DIAGNOSIS OF MENINGITIS

AMRITESH KUMAR
Assistant Professor (Microbiology)
1
What is Meningitis
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.

2
Why Diagnosing Meningitis is
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
3
On suspicion of Meningitis


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.
4
Basic Understanding on
Meningitis
On a broad
basis
Meningitis is
classified as
1 Purulent
Meningitis
2 Aseptic
Meningitis


5
What is Purulent
Meningitis

The CSF appears
typically turbid due
to the presence of
Leucocytes 100 to
several thousands /
mm
3
most of which
are Polymorph
nuclear leucocytes

6
Major Etiological agents of
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
7
Neonates and Infants
Meningitis

There is specific affinity of some pathogens
infecting Neonates and Infants
1 Coli forms
2 hemolytic streptococci
3 Pseudomonas
4 Salmonella and Listeria Monocytogenes
8
Iatrogenic Meningitis

Carelessly performed
Lumbar puncture
Accidental wound
infection in
neurosurgical wounds
Pyogenic
Staphylococcus
Streptococci
Coli form bacilli
Anaerobic cocci
Bacteriods

9
Aseptic Meningitis

In these conditions
CSF is clear or only
slightly turbid
contain moderate
number of
leucocytes 10
500 / mm
3
Majority
of cells are
lymphocytes, except
in early stages.
majority are caused
by viruses


10
Etiological agents of Aseptic
Meningitis
Enteroviruses
ECHO viruses
Coxsackie virus
Polio virus
Mumps virus
moderately
infective
Herpes simplex
Varicella zoster
Measles
Adenovirus
Arboviruses

11
CSF resembles - Aseptic Meningitis
in Several other Infections

Few conditions
associated with other
etiological agent
resemble aseptic
meningitis
Leptospirosis
( Serovars Canicola
icterohaemorrhagea
)
Fungi (
Cryptococcus
neoformans )
Amoeba
Naegleria,
Harmanella.

12
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
13
Tuberculosis Meningitis

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
/ mm
3
Majority are Lymphocytes
May form veil clot when
CSF is allowed to stand in a
undisturbed state.

14
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.

15
Cerebrospinal fluid
examination
16
Why specimen collection is
Important 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
17
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
18

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
19
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.
20
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.
21
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
22
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.
23
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.
24
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

25
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.
26
Laboratory Examination of
CSF

The specimens
should be examined
with naked eye
Look for Turbidity

Contamination with
Blood
Normal CSF appears
like water

27
Specimen Examination

CSF to be examined
for
Cell counts
Gram staining
Culturing
Estimation of
protein and
glucose

28
Cell counts in CSF
Microscopic
examination of
uncentrigured, well
mixed CSF is done in
slide counting
chamber.
Count the number of
Polymorphs

Lymphocytes
Erythrocytes
29
Normal cell counts
30

CSF normally contains 0- 5 leucocytes / mm
3
Mainly Lymphocytes
Newly born children contain up to 30/mm
3

Mainly polymorphs
In purulent Meningitis there are usually 100
300 leucocytes/mm3
In aseptic meningitis there are usually 10
500 leucocytes/mm
3
Mostly lymphocytes, though polymorphs may
predominate in the earliest stage of the illness.
In Tuberculosis meningitis there are usually
100 500 leucocytes/mm
3

Care in Counting the Cells

When counting the
cells, care must be
taken to identify the
RBC and rare
presence of
yeasts,
amoeba should
not be
mistaken for
leukocytes

31
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

32
Gram Staining of CSF

The CSF to be centrifuged to deposit the
cells and bacteria
The film made from the deposit to be
stained with Grams 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
33
Gram Staining technique
34
Gram Staining Procedure
35
Examination of Gram Stained
smear
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.

36
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
37
Culturing of CSF
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

38
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
39
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
40
Tests for Bacterial antigen
Detection
Co agglutination Tests
There are several
test kits available
commercially for
detection antigens
of
Meningococci
Pneumococci
H influenzae


41
Diagnosis of Viral Meningitis

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



42
Tuberculosis Meningitis -
Diagnosis

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
Jensens
Medium






43
AFB Staining Methods
Zeihl
Neelsens-
hot stain
Kinyouns-
cold stain

Modifications



44
Ziehl- Neelsen Procedure
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.

45
Ziehl- Neelsen Procedure
(continued)
46
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 Loefflers Methylene Blue stain (counter
stain). This stain adds blue color to non-acid fast
cells!! Leave Loefflers Blue stain on smear for 1
minute
11. Rinse slide. Blot dry.
12. Use oil immersion objective to view.

Ziehl-Neelsen
stain
4 5 6



7
1 2 3
47
How the Acid fast bacteria appear
48
Leptospiral Meningitis -
Diagnosis

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

49
Viruses - Meningitis

The following viruses
can cause Aseptic
meningitis
1 Echovirus
2 Coxsackie
3 Herpes virus
50
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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