Laboratory Diagnosis of

Meningitis
Dr.T.V.Rao MD
 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.
Why Diagnosing Meningitis is
Important

Diagnosing Meningitis is top priority in clinical

Medicine, in particular Bacterial meingitis, can
be a life threatening condition , the need for
appreciate antibiotic therapy at the earliest is a
priority.
Minimal Diagnostic faculties if done with
precision can reduce morbidity and mortality
 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.
 Basic Understanding on
Meningitis

 On a broad basis Meningitis is classified

as
1 Purulent Meingitis

2 Aseptic Meingitis
 What is Purulent Meningitis

The CSF appears typically turbid due

to the presence of Leucocytes 100 to
several thousands / mm3 most of which
are Polymorphonuclear leucocytes
 Major Aetiological agents of
Meningitis

1 Meningococcus
 2 Pneumococcus
 3 Haemophilus influenzae

On majority of the occasions the

pathogens pass from Respiratory tract via
blood stream and infect Meningtis
Can occur at any age
 Neonates and Infants
Meningitis

 Thereis 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

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

 When venous shunts are

implanted for therapeutic
purposes
Staphylococcus
epidermidis
or some other Saprophytic
bacteria which rarely
cause infection in normal
people can infect
meningis
 Aseptic Meingitis

 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
 Aetiological agents of Aseptic
Meningitis
 Enteroviruses
ECHO viruses
Coxsackie virus
Polio virus
Mumps virus
moderately infective
Herpes simplex
Varicella zoster
Measles –
Adenovirus
Arboviruses
 CSF resembles - Aseptic
Meningitis
 Few conditions
associated with other
etiological agent
resemble aseptic
meingitis
Leptospirosis
( Serovars Canicola
icterohaemorrhagea )
Fungi ( Cryptococcus
neofroms )
Amoeba – Naegleria,
Harmanella.
 Confusing CSF appereance
 When early treatment
is given in Bacterial
meingitis the Clinico
pathological
apperance appears
as Viral meingitis
 In viral Encephalitis
modereate Lympoyte
exduate is found as it
in Viral meingitis
 Tuberculosis Meningitis
 On many occasions
Tuberculosis present as
Aseptic meingitis, results
from Pulmonary or mesentric
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.
 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.
 Procedure to collect CSF
 The trained physician will
collect only 3-5 ml into a
labelled 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
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.
 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 Physcian 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.
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 leukocyte
 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.influenzae.
 If delay is anticipated
leave at Room
Temperature.
 Blood Culturing
A simultaneous blood
culture should be
collected in all
suspected cases of
Meningitis before the
Antibiotics are started
 Many cases of
Bacterial ( Pyogenic )
meningitis are
associated with
Bacteriaemia
Septic spots in Meningtis

 Patients having
Meiningococcal Meningtis
present with several
septic spots.
 The spots should be
scraped and fluid
examined for
demonostration of Gram -
ve cocci, on several
occaions bacteria are not
seen in CSF
 Laboratory Examination of CSF

 Thespecimens should be examined with

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

 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
Normal cell counts
 CSF normally contains 0- 5
leucocytes / mm3
Mainly Lymphocytes
Newly born children contain
upto 30/mm3
Mainly polymorphs
In purulent Meningtis there
are usually 100 – 300
leucocytes/mm3
In aseptic meningitis there are
usually 10 – 500
leucocytes/mm3
Mostly lymphocytes, though
polymorphs may predominate
in the earliest stage of the
illness.
In Tuberculosis meingitis
there are usually 100 – 500
leucocytes/mm3
 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
 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
leishman or Carol thionine and
examined under oil immersion
to asses the relative number of
two types of leucocytes

Dr.T.V.Rao MD
 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
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
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.
 Observe for the Presence of
One should be familiar with the
following bacteria for
successful reporting
Meningococci
Pneumococci
Haemophili
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
 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
Bacteriods 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
Pencillin, and Choramphenicol
 The antibiotic sensitivity pattern
can be reported at the earliest

Dr.T.V.Rao MD
Dealing with Growth in Robertson's
Cooked medium
 If the turbidity devlops in
RCM the broth should be
filmed and subcultured on
to Blood agar and
Chocolate agar plates
and incubated both
aerobically and
aerobically.

Dr.T.V.Rao MD
 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 upto 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
 Tests for Bacterial antigen
Detection
Co agglutination Tests
There are several test kits
avialble commercially for
detection antigens of
Meningococci
Pneumococci
H influenzae

Dr.T.V.Rao MD
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

Dr.T.V.Rao MD
 Viruses - Meningitis

 Thefollowing viruses
can cause Aseptic
meningitis
1 Echovirus
2 Coxsackie
3 Herpes virus
 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
 Other tests which support
diagnosis are Reduction of
Glucose, Protein is increased

Dr.T.V.Rao MD
Leptospiral Meningtis -
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
Antibiotic Selection in Meningitis

 In the past Benzyl Pencillin or Choramphenicol

are given in majority of cases suspected with
meningitis.
 In the recent past several new generation of
Cephalosporins are replacing past theraputic
ideas.
 It is important to isolate and identify the
causative agent.
 However it is essential to start a theraputic trail
with best clinical guess before the laboratory
results are available.
Problems in Devloping World
 It is much difficult to deal with
precision in Devloping
countries as many several
infrastructural facilities are not
available and Physicians have
work their best of the previous
clinical experiences.
 The wisdom of treating
Physicians still save several
lives in the third world
countries.

Dr.T.V.Rao. MD
Cr ea ted for be nef it of
Medica l and
par am edi cal Students
in De vlopi ng Wor ld
Dr.T.V.Rao MD
Email
doctortvrao@gmail.com