Laboratory diagnosis of meningitis

 

Preface

Meningitis is an inflammatory condition of the meninges (membrane surrounding brain and spinal cord) with an infectious aetiology.

            A typical case of meningitis usually presents with headache, high grade fever, projectile vomiting, nuchal rigidity and positive Kernig's & Brudzinski’s sign. Altered mental status, irritability are also common and early presentations in some cases.

Learning objectives:

1. Different types of meningitis and their etiologies.

2. Laboratory methods for analyses of CSF to evaluate a case of meningitis

            - Cytology

            - Chemical analysis

            - Bacteriological analyses by staining and culture

            - Antigen and antibody detection

3. CSF findings in different types of meningitis

Types of meningitis:         

            Meningitisis categorized into three different types depending upon their aetio-pathogenesis andthe cytology of the Cerebro Spinal Fluid (CSF).

1. Acute pyogenic meningitis:It is an acute inflammation of the meninges caused mainly by bacteria. The CSF will typically have large number of pus cells, predominantly Polymorphs.

2. Tuberculous meningitis: It is caused by Mycobacterium tuberculosis. It is one of the most common causes of meningitis in developing countries. The CSF cytology shows presence of large number of pus cells with predominant lymphocytes.

3. Aseptic meningitis: It is caused by viruses, fungi, parasites &some bacteria. The CSF cytology shows absent or few  pus cells with lymphocytic or mixed cellularity.

 

Causative agents of meningitis

A) Acute pyogenic meningitis

Bacteria causing meningitis vary according to the age groups

1. Newborn (neonatal meningitis):

            Group B beta haemolytic Streptococci (Streptococcus agalactiae): Usually acquired from vagina      while passage of baby through the birth canal.

Escherichia coli, Klebsiellapneumoniae, other coliform bacilli: Usually the source of bacteriais the digestive tract. 

            Pseudomonas aeruginosa, Listeria monocytogenes etc.

2. Children:

            Haemophilusinfluenzae type b (Hib): Common in young children (2 months to 5 years of age)

            Neisseria meningitidis (Meningococcus)

            Streptococcus pneumoniae (Pneumococcus)

3. Adults:

            Neisseria meningitidis (Meningococcus)

            Streptococcus pneumoniae (Pneumococcus)

            Listeria monocytogenes (less common)

4. Hospital acquired or iatrogenic meningitis:

            Various hospital strains i.e. Pseudomonas aeruginosa, Staphylococcus spp. may be introduced during invasive procedures like lumbar puncture, surgery or V-P shunts and may lead to meningitis.

 

B)Tuberculous meningitis: It is generally secondary to tuberculous lesions elsewhere in the body. It is caused by Mycobacterium tuberculosis.

 

C) Aseptic meningitis: It is associated with increased lymphocytes &pleocytosis in CSF with negative bacterial culture.

1. Viruses: Herpes simplex viruses (type I more common), Japanese encephalitis virus, Enteroviruses(i.e. Echo, Coxsackie, Polio) etc.

2. Fungi: Cryptococcus neoformans

3. Parasites:Toxoplasma gondii, Acanthamoeba, Naegleria (may cause pyogenic meningitis)

4. Bacteria:T.pallidum, Leptospira

5.Meningitis may be  one of the presentations of certain non-infectious diseases i.e. cancers, connective tissue disorders, sarcoidosis etc.

 

Laboratory diagnosis

Sample collection:

1.      The main specimen is CSF, which is analyzed to confirm clinical diagnosis, to find the type of meningitis and to evaluate aetiology.

2.      CSF is collected by lumbar puncture with strict aseptic precautions to prevent iatrogenic infection as wel as contamination of the specimen.

3.      Sample should be transported immediately to the laboratory& if possible hand delivered.

4.      If delay in transport of specimen is unavoidable, it should be kept at 37° C  or at room temperature and should never be kept in refrigerator. If CSF is to be processed only for viral studies, it can be refrigerated.

5.      Blood sample may be taken for culture examination. The bacteria causing pyogenic meningitis can usually be isolated from blood culture.

            To increase the isolation rate, nowadays, it is recommended that the CSF and blood should directly be collected separately in the bottles of automated culture system (Bactec or BacTAlert ) at bedside and transported immediately to the laboratory.

Laboratory examination of CSF:

CSF may be processed for,

1. Cytological study to confirm the diagnosis of meningitis and to evaluate its type and aetiology:

Total cell number up to 1-3 / cmm is considered normal in CSF. This will increase in case of meningitis.

a.     Acute pyogenic meningitis: Total cells rise up to 1000-10000/     

cmm; Predominant cells are neutrophils.                          

b.    Tuberculosis meningitis: Total cells rise upto 50-500/cmm;  

Predominant cells are lypmhocytes.

c.     Viral meningitis: Total cells rise upto 10-100/cmm; predominant

cells are lymphocytes.

            2. Biochemical tests

            In case of meningitis, total protein will increase and sugar will diminish than normal level.

            3. Microscopic examination for identification of microorganism

                        CSF is centrifuged and deposits are examined by following methods  

                     to identify the aetiological agent

                        a. Gram stain: to identify pyogenic bacteria

                        b. Z N stain: to detect M.tuberculosis

                        c. Negative stain(India ink or nigrosin): to detect Cryptococcus       

                        neoformans

d.    Wet film: to detect parasites like Naegleriafowlerii,

Acanthamoeba, Toxoplasma gondii

 

            4. Culture examination for isolation of pathogenic organism

            There are three ways to culture the CSF sample

1.    A set of culture plates including blood agar, chocolate agar and mac conkey agar are inoculated directly from sample and incubated at 37° C  in candle jar (or  CO₂incubator).
2.    CSF is centrifuged with precautions to avoid contamination and the deposit is inoculated in a similar set of culture plates.
3.   1 - 2 ml of CSF is inoculated in a liquid medium ( i.e. Glucose broth ) or in automated culture system bottle. This will allow growth of even minute number of bacteria if present. When the growth is indicated or observed in liquid medium, it is sub-cultured in similar set of plates to isolate them.

             The culture plates are observed for growth after 24-48 hours of incubation. The isolated bacteria are processed for their identification and antibiotic sensitivity testing.

 

            5. Detection of antigens or antibodies

             This is generally done from the supernatant of the centrifuged CSF sample.

            Detection of antigen:

a.     Capsular polysaccharide of aetiological agent may be detected by latex agglutination.

The following capsulated organisms may be detected by this method,

                        - N.meningitidis(Meningococci),

                        - Haemophilusinfluenzae,

                        - Streptococcus pneumoniae(pneumococci)

                        - Cryptococcus neoformans

                        b. Agglutination test may be useful to detect the surface antigens of

                        - Group B Streptococci or E.coli which are responsible for meningitis  

                       in neonates.

                       

            Detection of antibody:

Antibodies are demonstrable in the following infections, which can be detected by ELISA or immunofluroscence

                        - Herpes simplex virus

                        - Japanese encephalitis virus

                        - T. pallidum( neurosyphilis )

 

 

Exercise:

An 8 month child presented with fever, neck rigidity and projectile vomiting and suspected for meningitis.

Q.1     What are the microscopic findings in case of different kind of meningitis? Which stains are used to examine the smear prepared from centrifuge of CSF?

Q.2     Which culture media are used for plating?

Q.3     Which are the common agents suspected in this case?