How to Diagnose Meningitis in the Lab ?

in the lab ;
How to diagnose
Meningitis ??
BY
Dr. Nashwa Elsayed
Clinical Pathologist / Infection Control Specialist
MB.BCH
M.Sc. of Clinical & Chemical Pathology
Diploma in Infection Prevention & Control
Diploma in Health Care Quality Management

MENINGITIS
• Inflammatory process of meninges and
CSF within the subarachnoid space.
• Meningoencephalitis combines this with
inflammation of brain parenchyma.
• Most episodes of meningitis result from
hematogenous seeding of infection from
other sites to the meninges.
Dr. Nashwa Elsayed
How To Diagnose Meningitis ??

Dr. Nashwa Elsayed

** The infection occurs most often in
children, teens, and young adults.
**People at risk are older adults and
people who have long-term health
problems, such as a weakened
immune system.
Dr. Nashwa Elsayed

Classification of Meningitis
 Based on duration :
1) Acute : symptoms present within 24 hrs.
2) Sub acute : symptoms lasting from 1-7 days.
3) Chronic : symptoms lasting over 7 days
 Based on etiology:
1) Bacterial meningitis
2) Viral (aseptic) meningitis
3) Fungal meningitis
Dr. Nashwa Elsayed

Symptoms of Meningitis
Dr. Nashwa Elsayed

Symptoms in Infants
** They may have :
• Irritability
• Tiredness
• Poor Feeding
Dr. Nashwa Elsayed

Causes of Bacterial Meningitis
Premature babies and newborns to 3 months of age :
• E. coli and other coliforms
• group B Streptococci,
• Listeria monocytogenes,
• S. pneumoniae,
• H. influenza type b,
• N. meningitides
Age 3 months to adolescence :
• N. meningitides and
• S. pneumoniae
• H. influenza type B
Adults :
• N. meningitides
• S. pneumoniae (80% of all cases) of bacterial meningitis,
Age >50yrs
• increased risk of L. monocytogenes
Dr. Nashwa Elsayed

Diagnosis of Meningitis
• It is a Medical Emergency,
Why ??
• Bacterial meningitis, can be a life threatening
condition, need appropriate antibiotic therapy as
soon as possible.
• The diagnosis is made through the examination of
the spinal fluid collected through a spinal tap.
* This is crucial to identifying the Type & Organism.
Dr. Nashwa Elsayed

Every patient suspected of
having Meningitis should have
a specimen for CSF
examination in the laboratory
to establish or to rule out the
infection.
Dr. Nashwa Elsayed

Laboratory Investigations
• CSF Examination
• C-Reactive protein (CRP)
• ESR
• Full Blood Picture (CBC)
• Blood culture and other cultures (urine, abscess, and
middle ear).
• Serum electrolytes, BUN, Creatinine.
** In severe forms of meningitis, monitoring of blood electrolytes may
be important; hyponatremia is common in bacterial meningitis
Dr. Nashwa Elsayed

Laboratory Examination of the CSF
Dr. Nashwa Elsayed

CSF Sample Collection and Processing
• Lumbar puncture , shunts & cannulas
• Approximately 15 - 20 cc fluid collected
• Process within 1 hour without refrigeration -
STAT
Dr. Nashwa Elsayed

Laboratory Examination of the CSF
The first step to confirm the presence of
meningitis.
CSF should be examined for:
• Microbiology
• Biochemistry
• Cytological examination.
Dr. Nashwa Elsayed

Three tube set-up
Tube 1: Chemistry and Immunology
Tube 2: Microbiology
Tube 3: Cell count, differential, cytology
Dr. Nashwa Elsayed

Normal CSF profile
• Gross appearance: clear and colorless.
• Glucose: 40–85 mg/dL. (30 % blood glucose)
• Protein (total): 15–45 mg/dL.
• Leukocytes:
*WBC: 0–5/µL (adults / children);
up to 30/µL (newborns).
*Differential: 60–70% lymphocytes;
up to 30% monocytes and macrophages;
other cells 2% or less.
• Culture: sterile.
Dr. Nashwa Elsayed

CSF Profile in
Bacterial Meningitis
• Glucose: Normal to marked decrease. <40 mg/dL.
• Protein: (Marked increase) > 250 mg/dL.
• WBCs: >500 cells/µL (usually > 1000).
Early: May be < 100.
• Cell differential: Predominance of Neutrophils (PMNs)
• Culture: Positive
Dr. Nashwa Elsayed

Macroscopic Examination
• Normal CSF appearance is crystal clear and colorless
• Pathological processes can cause fluid to appear
cloudy, turbid, bloody, viscous, or clotted.
• The clarity of the fluid is of little clinical use, except
to provide an immediate indication of abnormality
of the CSF. A very useful point to remember is that a
large number of cells can be present without
affecting the clarity.
Dr. Nashwa Elsayed

Microscopic Examinations
• Cell counts:
RBC
Total Leukocyte
• Differential
• Cytology
Dr. Nashwa Elsayed

Method of CSF Cell Count
• Properly mix the CSF sample.
• Nine drops of CSF is diluted with one drop of CSF
diluting fluid (in the ratio 9: 1)
** CSF Diluting Fluid:
• Add 10 ml of glacial acetic acid and 0.2 grams of crystal violet to a 100-
ml volumetric flask. Dilute to the mark with distilled water.
• The counting chamber is covered with a cover slip.
• Charge the counting chamber with fluid and
allowed to stand for 5 min for the cells to settle.
• Cells are counted in all the nine squares.
Dr. Nashwa Elsayed

Calculation
• No. of cells =
Number of cells counted x 10
9
• (as Neubauer’s chamber has a depth of 0.1 mm and
total counting area is 9 sq. mm.)
Dr. Nashwa Elsayed

Cell counts
• “Normal” adult CSF: 0-5 cells/ml
• Mainly Lymphocytes.
 RBC count is of limited use, but can be used to:
correct CSF leukocyte counts & CSF protein values of
a traumatic tap CSF.
W = WBCf - WBCb x RBCf
RBCb
Dr. Nashwa Elsayed

Causes of increased cell count :
• Meningitis.
• Intracranial hemorrhage.
• Meningeal infiltration by malignancy.
• Multiple sclerosis.
Dr. Nashwa Elsayed

Differential WBC Count
• Performed on a stained (Wright’s stain) smear made
from CSF.
• It is recommended that stained smears be made even
when the total cell count is within normal limits.
• Count 100 cells in consecutive oil-power fields.
• Report percentage of each type of cell present.
Dr. Nashwa Elsayed

Predominant Cell in
Bacterial Meningitis
• Neutrophils
Dr. Nashwa Elsayed

Gram’s Stain
• Smear is made from the sediment and is air dried,
stain it with gram’s stain and observe it under oil
immersion.
Dr. Nashwa Elsayed

Latex Agglutination Test
There are several test kits available commercially for
detection antigens of :
• Meningococci
• Pneumococci
• H. influenzae
Dr. Nashwa Elsayed

Dr. Nashwa Elsayed
Negativereaction:
The suspension
remains
homogenous and
slightly milky in
appearance.
Positivereaction:
agglutination (or
visible clumping) of
the latex particles
and slight clearing
of the suspension
occurs within 2-10
minutes .

CSF
CULTURE & SENSITIVITY
Gold standard
** Indications:
1. Appearance of bacteria on gram stained smears.
2. Increased proteins or cell count.
• The deposited sediment plated on culture media
* Blood agar,
* Chocolate agar
• incubated with 5-10% Carbon dioxide
Dr. Nashwa Elsayed

Proper streaking and growth of N. meningitidis
on a Blood Agar Plate
Dr. Nashwa Elsayed

Dr. Nashwa Elsayed
Proper streaking and growth of S. pneumoniae
on a Blood Agar Plate

Dr. Nashwa Elsayed
Proper streaking and growth of H. influenzae on a
Chocolate Agar Plate

Direct antibiotic sensitivity detection
• When the organisms are numerous on Gram stained
film CSF can be directly inoculated into Blood agar
and Chocolate agar
• The commonly used effective antibiotic disks are
tested with sensitivity pattern,
• Commonly we can test Benzyl Pencillin, and
Choramphenicol
• The antibiotic sensitivity pattern can be reported
earlier.
Dr. Nashwa Elsayed

TREATMENT
• Antibiotics for bacterial meningitis vary
depending on the bacteria causing the
infection.
• Antibiotics are not effective in viral
meningitis.
• Other medications and intravenous fluids
will be used to treat symptoms such as brain
swelling, shock, and seizures.
Dr. Nashwa Elsayed

PREVENTION
• Meningococcal conjugate Vaccine.
• Meningococcal Polysacharride Vaccine.
• Haemophilus vaccine (HiB vaccine) in children.
• The pneumococcal conjugate vaccine is now a routine
childhood immunization and is very effective at
preventing pneumococcal meningitis.
• Household members and others in close contact with
people who have meningococcal meningitis should
receive preventive antibiotics.
Dr. Nashwa Elsayed

How to Diagnose Meningitis in the Lab ?

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