Mahen Kothalawala

CNS infections
Meningitis,
Encephalitis, Parameningeal abscesses (subdural empyema and

epidural abscess), Brain abscesses, & CSF shunt infections.

Meningitis
is an inflammatory response to bacterial infection of the

pia-arachnoid and CSF of the subarachnoid space

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

 Bacterial meningitis and other CNS infections are

considered infectious disease emergencies that can cause significant patient morbidity and mortality.

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%

`
Survivors end up with complications
Children suffers mostly with complications

Morbidity associated with complications

in children sensorineural hearing loss / Cranial nerve palsies epilepsy diffuse brain swelling In adults brain infarction,, hydrocephalus,

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

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.

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

Pathogenesis of Meningitis

Survival and Multiplication in the subarachnoid space

Crossing of the BBB and entry into the CSF

Bacteremia with intravascular survival

Nasopharyngeal colonization
Neisseria meningitides (meningococcus) and nasopharyngeal colonization with S pneumoniae (pneumococcus).

Nasopharyngeal epithelial cell invasion

Bloodstream invasion

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

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

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

 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

Specific Pathogens

Age group Age 0-4 weeks

Predominant Pathogen S agalactiae (group B streptococci) E coli K1 L monocytogenes S agalactiae E coli H influenzae S pneumoniae N meningitidis N meningitidis (worldwide epidemic strains A,B,C W135) S .pneumoniae H influenzae S pneumoniae N meningitidis H influenzae S pneumoniae N meningitidis L monocytogenes Aerobic gram-negative bacilli S pneumoniae N meningitidis L monocytogenes Aerobic gram-negative bacilli

Age 4-12 weeks

Age 3 months to 18 years Age 18-50 years

Age older than 50 years

Immunocompromised state

Intracranial manipulation, including neurosurgery

Staphylococcus aureus Coagulase-negative staphylococci Aerobic gram-negative bacilli, including Pseudomonas aeruginosa S pneumoniae H influenzae Group A streptococci Coagulase-negative staphylococci S aureus Aerobic gram-negative bacilli

Basilar skull fracture

CSF shunts

***Direct extension from the throat or nasal or ear colonization an give rise to post traumatic meningitis

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

 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

Clinical sign Kernig's sign Brudzinski's sign Nuchal rigidity sensitivity, 5%; likelihood ratio for a positive test result [LR(+)], 0.97) (sensitivity, 5%; LR(+), 0.97), (sensitivity, 30%; LR(+), 0.94)

Degree of meningeal inflamation 6 up to 100 Inbetween (>/=1000 WBCs/mL of CSF Clinical signs are unreliable Unreliable Nuchal rigidity shows diagnostic value- sensitivity 100% and negative predictive value 100%

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

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

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.

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

Specimens for diagnosis of CNS infections

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

0.5 cc preferred

Subdural empyema or epidural

Abscess material / irrigation

0.51.0 mL per culture request preferred

Collection and transportation

Specimen Cerebrospinal fluid (CSF) Container Sterile tube. Transport/Storage Conditions Room temperature. Ice/Refrigeration are detrimental to some bacteria and anaerobes. For PCR 4 Cfor <24 hr or freezing (20C) for longer periods. Room temperature for short periods (<1 hr). Refrigeration for longer times. Do not refrigerate if anaerobic culture is ordered.

Pus, irrigation fluid, and fluid aspirates

Sterile container. Anaerobic culture request requires transport in oxygen-free container.

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. Transport as soon as possible

Abscess material, fluid, and washes aspirated

Under anerobic conditions in to Nitrogen or CO , Large volumes-To a syringe itself- short time Or pre reduced aneerobic

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

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.

CSF Examinations
Macroscopy color,clotting etc
Complete count Differential count

Gram stain of direct smear

Culture Biochemistry sugar difference and proteins PCR when indicated

CSF Macroscopy

Color of CSF supernatant Purulent Yellow

Conditions or causes Pyogenic meningitis Blood breakdown products Hyperbilirubinemia CSF protein >=150 mg per dL (1.5 g per L) >100,000 red blood cells per mm3 Blood breakdown products High carotenoid ingestion Blood breakdown products Hyperbilirubinemia Purulent CSF Meningeal melanomatosis

Orange Pink Green Brown

Normal CSF values

Cell component Leukocytes Age Category Neonates 1 to 4 yr old 5 to puberty Erythrocytes Newborn Adults Protein Glucose Neonates Adults Normal Value 0 30 cells X 10 / L 0 20 X 10 / L 0 10 X 10 / L 0 675 X 10 / L 0 10 X 10 / L 0.7 g/l 0.2 0.4 g/l > 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)

differential diagnosis of various forms of meningitis

Diagnosis normal Acute pyogenic Chronic Aseptic (Viral) Pressure < 20 cm >20 cm variable < 20 cm Cells (10 / l) 1-2 >1000 > 1000 < 1000 PMN <1 > 50% Vary <50 Glucose ratio > .5 < .4 (>.2) < .4 > .4 Protein (g/ l) < 0.45 (15 45mg/dl) Lactate (mmol/l) <2

> 1(100 mg) > 4 > 0.45 Vary >2 <2

87% of Patients with meningitis 99% of Patients with meningitis More likely to have viral meningitis

1000 /mm WBCs 100 per mm3 100 per mm3

As CSF is hypotonic, WBCs lyse with the time.

Process, immediately

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

Presence of RBCs
Indicates intra cerebral ,SAH or traumatic tap
Presence of RBCs 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 10 /l - due to inflammation

Direct smear Gram stain

 a Gram stain of the cytospin CSF has a sensitivity of

90% if the LP is carried out before the administration of antibiotics.

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

Pneumococcus sterilization occurred within 4 hours

Up to 24 to 48 hrs CSF cellular and biocheical parameters remained unchained

Condition Bacterial meningitis

Diagnostic test

Sensitivity (%)

Specificity (%) 100 100

Cytospin Gram stain 6090 Culture 90

Antigen detection assays *

Tuberculous meningitis Acid-fast stain Culture PCR

50100
1022 3888 2785

100
100 100 95100

Effect of antibiotic treatment on cerebrospinal fluid. Am J Clin Pathol 1983; 80:386-387.

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

Utility of Gram stain for diagnosis of Pyogenic meningitis

Etiology All common etiologiesno previous antibiotics All common etiologiesantimicrobial therapy prior to lumbar puncture Streptococcus pneumoniae without antibiotics Neisseria meningitidis Haemophilus influenzae Listeria monocytogenes Sensitivity 75% to 90% 40% to 60% 90% 75% 86% <50%

 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

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.

Tuberculuos meningitis
AFB positive only in 3%
Cobweb formation is seen 2/3 cases Ratio of albumin to globulin changes can be used as

screening method(Nl ratio 6:1)Abnormal in TBM changes can be predicted with eletrophoresis (Modified Levinsons test

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.

 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.

Use of Culture

Culture media For routinely encountering pathogens Good quality Blood A, chocolate Agar either sheep or HBA Broth media

Incubation 24 to 48 hrs in 35% CO

Facultative anaerobes Anaerobes from cerebral abscesses

thioglycollate or chopped Extended meat broth, incubation- only when requested Use of lysis centrifugation method Only when requested

Yeast and fungi

********Culturing technique and media hardly ever changed over the years

Emerging issues

 Methods of rapid diagnosis

Emergence of antibiotic resistant Pathogens

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

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