Dr Mostafa Mahmoud, MD, PHDConsultant Microbiologist

Associate Prof of Microbiology & Immunology

INTRODUCTION• Cerebrospinal fluid

(CSF) is normally iswater - clear fluid,that is produced bythe choroid plexusinside the brain andcirculates in brainand spinal cord.

• Bacterial meningitis is the result ofinfection of the meninges (liningaround the brain).

• The examination of CSF from patientssuspected of having meningitis isalways considered to be a STATprocedure.

• CSF sample is the one of highemergency specimen for microbiologylab.

• CSF sample never to be refrigerated.

Most common organisms incriminated according to age group:

1- From neonates and up to two months of age:

- Lancefield group B streptococci.

- Escherichia coli (K1)

- Listeria monocytogenes

- Herpes simplex virus and

- Neisseria meningitidis.

- Candida for Premature neonates.

2- From children older than two months to young adults:

- N. meningitides

- Streptococcus pneumoniae,

- viruses (in particular enteroviruses) and

- Haemophilus influenzae type b. (incidence decreased after Hib vaccine).

3- From adults:

- S. pneumoniae,

- N. meningitidis,

- viruses and occasionally non-group b H. influenzae.

- Listeria monocytogenes (for patients > 60 years).

- Fungi have also been found to cause meningitis.

Specimen Collection and Transport

Spinal tap technique:- Patient lies on his or her side, with knees flexedand back arched to separate lumbar vertebrae.- The patient is surgically draped, area overlyinglumbar vertebrae is disinfected.- The space between lumbar vertebrae L3, L4 ispalpated with the sterile gloves forefinger- The spinal needle is carefully directed betweenthe spinous processes through the interspinousligaments into the spinal canal

CSF collection by lumbar puncture technique

Optimal Time of Collection

-Preferably before antimicrobial

therapy is started, but this must not

be delayed unnecessarily pending

lumbar puncture and CSF culture.

- If a delay in transport or processing

is anticipated, keep the specimen at

room temperature.

Adequate quantity and appropriate

number of specimens

- Ideally a minimum volume of 1 mL.

- For Mycobacterium species, as large 3 to 4 mL a

volume as possible.

- CSF is normally collected sequentially into three or

more separate containers which should be numbered

consecutively.

- Disposable sterile screw-capped plastic containers

should be used.

- Common practice is to send the first and last

specimens taken for microbiological examination and

the second specimen for protein (Chemistry).

Tubes for CSF collection

CSF Evaluation

• Tube 1: Cell count and differential

• Tube 2: glucose, protein (Chemistry)

• Tube 3: Cultures, gram stain, cytology, “other” studies (HSV PCR, West Nile, India ink, Crypto Antigen, VDRL, Lyme Ab, AFB, etc…)

• Tube 4: Cell count and differential

Normal CSF valuesParameter Age Normal range

Leucoytes Neonates

1-4yr old

5yr-puberty

Adults

0 - 30 cells x 106/L

0 - 20 cells x 106/L

0 - 10 cells x 106/L

0 - 5 cells x 106/L

Erythrocytes Newborn

Adults

0 - 675 cells x 106/L

0 - 10 cells x 106/L

Protein Neonates ≤6d

Others

0.7 g/L

0.2 - 0.4 g/L (<1% of serum

protein concentration)

Glucose > 60% of simultaneously

determined plasma concentration

(CSF: serum ratio > 0.6)

Specimen Processing

1. Examine the fluid for colour, turbidity

and the presence of visible blood or clots.

Record the observations

2. Perform bacterial antigens detection

(Serology) testing by heating the part of

the CSF sample is at 90 oC for 3 minutes,

•then centrifuged for 5 minutes and then

the supernatant is tested with:

H influenza, S. pneumonia, S. agalactia,

Klebsiella pneumonia, E coli

Direct Examination

- Prepare a slide from the CSF centrifuged

deposit as for the Gram stain,

-The sensitivity of the Gram stain may be

improved by serial drops being "built up" on the

slide after each drop has dried, to maximize the

amount examined.

- Care should be taken to ensure that the smeardoes not wash off during staining.

• allow to air dry.

• Fix in alcohol and stain with a stain suitable for WBC morphology.

• Heat fixation distorts cellular morphology.

• Gram Stain examination: Note the presence or absence of organisms and WBCs.

• DO NOT quantitate.

WBC’s

• Infection!

• PMN predominance: likely bacterial meningitis

• Lymphocytic predominance: viral vs. fungal vs. TB vs. malignancy.

Protein

• Normal: protein is excluded from CSF by blood-CSF barrier

• Increased: nonspecific

• Elevated in all infectious meningitis

–May remain elevated for months post-meningitis (viral or bacterial)

• Increased in malignancy and inflammatory conditions (ex. Guillain-Barre)

Glucose

Normal:

• Viral infection

Low glucose:

• Bacterial meningitis, TB, fungal

Really low:

• < 18 is strongly suggestive of bacterial meningitis

Typical Viral Meningitis

• CSF WBC elevated, but < 250 (PMNs in early disease, then lymphocytes)

• CSF protein elevated, but < 150

• Glucose > 50% of serum concentration.

Typical Bacterial Meningitis

• CSF WBC > 1000, PMN predominance

• CSF protein > 500 mg/dl

• CSF glucose < 45 mg/dl

Specimen Culture

• With a sterile pipette, inoculate each agarwith the centrifuged deposit.

• Allow inoculum to dry before spreading tominimize any antibiotic effect which may bepresent.

• For the isolation of individual colonies,spread inoculum with a sterile loop.

• Note: we are using tube number 3 forculture, serology, gram stain. Culture to bedone first of all.

Culture media, conditions of incubation

1- Sheep blood agar incubated at CO2 incubator (5-10%) at 35-37 oC.2- Chocolate agar incubated at CO2 incubator (5-10%) at 35-37 oC.3- MacConkey agar incubated in ordinary incubator at 35-37 oC.4- for special diagnosis include special media for TB or fungal infections.

Interpretation of Cultures

• Examine the Blood Agar and ChocolateAgar plates and the Fastidious AnaerobicBroth daily for 4 days incubation.

• If no growth on the culture plates but evidence of growth in Fastidious Anaerobic Broth,

• All isolates are to be identified

Susceptibility Testing:

• The following antibiotics are reported in case of Haemophilus ampicillin, third generation cephalosporins, chloramphenicol, and meropenam

Reporting Procedure

Appearance

• Report the appearance of the CSF and the presence of a clot if applicable

Microscopy

• Cell count

• Report numbers of PMNs and lymphocytes x 106 per litre or

• Report PMNs and lymphocytes as percentages of the total WBC (which is reported as x 106)

Gram stain

• Report the presence or absence of organisms and WBCs. Do not quantitate

• Report on organisms detected.

• India ink report if requested.

• Report on encapsulated yeasts detected.Microscopyfor Mycobacterium species and parasites.

Microscopy reporting time

• Urgent microscopy results to be telephoned or sent electronically when available. Written report, 16 -72 h.

• Culture– Negative Report: “No growth”.

– Positive Report: Report all isolates with appropriate sensitivities. Do not quantitate

– Report results ASAP by telephone to the ward/ordering physician for the following:

– All positive or STAT Gram stain

– Report result of Bacterial antigen test

– All positive culture.

– Culture reporting time:

• Clinically urgent culture results to be telephoned or sent electronically when available. Interim / final written report, 16 - 72 h stating, if appropriate, that a further report will be issued.

• CSF as panic results when:1- Positive serology testing for bacterial

antigens.

2- Positive Gram staining for any bacteria or fungus.

3- Positive culture results.

Antimicrobial susceptibility testing

• Report susceptibilities as clinically indicated.

Reporting To Infection Control Department

• All positive finding regarding Gram stainingor culture must be reported to the infectioncontrol physician and nurse at once.

• In cases of suspected meningococcal diseaseand contacts the isolation of N. meningitidisshould be reported immediately.