2. CNS infections
 Meningitis,
 Encephalitis,
 Parameningeal abscesses (subdural empyema and
epidural abscess),
 Brain abscesses,
&
 CSF shunt infections.

3. Meningitis
• is an inflammatory response to bacterial infection of the
pia-arachnoid and CSF of the subarachnoid space

4. Epidemiology
 Incidence is between 3-5 per 100,000
 More than 2,000 deaths annually in the U.S.

5. • Bacterial meningitis and other CNS infections are
considered infectious disease emergencies that can cause
significant patient morbidity and mortality.

6. Mortality/Morbidity
 Bacterial meningitis -uniformly fatal before the antimicrobial
era.
 overall mortality rate has decreased, but remains alarmingly
high - - Higher in developing countries
 Varies with the specific etiologic agent




S pneumoniae 19-26%
H influenzae - 3-6%
N meningitidis 3-13%,
L monocytogenes 15-29%

7. `
 Survivors end up with complications
 Children suffers mostly with complications

8. Morbidity associated with
complications
in children
In adults
sensorineural hearing loss / Cranial nerve
palsies
brain infarction,,
epilepsy
hydrocephalus,
diffuse brain swelling
hydrocephalus,
cerebral vein thrombosis,
cerebral palsy
More with H.influenzae meningitis
**** Severe morbidity is associated with H.influenzae meningitis and TB
meningitis due to fibrinous exudates

9. Viral meningitis
 Viral meningitis (without encephalitis) is less than 1%.
 In patients with deficient humoral immunity (eg,
agammaglobulinemia), enterovirus meningitis may
have a fatal outcome.

10. Meningococcal
Meningitis belt -
Faso, Chad, Ethiopia and Niger; in
2002, the outbreaks occurring in
Burkina Faso, Ethiopia and Niger
accounted for about 65% of cases

11. Pathogenesis of Meningitis
Survival and
Multiplication in
the subarachnoid
space
Nasopharyngeal
colonization
Neisseria meningitides (meningococcus)
and nasopharyngeal colonization with S
pneumoniae (pneumococcus).
Crossing of the
BBB and entry
into the CSF
Nasopharyngeal
epithelial cell
invasion
Bacteremia with
intravascular
survival
Bloodstream
invasion

12. Pathogenesis of Meningitis
eg, Naegleria fowleri,
Free living
amoeba in natural
resovoires
Meningitis
Nasopharyngeal
epithelial cell
invasion
Retrograde flow
to meninges
through the
olfactory bulb

13. Pathogenesis of Post traumatic/Neurosutgery Meningitis
Colonizing bacteria in
sinuses/auditory canal otitis
media, congenital
malformations, trauma, direct
inoculation during intracranial
manipulation
Spread along the
CSF fistulous
tract
Meningitis

14. Pathogenesis cont….
 With in the CNS, the infectious agents likely survive as
immunoglobulins, neutrophils, complement components
absent or activity limited
 replication of infectious agents remain uncontrolled triggers
the cascade of meningeal inflammation
 Increased CSF concentrations of TNF-alpha, IL-1, IL-6, and
IL-8 are characteristic findings in patients with bacterial
meningitis

15.  Treatment using rapidly bactericidal agents may
transiently worsen the patients condition due to rapid
release of pyrogenic substances in to CSF
 Increase of proinflammatory mediators

16. Specific Pathogens

17. Age group
Predominant Pathogen
Age 0-4 weeks
S agalactiae (group B streptococci)
E coli K1
L monocytogenes
Age 4-12 weeks
S agalactiae
E coli
H influenzae
S pneumoniae
N meningitidis
Age 3 months to 18
years
N meningitidis (worldwide epidemic strains A,B,C W135)
S .pneumoniae
H influenzae
Age 18-50 years
S pneumoniae
N meningitidis
H influenzae
Age older than 50 years
S pneumoniae
N meningitidis
L monocytogenes
Aerobic gram-negative bacilli
Immunocompromised
state
S pneumoniae
N meningitidis
L monocytogenes
Aerobic gram-negative bacilli

18. Intracranial manipulation, including
neurosurgery
Staphylococcus aureus
Coagulase-negative staphylococci
Aerobic gram-negative bacilli,
including
Pseudomonas aeruginosa
Basilar skull fracture
S pneumoniae
H influenzae
Group A streptococci
CSF shunts
Coagulase-negative staphylococci
S aureus
Aerobic gram-negative bacilli
***Direct extension from the throat or nasal or ear colonization an give rise to post
traumatic meningitis

19. Other causes
 Bacteraemic infectionof Salmonella, Brucella and





Staphylococcus aureus can cuase meningitis
Gram Negative meningitis in overwhelming infections
due to Strogyloides / Hyper infection due to
Strongyloides stercorhalis
Leptospira and Treponema
Protozoa – Acanthomoeba and Naeglaria fowleri
Fungi – Histoplasma and
Nematodes – Angyostrogilus cantonensis

20.  Clinical diagnosis unreliable- symptoms unreliable – specially extremes
of age
 The efficacy of treatment (CNS) infections -depends on the accuracy of
the etiologic diagnosis.
 requires the best specimen at the appropriate time,
 transporting it to the laboratory under optimum conditions,
 processing the specimen efficiently and timely manner,
 and selecting the tests necessary to identify the spectrum of possible
etiologies

21. Clinical sign
Kernig's sign
sensitivity, 5%; likelihood ratio for a positive test result
[LR(+)], 0.97)
Brudzinski's sign
(sensitivity, 5%; LR(+), 0.97),
Nuchal rigidity
(sensitivity, 30%; LR(+), 0.94)
Degree of meningeal
inflamation
≥ 6 up to 100
Clinical signs are unreliable
Inbetween
Unreliable
(>/=1000 WBCs/mL of CSF
Nuchal rigidity shows diagnostic value- sensitivity
100% and negative predictive value 100%

22. Diagnosis
 Should not be delayed
 Inform laboratory
 Initial report based on Cell count and Direct smear
 Cytospin method gives more positive yield than
traditional overlaying
 Gram stain can be considered as the gold standard

23. Diagnosis
 Is established by investigation of CSF obtained from




lumbar puncture,
Cysternal puncture or ventricular puncture or
fontanelle taps possible – not done routinely
Exclude raised intracranial pressure before performing
the procedure due to possibility of herniation
Place of CT/ MRI to exclude SOL
When, contraindication +, diagnosis established using
other means – Blood culture, WBC/DC, CRP together
with symptoms

24. Additional factors for success
 Communication between the clinician and laboratory-
about clinical notes, Patient condition, antibiotic
therapy, Patient delay and Doctor delay
 Seasonal prevalence of infectious diseases, for
enteroviruses and arboviruses,
 the epidemiology of emerging diseases such as West
Nile virus, and the immune status of the patient can
beis helpful.

25. Specimen collection and
transportation –timing
 All specimens should be collected prior to the
initiation of antimicrobials
 If therapy initiated – action to nullify it-Innoculating it
to broth media 1:5 ratio< specially for cerebral abcess

26. Specimens – for diagnosis of CNS
infections
Disease
Specimen
Meningitis
Cerebrospi • Mininmum of 1 mL
nal fluid /culture
(CSF)
• 1 to 2 mL [PCR]),
• 1 mL antibody test
Blood
Encephalit
is or brain
abscess
Quantity
5 to 10ml as for blood
culture
Note
For M. tuberculosis, and
dimorphic and filamentous fungi
require repeat CSF or large
volumes (10 to 20 mL) of
ventricular CSF.
0.5 to 1.0 mL per culture
request preferred
1 to 10 mL can be added to blood
culture medium ↓antimicrobial
effect- dilution of 1 :5 or 1:10
Tissue
Subdural
empyema
or epidural
Abscess
material or
irrigation
fluid
0.5 cc preferred
minced /gently ground. Mince
only if filamentous fungi
expected.
Abscess
material /
irrigation
0.5–1.0 mL per culture
request preferred
Small volumes of pus diluted
(ratio of 1:2) with sterile saline to
allow “washing” of material from

27. Collection and transportation
Specimen
Container
Transport/Storage Conditions
Cerebrospinal
fluid (CSF)
Sterile tube.
Room temperature. Ice/Refrigeration are
detrimental to some bacteria and
anaerobes. For PCR 4 °Cfor <24 hr or
freezing (−20°C) for longer periods.
Pus, irrigation
fluid, and
fluid aspirates
Sterile container. Anaerobic
culture request requires
transport in oxygen-free
container.
Room temperature for short periods (<1
hr). Refrigeration for longer times. Do
not refrigerate if anaerobic culture is
ordered.
Tissue,
debridement
material
Sterile container. Keep small
specimen portions moist with
sterile saline solution.
As for pus above. Small pieces of tissue
can be placed on sterile moist gauze to
facilitate location/identification by
laboratory personnel. Portion for PCR as
for CSF above.
Abscess
material,
fluid, and
washes
aspirated
Under anerobic conditions in
to Nitrogen or CO₂ ,
Large volumes-To a syringe
itself- short time
Or pre reduced aneerobic
Transport as soon as possible

28. Collection of CSF
 Cerebrospinal fluid collected by lumbar puncture is the
routine specimen for diagnosis of meningitis
 Strict aseptic techniques
 Three or four containers – depending on tests requested
 But should have separate tubes for Gram stain/culture,
Biochemistry and glucose level- accompanied by blood
sample for RBS
 Never to keep it in refrigerator

29. Which tube for microbiology?
 Any tube possible
 First tube- Theoretically risk of contamination -
epithelium or blood from skin and soft tissue
capillaries ruptured during the punctur
 In practice, total volume of fluid is more important
than the “tube” cultured.

30. CSF Examinations
 Macroscopy – color,clotting etc
 Complete count
 Differential count
 Gram stain of direct smear
 Culture
 Biochemistry – sugar difference and proteins
 PCR when indicated

31. CSF Macroscopy

32. Color of CSF supernatant
Conditions or causes
Purulent
Pyogenic meningitis
Yellow
Blood breakdown products
Hyperbilirubinemia
CSF protein >=150 mg per dL (1.5 g per L)
>100,000 red blood cells per mm3
Orange
Blood breakdown products
High carotenoid ingestion
Pink
Blood breakdown products
Green
Hyperbilirubinemia
Purulent CSF
Brown
Meningeal melanomatosis

33. Normal CSF values
Cell component
Age Category
Normal Value
Leukocytes
Neonates
0 – 30 cells X 10 ⁶ / L
1 to 4 yr old
0 – 20 X 10⁶ / L
5 to puberty
0 – 10 X 10⁶ / L
Newborn
0 – 675 X 10⁶ / L
Adults
0 – 10 X 10⁶ / L
Neonates
0.7 g/l
Adults
0.2 – 0.4 g/l
Erythrocytes
Protein
Glucose
> 60% of RBS value is considered
normal
•Bacterial or viral counts should be considered where leukocyte counts are
near the upper normal value
•5 WBCs per mm3 (normal value)

34. differential diagnosis of various
forms of meningitis
Diagnosis
Pressure
Cells
(10⁶ / l)
PMN
Glucose
ratio
Protein
(g/ l)
Lactate
(mmol/l)
normal
< 20 cm
1-2
<1
> .5
< 0.45 (15–
45mg/dl)
<2
Acute
pyogenic
>20 cm
>1000
> 50%
< .4
(>.2)
> 1(100 mg) > 4
Chronic
variable
> 1000
Vary
< .4
> 0.45
>2
Aseptic
(Viral)
< 20 cm
< 1000
<50
> .4
Vary
<2

35. 87% of Patients with meningitis
≥ 1000 /mm³ WBCs
99% of Patients with meningitis
≥100 per mm3
More likely to have viral meningitis
≤ 100 per mm3
 As CSF is hypotonic, WBCs lyse with the time.
 Process, immediately

36. Lymphocytes : PMN
 CSF, PMN:L ratio is unreliable for diagnosis of
meningitis
 Viral meningitis may show lymphocytosis – but
initially PMN predominates
 Neutropaenics – no or less PMN response

37. Presence of RBC’s
 Indicates intra cerebral ,SAH or traumatic tap
 Presence of RBC’s make interpretation of CSF analysis




difficult
But, rarely obscures it
Inspecting first and third lumbar puncture samples –
if RBC count different - Traumatic tap
WBC:RBC ratio of 1:500 to 1:1000 is considered normal
CSF obtain > 12 hrs post ICH may have WBC counts up
to 500 X 10⁶ /l - due to inflammation

38. Direct smear – Gram stain

39.  a Gram stain of the cytospin CSF has a sensitivity of
90% if the LP is carried out before the administration
of antibiotics.

40. Effects of antibiotics in CSF culture
and Direct smears
 In a retrospective review of 128 children with bacterial
meningitis, Kanegaye et al. (2001)
 compared 39 patients who received empiric
antimicrobial therapy before LP with 55 who
underwent LP before receiving antimicrobial therapy
Treatment Group - Bacterial
sterilization
Treatment group
Meningococcus – sterilization occurred
within 2 hrs
Up to 24 to 48 hrs CSF cellular and
biocheical parameters remained unchained
Pneumococcus – sterilization occurred
within 4 hours

41. Condition
Diagnostic test
Bacterial meningitis
Cytospin Gram stain 60–90
100
Culture
90
100
Antigen detection
assays *
50–100
100
Acid-fast stain
10–22
100
38–88
100
27–85
95–100
Tuberculous meningitis
Culture
PCR
Sensitivity (%)
Specificity
(%)
Effect of antibiotic
treatment on

42. Blood cultures
• 50 to 80% patients with meningitis has accompanied
bacteremias - blood cultures would be useful to isolation
of organisms – more than CSF growth
• Specially where LP is contraindicated
Blood cultures
Volume – 20ml or as
recommended by the
manufactures
•Collected before antibiotic therapy
•> 2 cultures taken from different sites or three cultures with
in 24 hrs
•Innoculate into broth medium at a ratio of >1:5
When suspecting – Dimorphic fungi or cryptococcus blod
should be colected to tube containing lysis solution – for lysis
centrifugation

43. Utility of Gram stain for diagnosis of Pyogenic
meningitis
Etiology
Sensitivity
All common etiologies—no previous antibiotics
75% to 90%
All common etiologies—antimicrobial therapy
prior to lumbar puncture
40% to 60%
Streptococcus pneumoniae –without antibiotics
90%
Neisseria meningitidis -
75%
Haemophilus influenzae
86%
Listeria monocytogenes
<50%

44.  Unfortunately the positivity rate of gram staining and
cultures remain low between 25- 40% as against the
rate of 80-85% from the developed world

45. Partially treated meningitis
 As the early symptoms and signs - non-specific, up to 50% receive
oral antibiotics.
 This delay the presentation to hospital &
CSF findings altered; - Gram stain and growth of organism may be
negative –
 Antibiotics rarely interfere with CSF protein/glucose and
molecular diagnosis (PCR).
 In partially treated meningitis – request for PCR and bacterial
antigens - not affected by prior antibiotic administration.

46. Tuberculuos meningitis
 AFB positive only in 3%
 Cobweb formation is seen 2/3 cases
 Ratioo of albumin to globulin changes can be used as
sxcrrening method(Nl ratio 6:1)Abnormal in TBM
changes can be predicted with eletrophoresis
(Modified Levinson’s test

47. Use of Bacterial Antigen Testing
 The use of rapid bacterial antigen detection in CSF and other body
fluids has come under question.
 Rarely does a positive result alter therapy, and test performance is
similar to that of the Gram's stain.
 Two contemporary approaches are advocated for bacterial antigen
testing.
 The first recommends testing only those specimens with abnormal CSF
parameters (cell count, protein, glucose).[35] This approach results in a
68% reduction in the number of antigen tests performed.
 Although positive CSF cultures occur when white blood cell count,
glucose, and protein values are within normal ranges, this is unusual
and does not justify testing all CSF for bacterial antigen.
 Another approach eliminates antigen testing, except in a few limited,
specific cases, such as prior antimicrobial therapy when culture results
are negative after 24 to 48 hours of incubation.

48.  latex particle agglutination tests , have similar
sensitivities to Gram stain or culture
 of doubtful benefit when used routinely,
 but sometimes identify organisms in patients with
partially treated bacterial meningitis and negative
Gram stain and culture.
 Cultures for bacteria and fungi should always be
performed, even in patients already treated with
antibiotics.

49. 
Use of Culture

50. Culture media
Incubation
For routinely encountering pathogens
Good quality Blood A,
chocolate Agar either
sheep or HBA -
24 to 48 hrs in 35% CO₂
Facultative anaerobes
Broth media
Anaerobes from cerebral abscesses
thioglycollate or chopped Extended
meat broth,
incubation- only
when requested
Yeast and fungi
Use of lysis
centrifugation method
Only when
requested
********Culturing technique and media hardly ever changed over the years

51. Emerging issues

52.  Methods of rapid diagnosis
 Emergence of antibiotic resistant Pathogens

53. PCR
 Broad range of PCRs – N.meningitidis,
H.influenzae,Streptococcus pneumoniae
 PCR of blood Buffy coat provide higher yield for N.
meningitidis
 Agents of Aseptic meningitis –Rapid RealTime PCR
for entero viruses available – results in 60 min

54. Antibiotic resistance
 worldwide increase in infection with penicillin and




cephalosporin resistant strains of S pneumoniae,
caused by either alteration in the penicillin binding
proteins (Mosaic PBP)
Incidence increasing Europe, South Africa, Asia, and the
United States.
American Academy of Pediatrics recommended
combination therapy, initially with vancomycin and either
cefotaxime or ceftriaxone for all children 1 month of age or
older with definite or probable bacterial meningitis.
N. meningitis less susceptible strains