CENTRAL NERVOUS

SYSTEM INFECTIONS
Haemophilus influenzae & Neisseria meningitidis

Terence L. Eday, RMT, MT(ASCPi)

CENTRAL NERVOUS SYSTEM
ANATOMY
Coverings and Spaces of the
CNS

•skull – cranial bones that cover

and encases the brain
•
•meninges – membranous
covering of the brain and spinal
cord; collective term for three
distinct layers
ØDura mater (outermost
membrane layer)
ØArachnoid
ØPia mater (innermost
membrane layer)
ANATOMY
Cerebrospinal Fluid (CSF)
Øenvelops the brain and spinal cord
Øcushion
Øprovide buoyancy for the bulk of the brain
Øcaries essential metabolites into the neural tissue and
cleanses the tissues of wastes
Øprovides a means by which the brain monitors changes
in the internal environment
Ø found in the subarachnoid space and within cavities
and canals of the brain and spinal cord
Øit is produced by the choroid plexus, a specialized
secretory cells
Ø
Ø
ROUTES OF INFECTION
1. Hematogenous spread
Ømost common way that the CNS becomes infected
Ø
2. Direct spread from an infected site
Øthe extension of an infection close to or contiguous with the
CNS; e.g. Otitis media, sinusitis, and mastoiditis
Ø
3. Anatomic defects in CNS structures

4. Travel along nerves leading to the brain (direct

intraneural)
Øleast common route of CNS infection; caused by e.i. Rabies
virus and herpes simplex virus
DISEASES OF THE CNS
•Meningitis
Øinfection within the subarachnoid space or throughout
the leptomeninges. It is divided into:
•Purulent Meningitis
•Aseptic Meningitis

ØPurulent meningitis, Px has a marked, acute

inflammatory exudate with large numbers of PMNs
Ø
ØAseptic meningitis, characterized by an increase of
lymphocytosis and pleocytosis
DISEASES OF THE CNS
•Meningitis
Øcan either be acute or chronic

ØAcute meningitis, are characterized by fever, stiff neck,

headache, nausea and vomiting, neurologic abnormalities,
and change in mental status

ØChronic meningitis, often occurs in Px who are

immunocompromised, and experience some or all of the
ff: fever, headache, stiff neck, nausea and vomiting,
lethargy, confusion, and mental retardation
DISEASES OF THE CNS
•Encephalitis/Meningoencephalitis
ØEncephalitis is an inflammation of the brain parenchyma
and is usually result of viral infection

ØMeningoencephalitis is concomitant meningitis that

occurs with encephalitis and the cellular infiltrate is more
likely to be lymphocytic
Ø
•Brain Abscess
CAUSATIVE ORGANISMS
•Bacterial Causes
Meningitis in children and young adults

ØNeisseria meningitidis
ØHemophilus influenzae
ØStreptococcus pnuemoniae
ØStaphylococcus aureus
ØE. coli
ØKlebsiella spp.
ØCitrobacter spp.
ØEnterobacter spp.
ØSerratia spp.
ØListeria monocytogenes
CAUSATIVE ORGANISMS
•Bacterial Causes
Meningitis in neonates
ØE. coli
ØStreptococcus agalactiae (group B)
ØListeria monocytogenes
ØKlebsiella spp.

Meningitis following trauma or surgery

ØPseudomonas aeruginosa
ØSalmonella spp.
ØBacillus cereus
ØStaph. epidermidis
ØProteus mirabilis
ØAerobacter spp.
ØPeptostreptococcus spp.
ØBacteroides spp.
CAUSATIVE ORGANISMS
•Bacterial Causes
Acute nonpurulent meningitis
ØMycobacterium tuberculosis
ØLeptospira spp.
ØMycoplasma spp.
ØChlamydia spp.
ØTreponema pallidum
Ø
Rare causes of meningitis
ØStreptococci other than Group B Edwardsiella tarda
ØFlavobacterium meningosepticum Acinetobacter spp.
ØCampylobacter fetus Brucella spp.
ØPasteurella spp. Actinomyces and Nocardia
ØNeisseria gonorrhea
ØAeromonas shigelloides
ØSerratia spp.
CAUSATIVE ORGANISMS
•Viral Causes
1. Enteroviruses - ECHO viruses
- Coxsackie viruses
- Polio viruses
2. Paramyxoviruses - mumps virus, measles virus
3. Herpes viruses - Herpes simplex virus
- Varicella – Zoster virus
4. Adenoviruses
5. Arbovirus - Flavi virus, e.g. Japanese B Ence. Virus
- Bunya virus
- Arena virus
- Other arbo virus
CAUSATIVE ORGANISMS
•Fungal Causes
1. Cryptococcus neoformans
2. Candida albicans
3. Aspergillus spp.
4. Mucor spp.
5. Histoplasma capsulatum
6. Coccidoides immitis
7. Blastomyces dermatitidis

Parasitic Causes
1.E. histolytica 7. Schistosoma spp.
2.Naegleria spp. 8. Strongyloides stercoralis
3.Acanthamoeba spp.
4.Toxoplasma gondii
5.Cysticercus cellulosae
6.Paragonimus westermani
LABORATORY DIAGNOSIS
•Specimen Collection
CSF is collected by lumbar puncture (spinal
tap)
LABORATORY DIAGNOSIS
•Specimen Collection
•Three or four tubes of CSF should be collected and immediately
labeled with the patient’s name. Tubes 3 or 4 is used for cell
count and differential.
LABORATORY DIAGNOSIS
•Specimen Collection
•A minimum of 5 to 10 mL is recommended for analysis.
•
•CSF should be hand-delivered immediately to the lab.
SPECIMENS SHOULD NEVER BE REFRIGERATED.

•If not rapidly processed, CSF should be incubated (35°C)

or left at room temperature.
•
•CSF for viral studies, specimens may be refrigerated for
as long as 23 hours after collection or frozen at -70°C if
a longer delay is anticipated.
CSF FOR VIRAL STUDIES SHOULD NEVER BE
FROZEN AT TEMPERATURES ABOVE -70°C
LABORATORY DIAGNOSIS
•Initial Processing
Macroscopic inspection
Appearance should be noted and recorded as: clear, hazy,
turbid, purulent, yellow, or blood-tinged, with fibrin web or
pellicle

Microscopic examination
•CSF should be centrifuged in a sterile tube (preferably a 15-mL
conical tube with screw cap) at 10000g for 5-10 minutes
•Supernatant is removed to a sterile tube, leaving approx. 0.5
mLof fluid in which suspended the sediment. The
supernatant can be used to test for the presence of
antigens or for chemistry evaluations
LABORATORY DIAGNOSIS
Direct Microscopy
•Examine one drop of the sediment microscopically (x400),
between a slide and coverslip, for:
- leukocytes (PMNs or lymphocytes)
- erythrocytes
- bacteria
- yeasts

•If C. Neoformans is suspected, do india ink.

•In areas where African trypanosomiasis occurs, examine
carefully for actively motile, flagellated trypanosomes
•In some instances, carefully examine for Naegleria fowleri
trophozoite.
LABORATORY DIAGNOSIS
LABORATORY DIAGNOSIS
Gram Staining
•Causative agent of bacterial meningitis may often be
observed in a Gram-stained smear
•
•Air-dry the smear, fix with gentle heat, and stain it by
Gram’s method. Examine at x1000 (OI) for at least 10
minutes, or until bacteria are found
•
Acid-fast stain
•Examination of an acid-fast-stained preparation of the
sediment or of the fibrin web is indicated when
tuberculous meningitis is suspected by the physician
LABORATORY DIAGNOSIS
CULTURE
CULTURE
Preliminary identification
•Growth on MacConkey agar is suggestive of
Enterobacteriaceae

•Colonies of Gram (+) cocci with a narrow zone of β-

haemolysis may be S. agalactiaeand should be confirmed
with the reverse CAMP test

•Flat colonies with a concave center and a slight green zone

of α-haemolysis are probably S. pneumoniae and should be
confirmed by optochin inhibition test.
CULTURE
Preliminary identification
•Gram (-) diplococci growing on blood and chocolate agar,
and giving rapidly oxidase test, may be considered to be
meningococci

•Colonies grow only on chocolate agar, and as satellite

colonies around the staphylococcal streak, H. influenzae is
considered
•
•colonies of Gram (+) rods with a narrow zone of β-
haemolysis on BA may be L. monocytogenes and need for
further confirmatory tests
CULTURE
Genera and Species to be considered
Current Name Previous Name
Moraxella catarrhalis Branhamella catarrhalis,
Neisseria catarrhalis
Neisseria gonorrhea
Neisseria meningitidis
Other Neisseria spp.
N. cinerea
N. lactamica
N. polysaccharea
N. subflava N. subflava, N. flava,
N. perflava
N. sicca
N. mucosa
N. flavescens
CULTURE
Neisseria meningitidis
•leading cause of fatal bacterial meningitis
•Gram (-) diplococci
•Oxidase positive
•Catalase positive
•DNAse negative
•produce acid from maltose
•has five main Groups: A, B, C, W135 and Y
N. MENINGITIDIS
Media of choice
Ø5% sheep blood agar
Øchocolate agar
ØThayer-Martin medium
Ømodified Thayer-Martin (MTM) medium
ØMartin-Lewis (ML) medium
ØNew York City (NYC) medium
ØTrans-Grow Medium

JEMBEC SYSTEM (BD)

CHOCOLATE AGAR
N. MENINGITIDIS
MEDIA Enrichment Antimicrobials
Thayer-Martin Martin Supplement
IsoVitaleX Collistin
Medium
Modified Thayer- IsoVitaleX Nystatin
Collistin
(MTM) (ML)
Martin Lewis Medium IsoVitaleX Vancomycin
Nystatin
Collistin
Medium
New York City (NYC) Lysed Horse Vancomycin
Anisomycin
Collistin
Medium blood, horse Trimethoprim
Increased conc
Nystatin
plasma, yeast of Vancomnycin
Vancomycin
dialysate Trimethoprim
N. MENINGITIDIS
Incubation Conditions and Duration
•agar plates should incubated at 35°C to 37°C for 72 hours in a
CO2-enriched, humid atmosphere.

•grows best under conditions of increased CO2 (3% to 7%), this

can be achieved by a candle jar, CO2 generating pouch, or CO2
incubator
 N. MENINGITIDIS
COLONIAL APPEARANCE AND OTHER CHARATERISTICS ON
CHOCOLATE AGAR*

Organism Appearance
Moraxella Large, nonpigmented or gray, opaque,
catarrhalis smooth; friable “hockey puck”
consistency; colony may be moved
Neisseria intact over
Small, surface
grayish white,orconvex,
agar
gonorrheae translucent, shiny colonies with either
smooth or irregular margins; may be up
to five different colony types on
Neisseria Medium, smooth, round, moist, gray to
primary plates
meningitidis white; encapsulated strains are
mucoid; may be greenish cast in agar
underneath
*Appearance on blood agar is the same oncolonies
CA except for pigmentation; colonies
are less opaque on blood agar
N. MENINGITIDIS
BIOCHEMICAL AND PHYSIOLOGIC CHARACTERISTICS OF
M. catarrhalis and Coccoid Neisseria spp.

Organism Modified Nutrient Agar Blood or

Thayer-Martin at 35°C Chocolate
Agar at 25°C
M. catarrhalis V + +

N. + - -
gonorrheae
N. + - -
meningitidis
IDENTIFICATION
Oxidase Test (Kovac’s Method)
Principle
To determine the presence of bacterial cytochrome oxidase
using the oxidation of the substrate tetramethyl-p-
phenylenediamine dihydrochloride to indophenol, a dark
purple-colored end product.

Positive: indicated by the development of a dark purple color

(presence of oxidase)
Negative: No color development
N. MENINGITIDIS
BIOCHEMICAL AND PHYSIOLOGIC CHARACTERISTICS OF
M. Catarrhalis and Coccoid Neisseria spp.

Organism Glucose Maltose Lactose Nitrate Gas from 0.1% Nitrite

Nitrate Reduction
Reduction

M. catarrhalis Neg. Neg. Neg. Pos. Neg. v

N. gonorrhoeae Pos.. Neg. Neg. Neg. Neg. Neg.

N. meningitidis Pos. Pos. Neg. Neg. Neg. v
N. MENINGITIDIS
BIOCHEMICAL REACTION OF N. meningitidis to Cystine
TrypticaseSoy Agar (CTA)

Glucose

Sucrose
Lactose

Maltose
N. MENINGITIDIS
Treatment
•Penicillin G is the drug of choice
•Chloramphenicol or a third-generation cephalosphorin such
as cefotaxime or ceftriaxone is used in persons allergic to
penicillins
•
Epidemiology, Prevention, & Control
•5 to 30% of the normal population may harbor
meningococci in the nasopharynx during interepidemics
•Rifampin, 600 mg orally twice daily for 2 days
(or minocycline, 100 mg every 12 hours)
•chemoprophylaxis is no longer reliable, due to appearance
of sulfonamide-resistant meningococci
•Vaccines are available but only protection against Group C
CULTURE
Genera and Species to be considered
Current Name Previous Name
Haemophilus influenzae

Haemophilus Haemophilus aegypticus

influenzae biogroup
aegyptius

Haemophilus ducreyi

Other Haemophilus spp.

H. parainfluenzae
H. parahaemolyticus
H. aprophilus
H. haemolyticus
H. segnis
H. paraphrophilus
CULTURE
Haemophilus influenzae
•small, gram-negative, pleomorphic
bacteria, require hemin and nicotine
adenine dinucleotide (NAD) for in
vitro growth (except for H.
aphrophilus)
•found on the mucous
membranes of the URT in
humans
•it is an important cause of
meningitis in children
•in young cultures (6-8 hours) on
enriched medium have a definite
capsule
•Pfeiffer’s bacillus
H. INFLUENZAE TYPE B
Antigenic Structure
• encapsulated H. Influenzae contains capsular
polysacharides
•
•capsular antigen of type b is a polyribose-ribitol phosphate
(PRP), major virulence factor
•
•can be typed by slide agglutination, coagglutination with
staphylococci or agglutination of latex particles

•a capsule swelling test with specific antiserum is analogous

to the quellung test for pneumococci
•
 CULTURE
Gram Stain
Organism Morphology (Gram’s Stain)

H. influenzae Coccobacilli or small rods

H. influenzae biotype agyptius Long, slender rods

H. haemolyticus Small coccobacilli or short rods but

H. parainfluenzae
H. parahaemolyticus
H. aphrophilus and
H. paraphrophilus
H. segnis
H. ducreyi
occasionally are seen as filamentous
Small pleomorphic rods or long filamentous
forms
Short to medium-length bacilli
Very short bacilli but occasionally are seen
as filamentous forms
Pleomorphic rods
Either slender or coccobacillary;
Describe appearing as “schools of fish”
CULTURE
Isolates from CSF or respiratory tract specimens that
1.are gram-negative rods or gram-negative coccobacilli,
2.
3.grow on chocolate agar in CO2 but not blood agar or
satellite around other colonies on blood agar, and
4.
5.are porphyrin-negative and nonhemolyticon rabbit or
horse blood may be identified as H. influenzae
CULTURE
Media of Choice
•Chocolate agar – provides hemin (factor X) and NAD (V
factor)
•Most strains will not grow on 5% sheep blood agar
•Grows on 5% sheep blood agar as tiny colonies that
grows very close to S. aureus colonies (V factor);
satelliting phenomenon
•

•horse blood-bacitracin agar may be used for isolation;

this is to prevent overgrowth by mucoid P. aeruginosa
•does not grow on MacConkey agar
•will grow in broths such as thioglycollate and BHI
•rabbit or horse blood agars for detecting hemolysin-
producing strains that will not grow on 5% sheep blood
Organism Mediu Appearance
Haemophilus aphrophilus m
CA Round; convex with opaque zone near center
H. ducreyi Selective Small, flat, smooth, and translucent to opaque at
48-72 hrs; colonies can be pushed intact across
agar surface

H. haemolyticus CA Resembles H. Influenzae except beta-hemolytic

on rabbit or horse blood agar

H. influenzae CA Unencapsulated strains are small, smooth, and

translucent at 24 hrs; encapsulated strains larger,
more mucoid colonies; mouse nest odor;
nonhemolytic on rabbit or horse blood agar

H. Influenzae biotype CA Resembles H. influenzae except colonies are

aegypotius smaller at 48 hrs

H. parahaemolyticus CA Resembles H. parainfluenzae except beta-

hemolytic on rabbit or horse blood agar
H. parainfluenzae CA Medium to large, smooth, and translucent;
nonhemolytic on rabbit or horse blood agar
H. paraphrophilus CA Resembles H. aphrophilus
H. segnis CA Convex, grayish white, smooth or granular at 48
hrs
IDENTIFICATION

Satelliting phenomenon of H. influenzae around S.

aureus streak
 IDENTIFICATION
Hemophilus isolates may also be identified to species
using rapid sugar fermentation tests
Organism X Factor V Factor β-hemolytic on Lactose Mannose
RBA Fermentation Fermentation

H. influenzae*
+ + - - -
H. Influenzae
biotype aegyptius* + + - - -
H. ducreyi
+ - - - -

*Cannot be differentiated biochemically

IDENTIFICATION
X and V Factor Test

Principle
Members of the genus Haemophilus require accessory
growth factors in vitro. Some Haemophilus spp. require X
factor (hemin) alone, V factor (NAD, nicotinamide-adenine
dinucleotide) alone, or a combination of both

Positive: Growth around the XV disk only shows a

requirement for both factors. Growth around the V disk, no
growth around the X disk, and light growth around the XV
disk shows a V factor requirement.

Negative: Growth over entire surface of the agar indicates

no requirement for either X or V factor.
IDENTIFICATION
X and V Factor Test

Quality Control
Positive: Haemophilus influenzae
will show a halo of growth around
the XV disk; the rest of the agar
surface will show no growth.
Haemophilus parainfluenzae will
show a halo growth around the XV
and V disks.

Negative: Haemophilus aphrophilus

will grow over the entire surface of
the plate. Neither X, nor V, nor XV
factors are necessary for growth.
SEROLOGY
BD Directigen™
Meningitis Individual Tests
•Presumptive latex slide agglutination tests for the direct
qualitative detection of antigens to H. influenzae type b, S.
pneumoniae, N. meningitidis Group B and E. coli K1 in CSF
and serum.

•In addition, the test kit provides confirmation and

serogrouping capabilities from suspected colonies of H.
influenzaetype b, S. pneumoniae, Group B Streptococcus,
and N. meningitidis Group B
ANTIMICROBIAL THERAPY AND
SUSCEPTIBILITY TESTING
Organism Therapeutic Options Potential Resistance to Comments
Therapeutic Options
Hemophilus Usually ceftriaxone or Beta-lactamase- •Resistance to
influenzae cefotaxime; for localized mediated to third gen
infections several ampicillin is cephalosporin
cephalosporins, beta- common; beta- has not been
lactam/beta-lactamase inhibitor lactam resistance is documented
combinations, macrolides, rare (less than 1%) •Testing to guide

trimethoprim/sulfamethoxazole, & therapy is not

certain fluoroquinolones are routinely needed
effective
PREVENTION AND CONTROL
•can be prevented by administration of Haemophilus b
conjugate vaccine to children
•
•Rifampin chemoprophylaxis is recommended for all
household contacts of index cases of H. Influenzae type b
meningitis
REFERENCES
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Microbiology, 24th Edition. 2007. 21:302-304, 19:280-283.
3.Henry, J.B., Clinical Diagnosis and Management by Laboratory
Methods, 20th Edition. 2001. W.B. Saunders Company. 57:1258-
1259.
4.Forbes, B.A. et al., Bailey & Scott’s Diagnostic Microbiology,
12th Edition. 2007. Mosby Elsevier. 34:403-409, 42:447-453.
5.BACTERIOLOGY. Subcourse MD0856, 200 Edition. U.S. Army
Medical Department Center and School. Fort Sam Houston,
Texas.
6.Material Safety Data Sheet – Infectious Substances. Public
Health Agency of Canada. April 2001.
7.Bacterial Meningitis Fact Sheet. www.meningitis-trust.org.uk.
2001
8.Meningococcal, HIB and Pneumococcal Meningitis Fact Sheet.
www.meningitis-trust.org.uk. 2001.
9.Basic Laboratory Procedures in Clinical Bacteriology. 2nd
Edition. 2003. Cerebrospinal Fluid. p 25-29.
THANK YOU!
AND
GOD BLESS!