How to Diagnose Meningitis in the Lab ?

in the lab ;

How to diagnose

Meningitis ??BY

Dr. Nashwa ElsayedClinical 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 ??

** 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.


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 meningitis2) Viral (aseptic) meningitis3) Fungal meningitis


Symptoms of Meningitis


Symptoms in Infants

** They may have :• Irritability• Tiredness• Poor Feeding


Causes of Bacterial MeningitisPremature 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


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.


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

How To Diagnose Meningitis ??

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


Laboratory Examination of the CSF


CSF Sample Collection and Processing

• Lumbar puncture , shunts & cannulas

• Approximately 15 - 20 cc fluid collected

• Process within 1 hour without refrigeration -STAT


Laboratory Examination of the CSF

The first step to confirm the presence of meningitis.

CSF should be examined for:• Microbiology• Biochemistry• Cytological examination.


Three tube set-up

Tube 1: Chemistry and Immunology

Tube 2: Microbiology

Tube 3: Cell count, differential, cytology


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.


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


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.


Microscopic Examinations

• Cell counts:RBCTotal Leukocyte

• Differential • Cytology


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.


Calculation• No. of cells =

Number of cells counted x 109

• (as Neubauer’s chamber has a depth of 0.1 mm and total counting area is 9 sq. mm.)


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 RBCfRBCb


Causes of increased cell count :

• Meningitis.• Intracranial hemorrhage.• Meningeal infiltration by malignancy.• Multiple sclerosis.


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.


Predominant Cell in Bacterial Meningitis

• Neutrophils


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.


Latex Agglutination Test

There are several test kits available commercially for detection antigens of :

• Meningococci • Pneumococci• H. influenzae



Nega

tive r

eacti

on: The suspension

remains homogenous and slightly milky in appearance.

Posit

ive re

actio

n: 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


Proper streaking and growth of N. meningitidison a Blood Agar Plate



Proper streaking and growth of S. pneumoniaeon a Blood Agar Plate


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.


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.


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.


Thank You

How to Diagnose Meningitis in the Lab ?

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