LABORATORY DIAGNOSIS OF MENINGITIS

Definition

Meningitis• Inflammation of the subarachnoid space or

throughout the leptomeninges

Types of Meningitis

• Purulent meningitis • Acute• Chronic

• Aseptic meningitis-no growth of bacteria in CSF culture

• Acute Meningitis-Onset of meningeal symptoms over the course of hours to several days

• Chronic Meningitis-Insidious onset

Signs of meningoencephalitis,symptoms for atleast 4 weeks

Etiology:PURULENT MENINGITIS:

Hemophilus influenzaeStreptococcus pneumoniaeNeisseria meningitidisGroup B StreptococcusListeria monocytogenesEscherichia coliKlebsiella speciesPseudomonas speciesStaphylococcus aureusS. epidermidis

• Naegleria fowleri (Primary amoebic meninigoencephalitis)• Acanthamoeba species

Etiology contd…….

• ASEPTIC MENINGITIS:• Viruses:– Enteroviruses (Echovirus, poliovirus, coxackieviruses)– Mumps– Herpes simplex– Varicella-zoster– Measles– Adenovirus– Arbovirus

Bacteria:Leptospira interrogans serovars

icterohemorrhagiae & canicolaTreponema pallidumMycobacterium tuberculosis

Fungi:Cryptococcus neoformans

Bacteria causing purulent meningitis related to age

• Premature newborns:– Escherichia coli, Klebsiella spp, Enterobacter spp, Proteus

spp,Listeria monocytogenes• Infants:– S. agalactiae, L. monocytogenes, H. influenzae, S.

pneumoniae• Children:– N. meningitidis, S. pneumoniae, H. influenzae

• Adolescents:– N. meningitidis

• Adults:– S.pneumoniae

• Old age- L.monocytogenes

Laboratory Diagnosis

Specimen collection & transport

CSF collected by inserting sterile wide-bore needle into subarachnoid space between L4 & L5 lumbar vertebrae & CSF allowed to drip into dry sterile container.

2 tubes of CSF should be collected ( 1 for culture, 2nd for other investigations)

Volume of CSF collected critical for detection of certain microorganisms (eg: mycobacteria, Fungi).

Minimum of 5-10ml recommended for collection. Processing too little specimen lowers sensitivity.

Precautions to be taken:

• CSF sample to be delivered immediately to laboratory

• CSF sample never to be refrigerated• If not rapidly processed, CSF to be incubated

at 350C or at room temperature.

• Exception: CSF sample for viral studies may be refrigerated for as long as 23 hours or frozen at -700C if longer delay anticipated.

Guidelines for interpretation of results following hematologic & chemical analysis of CSF

Clinical setting

Leukocytes/mm3

Predominant cell type

Protein Glucose

Normal 0-5 None 15-50 mg/dl 45-100 mg/dl

Viral infection 2-2000 (mean of 80)

Mononuclear Slightly elevated (50-100 mg/dl) or normal

Normal

Purulent infection

5-20,000 ( mean of 800)

PMN Elevated (>100 mg/dl)

Low (<45 mg/dl)

Tuberculosis & fungi

5-2000 ( mean of 100)

Mononuclear Elevated (>50 mg/dl)

Normal or low

Microbiological investigations:

• Microscopy• Culture• Antigen detection• Molecular methods• Miscellaneous tests

MicroscopyStained smear of sediment:

Gram stain (must on all CSF sediments)Acridine orange fluorochrome stain ( for faster

examination)Wet preparation:

To detect motile amoebaPhase contrast microscopy or light microscopy

Acid fast stain:When tubercular meningitis suspectedAFB difficult to detect in CSF

• India ink preparation:– To detect cryptococcal

meningitis– Presence of capsulated

budding yeast cells

Culture:Usually done for all CSF samples received.

CSF sample must be cultured as soon as possible.

If delay anticipated, CSF kept at 35-370 C.

Inoculate specimen on chocolate agar, blood agar & incubate in 5% to 10% CO2at 35-370 C upto 72 hours.

Inoculate specimen on MacConkey agar, incubate aerobically at 35-370 C overnight.

Antimicrobial sensitivity tests to be done for positive cultures.

Culture contd….

• For CSF fungal cultures, inoculate CSF sediment onto Sabouraud dextrose agar & incubate aerobically at 350 C for 4 weeks.

• Suspected TB meningitis-LJ medium Liquid media Automated culture systems

• Naegleria or Acanthamoeba speciesBacterial overlay on non-nutrient agar used for detection

Antigen testing:

• Bacterial antigen testing (BAT):– For rapid detection of antigen from CSF.– Useful in previously treated patients & gram stain

negative CSF specimens with abnormal parameters.– Latex agglutination kits to detect bacterial antigen

directly from CSF– Less than 100% sensitivity & specificity.

• Latex agglutination test for cryptococcal antigen. (CALAS-Cryptococcal Latex Agglutination System)

Bacterial antigen testing

Molecular methods:

Polymerase chain reaction for:Mycobacterium tuberculosisHerpes simplex virusEnteroviruses(RT-PCR)

Miscellaneous tests:Adenosine deaminase assayCSF lactate determinations

Points to Remember• Inflammation of meninges• Purulent/Aseptic ; Acute/Chronic• Bacteria,Viruses,Fungi,Parasites• CSF sample collection-Never refrigerate• Biochemical & cytological parameters• Staining of CSF-Gram staining• Culture• Antigen detection• PCR