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DIAGNOSIS OF MENINGITIS
SKILLS FOR RESIDENTS

Dr.T.V.Rao MD
 Why Skill Based Learning for Residents
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The Indian Medical Curriculum is in for Rapid changes

for making the MBBS Doctors competent to perform
several life saving procedures learned to the greater
perfection. This programme is created that young
residents to learn the life saving diagnosis of
Meningitis by doing a Lumbar puncture and simple
observation in emergency hours with interactive
observation.

Dr.T.V.Rao MD
 What is Meningitis
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Meningitis is an infection
of the coverings around
the brain and spinal cord.

The infection occurs most
often in children, teens,
and young adults. Also at
risk are older adults and
people who have long-
term health problems, such
as a weakened immune
system.
 Why Diagnosing Meningitis is
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Important

Diagnosing Meningitis is top priority in clinical

Medicine, in particular Bacterial meningitis, can be
a life threatening condition , the need for
appreciate antibiotic therapy at the earliest is a
priority.
Even with Minimal Diagnostic faculties if done with
precision can reduce morbidity and mortality
 On suspicion of Meningitis
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Every patient suspected of

having Meningitis should have a
specimen of CSF examination in
the laboratory to establish the
infection and to rule out
infection.
 Basic Understanding on
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Meningitis

On a broad basis
Meningitis is
classified as
1 Purulent
Meningitis
2 Aseptic
Meningitis
 What is Purulent Meningitis
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The CSF appears

typically turbid due to
the presence of
Leucocytes 100 to
several thousands / mm3
most of which are
Polymorph nuclear
leucocytes
 Major Etiological agents of
8 Meningitis

1 Meningococcus

2 Pneumococcus

3 Haemophilus influenza
On majority of the occasions the pathogens pass from
Respiratory tract via blood stream and infect Meningitis
Can occur at any age
 Neonates and Infants
9 Meningitis

There is specific affinity of some pathogens infecting

Neonates and Infants
1 Coli forms
2 ß hemolytic streptococci
3 Pseudomonas
4 Salmonella and Listeria Monocytogenes
 Iatrogenic Meningitis
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Carelessly performed Lumbar

puncture

Accidental wound infection
in neurosurgical wounds
Pyogenic Staphylococcus
Streptococci
Coli form bacilli
Anaerobic cocci
Bacteriods
 Aseptic Meningitis
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In these conditions CSF is

clear or only slightly
turbid contain moderate
number of leucocytes
10 – 500 / mm3
Majority of cells are
lymphocytes, except in
early stages. majority
are caused by viruses
 Etiological agents of Aseptic Meningitis
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Enteroviruses
ECHO viruses
Coxsackie virus
Polio virus
Mumps virus
moderately infective
Herpes simplex
Varicella zoster
Measles –
Adenovirus
Arboviruses
 CSF resembles - Aseptic Meningitis in
Several other Infections
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Few conditions associated

with other etiological
agent resemble aseptic
meningitis
Leptospirosis
( Serovars Canicola
icterohaemorrhagea )
Fungi ( Cryptococcus
neoformans )
Amoeba – Naegleria,
Harmanella.
 Confusing CSF appearance

When early treatment is
given in Bacterial
meningitis the Clinico
pathological appearance
appears as Viral meningitis

In viral Encephalitis
moderate Lymphocyte
exduate is found as it in
Viral meningitis

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 Tuberculosis Meningitis
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On many occasions Tuberculosis

present as Aseptic meningitis,
results from Pulmonary or
mesenteric tuberculosis

Can be associated with Miliary
tuberculosis.

Cell counts on CSF will reveal 100
– 500 leucocytes / mm3

Majority are Lymphocytes

May form veil clot when CSF is
allowed to stand in a undisturbed
state.
 WHY MICROBIOLOGICAL DIAGNOSIS IS
LIFE SAVING
Information derived from the results has impact on :

Diagnosis of infectious diseases

Antibiotic prescribing

Formulation of local antibiotic policy

Public health impact eg
Meningococcal infection.

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 17

Cerebrospinal fluid
examination
 Why specimen collection is Important
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in Microbiology
Specimen collection in Microbiology to isolate and
identify the causative agents forms back bone of
the investigative procedures.
In developing world, lack of awareness and casual
attitude among junior staff hampers the definitive
diagnosis.
Specific procedures in collecting specimens will
certainly improve the quality of services of
Microbiology Departments
 Some tips better Diagnosis

Laboratory investigation should start as early as possible

Specimens obtained early, preferably prior to

antimicrobial treatment likely to yield the infective

pathogen

Before doing anything, explain the procedure to patient

and relatives

When collecting the specimen, avoid contamination

Take a sufficient quantity of material

Follow the appropriate precautions for safety
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 An Ideal Request form
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 Name xxxx Age Sex

 IP/ OP No xyz Time Date
 Ward xx123 Urgent / Routine
 Nature of specimen CSF
 Investigation needed xxxx
 xxxx

Doctor/Staff
Contact No 1234567
 Why Proper written Request
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 Your request is a legal document.

 Identifies all the outcome of test.

 No interchange of results.

 Short forms are dangerous

 Signature of the Doctor / Nurse is essential in

legible form, can help to contact in case of

results which can save a patient.
 Specimen collection for
CSF Examination

 Lumbar puncture to
collect the CSF for
examination to be
collected by Physician
trained in procedure
with aseptic
precautions to prevent
introduction of
Infection.

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 Procedure to collect CSF

 The trained physician will

collect only 3-5 ml into a
labeled sterile container
 Removal of large volume of
CSF lead to headache,
 The fluid to be collected at the rate
of 4-5 drops per second.
 If sudden removal of fluid is
allowed may draw down
cerebellum into the Foramen
magnum and compress the Medulla
of the Brain

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CSF needs a New and Sterile container

 Fresh sterile screw capped

container to be used.
 Reused containers, not to
be used, contamination
from the previous
specimens misrepresent
the present specimen.

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 Lumbar puncture for CSF collection

 The best site for puncture is inter

space between 3 and 4 lumbar
vertebrae
( Corresponds to highest point of iliac crest )

The Physician should wear sterile

gloves and conduct the
procedure with sterile
precautions, The site of
procedure should be disinfected
and sterile occlusive dressing
applied to the puncture site after
the procedure.

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Transportation to Laboratory
 The collected specimen
of CSF to be dispatched
promptly to Laboratory ,
delay may cause death
of delicate pathogens,
eg Meningococci and
disintegrate leukocytes

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 Preservation of CSF

 It is important when there is

delay in transportation of
specimens to Laboratory
do not keep in
Refrigerator, which tends
to kill H. Influenza
 If delay is anticipated leave
at Room Temperature.

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 Laboratory Examination of CSF
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The specimens should be

examined with naked eye
Look for Turbidity
Contamination
with Blood
Normal CSF appears like
water
 Specimen Examination
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CSF to be examined for

Cell counts
Gram staining
Culturing
Estimation of
protein and glucose
 Cell counts in CSF

Microscopic examination of
uncentrigured, well mixed
CSF is done in slide
counting chamber.

Count the number of
Polymorphs
Lymphocytes
Erythrocytes

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 Normal cell counts
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CSF normally contains 0- 5 leucocytes / mm3

Mainly Lymphocytes
Newly born children contain up to 30/mm3
Mainly polymorphs
In purulent Meningitis there are usually 100 – 300
leucocytes/mm3
In aseptic meningitis
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there are usually 10 – 500
leucocytes/mm
Mostly lymphocytes, though polymorphs may
predominate in the earliest stage of the illness.
In Tuberculosis3 meningitis there are usually 100 – 500
leucocytes/mm
 Care in Counting the Cells
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When counting the cells,

care must be taken to
identify the RBC and
rare presence of
yeasts, amoeba
should not be
mistaken for
leukocytes
 Differential Leukocyte counts

If there is any difficulty in
differentiating polymorphs and
lymphocytes in the counting
chamber
Make a film of cellular deposit
after specimen has been
centrifuged
Stain with
Methylene blue
leishmans or Carol thionine
and examined under oil
immersion to asses the
relative number of two
types of leucocytes

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 Gram Staining of CSF
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The CSF to be centrifuged to deposit the cells and

bacteria

The film made from the deposit to be stained with
Gram’s method

Make a thick smear with of area spread 10 mm in
diameter encircle by a scratch on the surface of the
slide

If the CSF appears turbid make a thin film

All the smears are dried and fixed on heat
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Gram Staining technique
 Gram Staining Procedure

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 Examination of Gram Stained smear
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A careful search for

Bacteria to be made in
particular where there
are plenty of leucocytes

At least keen observation
to be done for 10 mt
before reporting a
negative smear.
 Observe for the Presence of
One should be familiar with the
following bacteria for successful
reporting
Meningococci
Pneumococci
Haemophilus
Coli form bacilli
Streptococci
Listeria
All the results are promptly reported to
treating Physician
When variety of bacteria are found
specimens may be contaminated.
May need a fresh specimen for
examination

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 Culturing of CSF
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The deposited sediment

plated on culture media
Blood agar,
Chocolate agar
incubated with 5-10%
Carbon dioxide
A part of the specimen
inoculated into Robertson's
cooked medium
In suspected cases of Brain
abscess Bacteroides and
anaerobic cocci are
cultured in anaerobic
medium
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
Penicillin, and
Chloramphenicol

The antibiotic sensitivity
pattern can be reported at
the earliest

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 Biochemical testing for Infections

CSF should be tested for quantization
of
Glucose and Protein
Normal CSF contain
2.2 to 4mmol/liter correlates to 60%
of the plasma levels
Protein is present at concentration of
0.15 to 0.4 grams/liter
It can be higher in neonates can be up
to 1.5 grams / liter
In pyogenic meningitis Protein
concentration is increased and
Glucose concentration decreased.
In aseptic meningitis Glucose
concentration is normal and protein
concentration raised

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Tests for Bacterial antigen Detection
Co agglutination Tests
There are several test
kits available
commercially for
detection antigens of
Meningococci
Pneumococci
H influenzae

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 Diagnosis of Viral Meningitis
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The virus are to be

isolated from CSF

Presence of Viral
antibodies by paired
sampling of serum

In few viral infections the
virus can be isolated from
Throat swabs
Specimens of feces
 Tuberculosis Meningitis -Diagnosis
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CSF should be
tested for presence
of Acid fast bacilli
by simple Ziehl
Neelsen method

The deposit of the
concentrate can be
inoculated onto
Lowenstein
Jensen’s Medium
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AFB Staining Methods

Zeihl Neelsen’s-
hot stain

Kinyoun’s-cold

stain
Modifications
 Ziehl- Neelsen Procedure
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Make a smear. Air Dry. Heat Fix.

2. Flood smear with Carbol Fuchsin stain

Carbol Fuchsin is a lipid soluble, phenolic compound, which is able to penetrate
the cell wall
3. Cover flooded smear with filter paper
4. Steam for 10 minutes. Add more Carbol Fuchsin stain as needed
5. Cool slide
6. Rinse with DI water
7. Flood slide with acid alcohol (leave 15 seconds). The acid alcohol
contains 3% HCl and 95% ethanol, or you
can declorase with 20% H2 S04

The waxy cell wall then prevents the stain from being removed by the acid alcohol
(decolorizer) once it has penetrated the cell wall. The acid alcohol decolorizer will
remove the stain from all other cells.
 Ziehl- Neelsen Procedure (continued)
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8. Tilt slide 45 degrees over the sink and add acid alcohol
drop wise (drop by drop) until the red color stops
streaming from the smear
9. Rinse with DI water
10. Add Loeffler’s Methylene Blue stain (counter stain). This
stain adds blue color to non-acid fast cells!! Leave
Loeffler’s Blue stain on smear for 1 minute
11. Rinse slide. Blot dry.
12. Use oil immersion objective to view.
 1 2 3

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4 5 6

7
Ziehl-Neelsen
stain
 How the Acid fast bacteria appear
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 Leptospiral Meningitis - Diagnosis
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On few occasions in endemic

areas Leptospira can produce
meningitis

Rarely Leptospira can be seen
in CSF under Dark ground
microscopy

Cane cultured on Korthoff
other Leptospiral medium
 Viruses - Meningitis

The following viruses can

cause Aseptic meningitis
1 Echovirus
2 Coxsackie
3 Herpes virus

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 Tele contact is crucial in serious patients

 When the patient is

serious, write a
Tele contact
number which
can help in prompt
delivery of results

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The Programme is created by Dr.T.V.Rao

MD for ‘e’ learning for Young Resident
Doctors in the Developing World

Email
doctortvrao@gmail.com