CSF Culture, and Special stains

What sample is needed for Cerebrospinal Fluid Analysis?

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

How will you define Meningitis?

1. Meningitis is the inflammatory process involving the protective layers surrounding the brain and spinal cord, the meninges.
2. Symptoms include fever, stiff neck, severe headache, nausea, vomiting, and sensitivity to light.

How will you perform the Cerebrospinal Fluid (CSF) culture?

1. The diagnosis of acute bacterial meningitis depends on the isolation of the organism.
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. The 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 more common than meningococci.
   1. The gram-negative bacilli follow these.
7. Staphylococci are reported in 4% to 7% of the cases, and most are associated with brain surgery, septicemia, or endocarditis.
8. Any organism, even fungi, can infect the brain.

How will you find the organism in the Cerebrospinal Fluid?

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 for diagnosing meningitis caused by Gram-negative bacteria.
8. Serologic tests are done to identify the microorganism.
   1. CSF serological tests can diagnose neurosyphilis.

How will you prepare the sediment for stains from Cerebrospinal Fluid (CSF)?

1. CSF can be centrifuged, and the sediment is stained for Gram stain
2. Centrifuge CSF at 1500 g for 15 minutes, and prepare the slides from the sediments.
3. The cytocentrifuge will provide a highly concentrated sample.
4. The most common stain used is the Gram stain..
5. Even with the concentrated CSF, 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.

What do you know about the Gram stain of Cerebrospinal Fluid (CSF)?

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 is characterized by the presence of bacteria, whether they are cocci or bacilli, and whether they are gram-positive or gram-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 in newborns).
   7. Tuberculous meningitis (advise Acid-fast stain or fluorescent antibody stain).
   8. The fungi are Gram-stain positive or require India Ink preparation.

How will you interpret the culture and Sensitivity of Cerebrospinal Fluid?

1. Culture can identify the organism, and the most common organisms 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 identify around 85% of the bacteria in cases of suspected bacterial meningitis.
4. Meningococci grow best in a high CO2 atmosphere.
5. TB culture needs 4 to 6 weeks.

How will you identify Tuberculous bacilli in Cerebrospinal Fluid?

1. CSF and sputum smears are stained for Acid-fast bacilli.
   1. These are red, rod-shaped bacteria.

2. The conventional culture of T. bacilli requires 4 to 6 weeks of incubation.
3. The BACTEC method is a culture technique in which the growth medium for culturing M. tuberculosis contains a substrate labeled with radioactive carbon. (¹⁴C).
   1. The bacteria utilize this substrate and produce radioactive carbon dioxide (14CO2) from it.
   2. This ¹⁴CO2 is detected and quantified.
   3. This will give quick identification of mycobacteria.
4. The polymerase chain reaction (PCR) method has also been developed.
   1. This allows the amplification of genomes, which the genetic DNA probe can detect.
   2. Mycobacterium is detected in 36 to 48 hours.

How will you perform the Cerebrospinal Fluid (CSF) protein electrophoresis?

What are the indications for CSF electrophoresis?

1. Electrophoresis is used to detect any abnormalities in proteins and immunoglobulins.
2. This helps to diagnose:
   1. Multiple sclerosis.
   2. Neurosyphilis.
   3. Autoimmune diseases.

What will you see in Multiple Sclerosis (MS) in Cerebrospinal Fluid?

1. It is a chronic demyelinating disease characterized by relapses, a remitting course, and episodes separated in time and anatomic location within the central nervous system.
   1. Recurrent illness of unpredictable length and severity.
   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 paresthesia 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.
   4. There is increased IgG production.
   5. There is an increased IgG in 60% to 80% of the cases.
3. Increased IgG: albumin ratio:
   1. The normal ratio is <25%.
   2. Approximately 70% of MS patients have an elevated IgG/albumin ratio.
   3. This IgG/albumin ratio is more specific than the IgG alone.
4. The gamma region has a discrete, sharp band called the oligoclonal band.
   1. The presence of an oligoclonal band is a significant laboratory finding.
   2. Without an oligoclonal band, if IgG is raised, it has no significance.
   3. Total protein levels are mildly elevated in 25% to 50% of cases.
   4. If the total protein level is greater than 100 mg/dL, the diagnosis of MS is less likely.

5. The oligoclonal band may be seen in HIV.
6. Electrophoresis differentiates CSF from serum, as there is an additional band of transferrin in CSF that is not present in serum.

How will you summarize various types of meningitis?

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

How will you differentiate various types of meningitis?

Questions and answers: