CSF MICROBIOLOGICAL EXAMINATION – I

MICROBIOLOGICAL
EXAMINATION – I
Hussein A. Abid
Laboratory Medicine Specialist
Iraqi Medical Laboratory Association
Scientific Affairs and Cultural Relations Section
Scientific Affairs and Training
Second lecture
24/09/2018 (Monday)

CSF ANALYSIS
Aims of the test:
• Diagnosis of bacterial or fungal meningitis by
microscopic examination and culture with
identification and susceptibility test of the
isolated organism.
Cerebrospinal Fluid (CSF) Processing In Medical Laboratory
2

RECEPTION UNIT
• The technologists will handle the CSF specimens
completely by the following procedure of “SPECIMEN
HANDLING AND STORAGE”.
• Specimen containers and requisitions will be delivered to
the clinical microbiology laboratory immediately after
collection. Upon receipt, the staff will check requisitions for
completeness.
• Specimens will be stored properly (at room temperature)
until they are picked-up by the microbiology staff.
• The staff will assign numbers for the specimens and indicate
them on the original requisitions.
3

LABELING AND LOGBOOK
• Upon receiving the specimen and requisition with
complete data, record it in the microbiology log-
book in numeral order.
• The number assigned to the specimen is written
on the specimen container and the requisition
form, culture media containers, and culture media
plates. In addition, date and time of processing
should be written clearly on all culture plates,
tubes, slides or whatever used in the processing of
the specimen.
4

SPECIMEN REJECTION CRITERIA
In general, specimens for the microbiology lab are
unacceptable if any of the following conditions
apply:
1. The information on the label doesn’t match the
information on the request form.
2. The specimen was transported in an improper
container or at wrong temperature.
3. The quantity of the specimen is insufficient to carry
out all the required examination.
4. Leaking specimen.
5

SPECIMEN REJECTION CRITERIA
• Every effort should be made to contact the
physician or unit if a specimen is rejected.
• The physician will be informed about the
reason/s for specimen rejection.
6

POSSIBLE PATHOGENIC BACTERIA
Gram negative (–ve)Gram positive (+ve)
• Neisseria meningitidis
• Haemophilus influenzae (type B)
• Escherichia coli *
• Pseudomonas aeruginosa *
• Proteus spp.*
• Salmonella serovars
• Streptococcus pneumoniae
• Streptococcus agalactiae *
• Listeria monocytogenes *
Other bacteria
• Mycobacterium tuberculosis
• Treponema pallidum
7
NOTE:
1. (*) mainly isolated from neonates.
2. Bacteria may also be found in the CSF when there is a brain
abscess, e.g. Bacteroides species and other anaerobes.

OTHER MICROORGANISMS
FungiParasites
• Cryptococcus neoformans
(mainly in AIDS patients)
• Aspergillus spp. (less common)
• Trypanosoma spp.
• Naegleria fowleri
• Angiostrongylus cantonensis
(larvae, rarely)
• Dirofilaria immitis (CSF usually
contains eosinophils).
• Also Toxoplasma gondii (mainly
in AIDS patients).
Viruses
• Particularly Coxsackie viruses, Echovirus, and Arboviruses. Also,
Herpes Simplex 2 virus (HS2-V), Varicella-Zoster virus (VZ), and
Lymphocytic-Choriomeningitis virus (LCM).
• Rarely Polioviruses may be isolated from CSF.
8

NOTES ON PATHOGENS
• Inflammation of the meninges (membranes that cover the
brain and spinal cord) is called meningitis.
• Pathogens reach the meninges in the blood stream or
occasionally by spreading from nearby sites such as the
middle ear or nasal sinuses.
• Fever, headache, neck stiffness, and intolerance of light are
typical symptoms of acute bacterial meningitis.
• In children, vomiting, convulsions and lethargy are
common. A haemorrhagic rash is associated with
meningococcal meningitis.
9

NOTES ON PATHOGENS
Meningitis is described as:
 Pyogenic (purulent), when the CSF contains mainly
polymorphonuclear neutrophils (PMN, pus cells), as in acute
meningitis caused by N. meningitidis, H. influenzae, and S.
pneumoniae. Pus cells are also found in the CSF in acute
amoebic meningoencephalitis.
 Lymphocytic, when the CSF contains mainly lymphocytes, as
in meningitis caused by viruses, M. tuberculosis, and C.
neoformans. Lymphocytes are also found in the CSF in
trypanosomiasis meningoencephalitis, and neurosyphilis.
10

NOTES ON PATHOGENS
• In developing countries, meningitis epidemics are usually
caused by N. meningitidis serogroups A and C and only
occasionally by group B and other serogroups. Outbreaks are
common in sub-Saharan Africa (meningitis belt) with most
group A meningococcal meningitis epidemics occurring in the
hot dry season.
• In recent years meningococcal meningitis has risen to
epidemic proportions in some countries of South America, the
Middle East, and Asia. Rarely, epidemic meningitis is caused
by S. pneumoniae but endemic pneumococcal meningitis is
common and has a high fatality rate.
11

NOTES ON PATHOGENS
• In developing countries, neonatal meningitis is caused
mainly by S. pneumoniae (about one third of cases),
Salmonella serovars and other enteric bacteria, N.
meningitidis, and H. influenzae. Streptococcus agalactiae
(Group B strep.) is a rare cause.
• Haemophilus meningitis occurs mainly in infants and young
children below 5 years with a high incidence below 2 years.
• C. neoformans is mainly an opportunistic pathogen, causing
life-threatening meningo-encephalitis in those with AIDS
and other conditions associated with immunosuppression.
12

NOTES ON PATHOGENS
• In parts of sub-Saharan Africa and other areas of high HIV
prevalence, cryptococcosis has been reported in up to 30%
of AIDS patients.
• Syphilitic meningitis may occur in secondary syphilis but it is
usually a complication of late syphilis.
• N. fowleri causes primary amoebic meningoencephalitis, a
rare and usually fatal disease.
13

REVIEW
Specimen collection:
• Only well-trained physician should collect a sample.
Quantity of specimen:
• About 3 mL of CSF is sufficient for culture.
Time relapse before processing the sample:
• CSF is an emergency specimen and should be processed
immediately.
Storage:
• Room temperature conditions.
14

NOTES
In a hospital with a microbiology laboratory:
• IMPORTANT: Advise the laboratory before performing a
lumbar puncture so that staff are prepared to receive and
examine the specimen immediately.
• A delay in examining CSF reduces the chances of isolating
a pathogen. It will also result in a lower cell count due to
WBCs being lysed, and to a falsely low glucose value due to
glycolysis.
• When trypanosomes are present, they will be difficult to find
because they are rapidly lysed once the CSF has been
withdrawn.
15

NOTES
• Cerebrospinal fluid must be examined without
delay, and the results of tests reported to the
medical officer as soon as they become
available, especially a Gram smear report.
• The fluid should be handled with special care
because a lumbar puncture is required to
collect the specimen.
16

SAMPLE PROCESSING (1st day)
1. Appearance description: (color, appearance, clot..)
2. Staining: (Gram, Giemsa, India ink..)
3. Culturing procedure:
• As a general rule in CSF and body fluid specimens
for culture, centrifuge clear specimen and inoculate
plates and do staining from sediments. While turbid
specimens may not be centrifuged.
• CSF cultured on two blood agar plates (aerobically
and anaerobically), chocolate agar (CO2),
MacConkey agar, fluid thioglycollate.
17

FIRST DAY PROCESSING
1. Report the appearance of the CSF as soon
as the CSF reaches the laboratory. Report
whether the fluid:
 is clear, slightly turbid, cloudy or definitely
purulent (looking like pus)
 contains blood
 contains clots
 pH
18

• Normal CSF appears clear and colorless.
• Purulent or cloudy CSF: indicates presence of pus cells,
suggestive of acute pyogenic bacterial meningitis.
• Blood in CSF: this may be due to a traumatic (bloody)
lumbar puncture or less commonly to hemorrhage in the
central nervous system. When due to a traumatic lumbar
puncture, tube #1 will usually contain more blood than tube
#2.
• pH determination: pH can be measured by using pH paper
or using pH meter.
19

• Xanthrochromia: pink, orange, or yellow
discoloration due to RBC lysis, hemoglobin
breakdown, oxyhemoglobin, bilirubin, increased
protein, carotenoids or melanin.
• Clots in CSF: indicates a high protein
concentration with increased fibrinogen, as can
occur with pyogenic meningitis or when there is
spinal constriction.
20

21

Depending on the appearance of CSF, proceed
as follows:
A) Purulent or cloudy CSF: suspect pyogenic
meningitis and test the CSF as follows:
 Immediately make and examine a Gram stained
smear for bacteria and polymorphonuclear
neutrophils (pus cells). Issue the report without
delay.
 Culture the CSF
22

B) Slightly cloudy or clear CSF: test the CSF as follows;
• Perform a cell count and note whether there is an increase
in white cells and whether the cells are mainly pus cells or
lymphocytes.
• When cells predominantly pus cells (neutrophils):
 Examine a Gram stained smear for bacteria.
 Examine a wet preparation (sediment from centrifuged
CSF) for motile amoebae which could be Naegleria
(rare).
 Culture the CSF.
23

• When cells predominantly lymphocytes: this could indicate viral meningitis,
tuberculous meningitis, cryptococcal meningitis, trypanosomiasis encephalitis, or
other condition in which lymphocyte numbers in the CSF are increased. Perform
the following tests;
 Measure the concentration of protein or perform a Pandy’s test. The CSF
protein is raised in most forms of meningitis and meningoencephalitis.
 Measure the concentration of glucose. This is helpful in differentiating viral
meningitis in which the CSF glucose is usually normal from tuberculous
meningitis and other conditions in which the CSF glucose is reduced.
 Examine a wet preparation for encapsulated yeast cells that could be C.
neoformans.
 Examine a wet preparation for trypanosomes and a Giemsa stained smear for
morula (Mott) cells when late stage trypanosomiasis is suspected.
24

• Report the CSF as ‘Normal’: when it appears clear,
contains no more than 5 WBC × 10 6/L, and the protein
concentration is not raised (or Pandy’s test is
negative).
• NOTE: A CSF begins to appear turbid when it contains
about 200 WBC × 106 /L.
25

26

CSF MICROBIOLOGICAL EXAMINATION – I

Recommended

Recommended

More Related Content

What's hot

What's hot (20)

Similar to CSF MICROBIOLOGICAL EXAMINATION – I

Similar to CSF MICROBIOLOGICAL EXAMINATION – I (20)

More from Hussein Al-tameemi

More from Hussein Al-tameemi (20)

Recently uploaded

Recently uploaded (20)

CSF MICROBIOLOGICAL EXAMINATION – I