26.

Laboratory diagnosis of Meningitis

Competency
MI 5.3 Identify the microbial agents causing meningitis

Specific Learning Objectives

At the end of this practical, the students will be able to:

 Enlist pathogens causing infections in Central Nervous System.
 Enlist the appropriate sample to be collected in case of CNS infection
 Describe collection, transport and storage of CSF.
 Identify the microbial agents causing meningitis based upon the macroscopic and
microscopic findings of CSF.
 Choose the appropriate laboratory investigations to be performed for diagnosis of CNS
infections.
 Interpret the results of the laboratory tests performed in diagnosis of the CNS infections.
Exercise 26.1:
Clinical Case: A febrile drowsy and irritable child

A 12-year-old girl was admitted to the hospital with complaints of high grade fever, headache,
vomiting, altered mental status, seizure. On examination there was neck rigidity and signs of
meningeal irritation were present. Biochemical and cytological analysis of CSF sample revealed
glucose level (40mg/dl), CSF pressure (100mm H2O), protein (60mg/dl) and cell count (25µl),
which is predominantly lymphocytic. No organisms were detected in Gram staining and India
ink staining of CSF.

1. What is the most likely clinical diagnosis in this child? Give reasons
History of high grade fever, headache, vomiting, altered mental status, seizure
On examination there was neck rigidity and signs of meningeal irritation were
present.
Lab investigation revealed Biological and cytological analysis of CSF sample
is suggestive of viral meningitis.
Biochemical analysis: Normal glucose level, mildly elevated protein.
Cytological analysis: Mildly increased cell count which is predominantly
lymphocytic. No organism detected in Gram staining and India ink of CSF.
Based on this a presumptive diagnosis of aseptic meningitis can be made

2. Suggest investigations to be carried out to confirm the diagnosis

Serological tests: ELISA and Immunochromatographic card test can be used.
Molecular methods: Multiplex PCR and Multiplex real time PCR can be used
for simultaneous detection of viral meningitis

3. Keeping the age of patient, enlist probable pathogens in the order of priority.
1. Ent e rov i r us es ( m os t c om m o n )
2. He r pe s s i m p l ex
3. Ar bo vi rus es

4. Describe the precautions to be taken while collection and transportation of CSF.

CSF collection: Lumbar puncture under strict aseptic conditions. It is divided into three sterile
containers; one each for cell count, biochemical analysis and bacteriological examination.

CSF transport: Should be examined immediately

When the bacteriological examination (culture) is required - CSF should never be refrigerated
- if a delay is expected - kept in an incubator at 37°C.
For molecular diagnosis - CSF can be kept inside the freezer.

5. Describe microbiological investigations for diagnosis of meningitis.

STAINING METHODS:
Gram Stain: After centrifugation or heaped smear
 Ziehl-Neelsen staining
 India ink preparation - for detection of capsule of Cryptococcus neoformans
 Wet mount preparation - Trophozoite of parasites such as Naegleria
 Bacteriological culture: Enriched media like chocolate agar and blood agar
  Brain Heart Infusion (BHI) broth: CSF is enriched
 Blood culture
 Biochemical tests
 Antimicrobial susceptibility test
 Fungal culture: It is carried out by inoculating the CSF on SDA (Sabouraud’s
dextrose agar) or BHI agar
 Viral culture: Can done by inoculating the CSF onto appropriate cell lines
 Serological Test
 Antigen and Antibody Detection
- Antibody detection in serum may be useful for the diagnosis of underlying viral
etiology
- For example - detection of serum antibody against herpes simplex virus
 Bacterial Endotoxin Detection - Limulus lysate assay
 Molecular Methods
- Tubercular meningitis - PCR (Mycobacterium tuberculosis specific genes - IS
6110 gene)
Exercise 26.2:
Clinical Case: An AIDS patient with fever, headache and stiff neck

A 45-year-old HIV infected male presented to the emergency department with on

and off fever and severe headache for 1month duration. On examination, there was
neck rigidity and signs of meningeal irritation. His cerebrospinal fluid sample was
collected by lumbar puncture and sent for microbiological investigation, such as
CSF smear (India Ink preparation) and culture on SDA. India ink staining showed
clear refractile capsules surrounding spherical budding yeast cells and creamy
mucoid colonies on SDA.

1. What is the most probable diagnosis, Justify your answer?

History of HIV infected adult patient with intermittent fever and chronic headache for 1
month gives clue that the patient is retro positive with immunocompromised status.
On examination, there was neck rigidity and signs of meningeal irritation.
Lab investigations India ink staining showed clear refractile capsules surrounding
spherical budding yeast cells and creamy mucoid colonies on SDA.

2. Draw a labeled diagram of this organism as seen in Indian ink preparation

3. Describe other microbiological investigations that can be done to diagnose this

disease.

 Negative staining: Modified India ink stain and nigrosin stain - demonstrate the
capsule
 Gram staining - gram-positive round budding yeast cells
 Other stains:
- Mucicarmine stain: It stains the carminophilic cell wall of C. neoformans
- Masson-Fontana stain: It demonstrates the production of melanin
 - Alcian blue stain to demonstrate the capsule.
 Capsular Antigen detection: from CSF or serum by latex agglutination test
 SDA without antibiotics, blood agar or chocolate agar and incubated at 37°C
 Blood inoculated in biphasic blood culture bottles
 Colonies - mucoid creamy white and yeast like
 Confirmation of Cryptococcus species :
- Niger seed agar and bird seed agar
- Growth at 37°C
- Urease test is positive
- Assimilation of inositol and nitrate
- Mouse pathogenicity test
-
4. Name the media that will be used for isolation of the organism.
 SDA without antibiotics, blood agar or chocolate agar and incubated at 37°C
 Niger seed agar and bird seed agar
Exercise 26.3:
Clinical Case: A case of fever, headache and stiff neck

A 7 year old girl was admitted to the hospital with complaints of high grade fever, altered
mental status and neck rigidity. On examination, it was found that there was inability to
straighten the leg when the hip is flexed to 900. CSF sample was collected by lumbar
puncture in a sterile container and sent to the laboratory for culture. CSF on Gram stain
reveals plenty of pus cells and short gram negative pleomorphic bacilli.

1. Name the possible pathogen

History is suggestive of meningitis and gram negative pleomorphic bacilli in
Gram stain suggests that the probable organism is Haemophilus influenzae.

2. Name the media that will be used for isolation of the organism.
 Blood agar with S. aureus streak line: satellitism
 Chocolate agar: grows well
 Fildes agar and Levinthal’s agar – Transparent media - iridescent colonies
 Haemophilus selective medium: contains bacitracin & sucrose

3. Enlist the types of infection seen in CNS with their key etiological agents
and mention the differences between CSF findings in various types of
CNS infections

Characteristics Normal individual Pyogenic Tuberculous Viral meningitis

meningitis meningitis
CSF pressure (mm of Normal (50–150) Highly elevated Moderately Slightly elevated/normal
water) (>180) elevated
Total leukocyte 0–5 100–10,000 10–500 25–500
count (per mm3)
Predominant cell Lymphocytes Neutrophils Lymphocytes Lymphocytes
type
Glucose (mg%) 40–70 <40 mg/dL 20–40 mg/dL Normal
(decreased to (slightly
absent) decreased)
Total proteins (mg%) 15–45 >45 mg/dL (usually 100–500 20–80 mg/dL (normal
>250; markedly mg/dL or slightly elevated)
increased) (moderate to
markedly
increased)
 4. From another patient with similar complaints CSF culture yielded growth. A heat-
fixed smear prepared from the colonies grown from the specimen is provided.
Perform the Gram stain, focus under the microscope, record your observations
and interpret the results

Observations Pink coloured rod shaped and oval shaped structures are
seen
Inference Gram negative coccobacillary forms morphologically
resembling Haemophilus influenzae are seen
Example
27. Laboratory diagnosis of Encephalitis

Competency
MI5.2: Describe the etiopathogenesis, clinical course and discuss the laboratory diagnosis of
encephalitis
Specific Learning Objectives

At the end of this practical, the students will be able to:

 Enlist pathogens causing encephalitis
 Enlist the appropriate sample to be collected in case of encephalitis
 Choose the appropriate laboratory investigations to be performed for diagnosis of
encephalitis

Exercise27.1:
Clinical case: A case of fever and seizure

A 25-year-old man is bitten by a street dog, which was barking excessively and very agitated in
behaviour. Four days later, the dog was found dead. Brain biopsy of the dog was done and was sent
for histopathological staining which showed intracytoplasmic eosinophilic inclusion bodies. The
electron microscopy of causative agent is bullet shaped.

1) Suggest investigations that need to be done to confirm the probable

 History of dog bite (without provocation) with abnormal behaviour and dog death within 4 days.
 Dog brain biopsy: showed intracytoplasmic eosinophilic inclusion bodies ( negri bodies)
 Electron microscopy: Bullet shaped rabies virus
 Direct immunofluorescence test (direct-IF); also called as direct fluorescent antibody (DFA) test
 Immunohistochemistry
 Antibody detection
 Reverse transcription-polymerase chain reaction (RT-PCR) can be used to amplify genes
of rabies virus RNA from fixed or unfixed brain tissue.

2) How do you differentiate a case of encephalitis from meningitis?

Meningitis Encephalitis
Definition Inflammation of the thin membranes that surround Inflammation of the
the brain and spinal cord called the meninges brain parenchyma.
(mostly of the pia mater and arachnoid mater)
Causative Agent Bacteria, Viruses and Fungi Mostly viruses
 Microorganisms The most common are echovirus, poliovirus, and Viruses (herpes
coxsackie. virus, rabies virus,
Bacterial meningitis develops under the action arboreal viruses,
of Streptococcus, Neisseria, Haemophilus, Listeria, cytomegalovirus,
and other bacteria. etc.);
Bacteria – often
occurring as
meningoencephalitis
caused by
meningococci,
pneumococci,
Hemophilus,
tuberculous bacteria,
etc.)
Fungi or parasites
such as leptospirosis,
toxoplasmosis,
trichinellosis, etc.

Symptoms Headache is most common, along with vomiting or Headache, joint

nausea, skin rash or discoloration of skin, high pain, irritability,
fever, stiff neck, confusion, double vision. fever, lethargy,
seizures, behavioral
changes.
Diagnosis Physical examination, Clinical presentation
Followed by CBC with differential, C-reactive and supported by
protein, and blood for Gram stain and culture. spinal fluid analysis
and neuroimaging
abnormalities, CSF,
PCR.

3) Enlist the infective causes of encephalitis and their supportive laboratory findings.

Acute viral encephalitis

Herpesviruses
 Herpes simplex virus (HSV-1>HSV-2): The most common cause of sporadic encephalitis
 Cytomegalovirus (in immunocompromised host)
 Human herpesvirus 6
 Varicella-zoster virus
 Epstein-Barr virus
Arboviruses: Important ones in India are:
 Japanese encephalitis virus (the most common cause of epidemic encephalitis in India)
 West Nile virus (the most common cause of epidemic encephalitis in USA)
Rabies virus: Causes encephalitis secondary to dog bite

Nipah and Hendra viruses

Rare causes: Enteroviruses and mumps virus
26. Laboratory diagnosis of Meningitis
Suggested Teaching-Learning Methods
 Small Group Discussion: Using suitable Case Scenario or laboratory reports of patients
diagnosed with specific CNS infection (Eg Pyogenic meningitis in a child, an AIDS
patient with chronic headache)
 Demonstration of microscopic slides of CNS pathogens like
 Meningococci, Pneumococci, H.Influenzae , Toxoplasma gondii, Trypanosoma
 India ink preparation of Cryptococcus neoformans

Exercise 26.1: Aseptic Meningitis

Clinical Case: A febrile drowsy and irritable child
Key points for case scenario: boy, two days history of fever, lethargy, irritability and poor
feeding, febrile and drowsy, no neck stiffness

Exercise 26.2: Cryptococcal meningitis

Clinical Case: An AIDS patient with fever, headache and stiff neck
Key points for case scenario: Adult, headache, fever, vomiting and stiff neck, CSF: low
glucose, high protein and PMNs <300/mm3, Indian ink- capsulated, budding yeast cells

Exercise 26.3: Pyogenic meningitis

Clinical Case: A case of fever, headache and stiff neck
Key points for case scenario: Child, high grade fever, vomiting, altered consciousness and
neck stiffness, CSF- plenty of pus cells and short gram negative pleomorphic bacilli

Exercise27.1: Encephalitis
Clinical case: A case of fever and seizure
Key points for Case scenario: Child, seizures, drowsy, fever, headache and irritable, neck
stiffness absent, rashes, probable clinical diagnosis of encephalitis