be taken to confirm an ante-mortem suspected diagnosis of meningococcal disease, or may be a way of establishing the cause of illness and death in cases with undiagnosed infection. These specimens may be especially useful for sudden, unexplained deaths, especially in infants and young adults. Spleen and heart blood cultures can be submitted and processed similar to routine blood cultures, especially if performed as soon as possible after death (ideally within 15 hours). Post- mortem CSF can also be submitted to a microbiology laboratory for processing. Ideally specimens should be taken at the start of the post-mortem examination, and every effort should be made to avoid contamination. Non-culture diagnostic tests may be very important if meningococcal disease is suspected, and specimens should be submitted for PCR in addition to culture (see above for details).