CSF Culture, Special stains

Sample 

1. The analysis is done in the CSF fluid.
2. CSF is collected in three tubes:
   1. The second tube is recommended for the culture or the third tube for culture and special studies.
   2. Never use the first tube for the culture.
3. Transport the CSF immediately to the lab; culture should be done as the sample reaches the lab.

 CSF for culture and its basis

1. The diagnosis of acute bacterial meningitis depends upon the organisms’ isolation.
2. In infants under the age of 1 to 2 months, group B-streptococci are most common. This is followed by Escherichia coli.
   1. The third common organism is Listeria monocytogenes.
   2. Then other organisms are Enteric gram-negative bacteria.
3. For children from age 3 months to 5 or 6 years, the most common organism is H.influenzae.
   1. The second common organism is Meningococci.
   2. This is followed by the Pneumococci.
4. For older children and adults, the most common is Meningococci.
   1. It is followed by Pneumococci.
5. In Adults, the most common are Meninogococci and Pneumococci.
6. In old people, pneumococci are most common than meningococci.
   1. These are followed by the gram-negative bacilli.
7. Staphylococci are reported in 4% to 7% of the cases, and most are associated with brain surgery, septicemia, or endocarditis.
8. The brain can be infected by any kind of organism, even fungi.

Lab diagnosis of causative organism:

1. Gram stain.
2. Acid-fast stain.
3. Fluorescent antibody stain.
4. India Ink preparation.
5. Latex agglutination.
6. Enzyme-linked immunoabsorbent assay (ELISA).
7. The Limulus lysate test is useful to diagnose meningitis caused by gram-negative bacteria.
8. Serologic tests are done to identify the microorganism.
   1. CSF serological tests can diagnose neurosyphilis.

Procedure to prepare the sediment for stains:

1. The most common stain used is the Gram stain.
2. CSF can be centrifuged, and the sediment is stained for gram stains.
3. Centrifuge CSF at 1500 g for 15 minutes, and prepare the slides from the sediments.
4. Cytocentrifuge will provide a highly concentrated sample.
5. Even with the concentrated CSF still, 10% of the cases of gram stain and culture are negative.
6. Take blood culture and CSF culture because causative bacteria will also be present in the blood.
7. Screening of the gram stain of CSF is very difficult and may have false-negative results.

Gram stain:

1. It gives a preliminary diagnosis.
   1. Gram stain yield is 70% of the suspected cases.
   2. A negative gram stain does not rule out bacterial meningitis.
2. Gram stain will differentiate:
   1. Bacterial meningitis with the presence of bacteria, whether it is cocci or bacilli, and as gram-positive or negative.
   2. Viral meningitis will be negative.
3. The most common causative agents are H. Influenzae in children and Neisseria and Streptococcus in adults.
4. The most common organisms are:
   1. Streptococcus pneumonia (gram-positive cocci).
   2. Hemophilus influenza (gram-negative rods, pleomorphic).
   3. Escherichia coli (gram-negative rods).
   4. Neisseria meningitidis (gram-negative cocci).
   5. Listeria monocytogenes (gram-positive rods, common in newborns).
   6. Streptococcus agalactiae (gram-positive cocci, common newborns).
   7. Tuberculous meningitis (advise Acid-fast stain or fluorescent antibody stain).
   8. The fungi are gram stain positive or advise India Ink preparation.

Culture and Sensitivity

1. Culture can identify the organism, and the most common organism found are:
   1. Bacteria.
   2. Fungi.
   3. T.Bacilli.
2. Culture needs 24 to 48 hours to get a result. This may take up to 72 hours to get sensitivity.
3. Culture can recognize around 85% of the bacteria in case of suspected bacterial meningitis.
4. Meningococci grow best in a high CO2 atmosphere.

Identification of Tuberculous bacilli:

1. CSF and sputum smears are stained for the Acid-fast bacilli.
   1. These are red, rod-shaped bacteria.
2. The conventional culture of the T bacilli takes 4 to 6 weeks of incubation.
3. BACTEC  method is a culture technique in which the growth medium for the culture of M.tuberculosis has added a substrate labeled with radioactive carbon. (¹⁴C).
   1. This substrate is used by the bacteria and produces radioactive carbon dioxide (14CO₂), from the substrate.
   2. This ¹⁴CO2is detected and quantitated.
   3. This will give quick identification of mycobacteria.
4. The polymerase chain reaction (PCR) method is also developed.
   1. This allows the amplification of genomes which can be detected by the genetic DNA probe.
   2. Mycobacterium is detected in 36 to 48 hours.

CSF protein electrophoresis

1. Indication:

   1. Electrophoresis is done to find any abnormality of the proteins and immunoglobulins.
2. This helps diagnose:
   1. Multiple sclerosis.
   2. Neurosyphilis.
   3. Autoimmune diseases.

Multiple sclerosis (MS)

1. It is a chronic demyelinating disease characterized by relapses, remitting course with the central nervous system episodes separated in time and anatomic location.
   1. Recurrent illness of unpredictable length and severity of the disease.
   2. A multifocal demyelinating process in the cerebral hemisphere.
   3. The involvement of white matter results in various combinations of weakness, ataxia, vision problems, and paresthesias and frequently ends in paralysis.
2. In Multiple sclerosis, typical findings are:
   1. In the early stages, routine CSF examination changes are nonspecific.
   2. Later on, changes are similar to aseptic meningitis.
   3. Increased total proteins, and this is mainly gamma globulin.
      1. There is increased IgG production.
      2. There is an increased IgG in 60% to 80% of the cases.
      3. Increased IgG: albumin ratio.
         1. The normal ratio is <25%.
         2. Around 70% of MS patients have raised IgG/albumin ratio.
         3. This IgG/albumin ratio is more specific than only IgG.
      4. The gamma region has a discrete sharp band called the oligoclonal band.
         1. The presence of the oligoclonal band is an important lab. finding.
         2. In the absence of an oligoclonal band, if IgG is raised has no significance.
         3. Total proteins are mildly elevated in 25% to 50% of the cases.
         4. If the total protein is >100 mg/dL, then the diagnosis of MS is less likely.

3. The oligoclonal band may be seen in HIV.
4. Electrophoresis differentiates CSF from serum, where there is an extra band of transferrin in CSF and not in the serum.

Table of various types of meningitis:

Disease	Pressure	Gross	Glucose mg/dL	Protein mg/dL	TLC  count/cmm	Type of Cells
Normal	70-200	Clear(C.L)	45-85	10-45	0-8	MN
Bact. Meningitis	Increased	Turbid	0-25 mg/dL	50-500	1000-2000+	Polys
Viral meningitis			Normal	<100 mg/dL	5 to 300	Lymphocytes
Tub. Meningitis	Increased	clear/cobweb	15-45 (Decreased)	50-100	100-300	Lympho
Fungal meningitis	Increased		Normal to Decreased	50 to 300	40 to 400	Lymph0 + monocytes
Poliomyelitis	Increased	clear	normal	50-200	50-250	poly/lympho
Subarachnoid Hemorrhage	increased	bloody	normal	50-100	normal	RBCs

MN = Mononuclear cells.
TLC = Total leucocytic count.
Mening. = Meningitis.
Lympho = Lymphocytes.
Haem = Haemorrhage.

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