DIAGNOSIS OF MENINGITISSKILLS FOR RESIDENTS

Dr.T.V.Rao MD

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Why Skill Based Learning for Residents2

� The Indian Medical Curriculum is in for Rapid changes

for making the MBBS Doctors competent to perform

several life saving procedures learned to the greater

perfection. This programme is created that young

residents to learn the life saving diagnosis of

Meningitis by doing a Lumbar puncture and simple

observation in emergency hours with interactive

observation.

� Dr.T.V.Rao MD

What is Meningitis

� Meningitis is an infection of the coverings around the brain and spinal cord.

� The infection occurs most often in children, teens, and young adults. Also at risk are older adults and people who have long-term health problems, such as a weakened immune system.

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Why Diagnosing Meningitis is Important

Diagnosing Meningitis is top priority in clinical Medicine, in particular Bacterial meningitis, can be

a life threatening condition , the need for appreciate antibiotic therapy at the earliest is a

priority.

Even with Minimal Diagnostic faculties if done with

precision can reduce morbidity and mortality

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On suspicion of Meningitis

Every patient suspected of having Meningitis should have a specimen of CSF examination in the laboratory to establish the infection and to rule out

infection.

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Basic Understanding on

Meningitis

� On a broad basis

Meningitis is

classified as

1 Purulent

Meningitis

2 Aseptic

Meningitis

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What is Purulent Meningitis

The CSF appears typically turbid due to the presence of Leucocytes 100 to several thousands / mm3

most of which are

Polymorph nuclear

leucocytes

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Major Etiological agents of

Meningitis

� 1 Meningococcus

� 2 Pneumococcus

� 3 Haemophilus influenza

On majority of the occasions the pathogens pass from Respiratory tract via blood stream and infect Meningitis

Can occur at any age

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Neonates and Infants

Meningitis

� There is specific affinity of some pathogens infecting Neonates and Infants

1 Coli forms

2 ß hemolytic streptococci

3 Pseudomonas

4 Salmonella and Listeria Monocytogenes

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Iatrogenic Meningitis

� Carelessly performed Lumbar puncture

� Accidental wound infection in neurosurgical wounds

Pyogenic Staphylococcus

Streptococci

Coli form bacilli

Anaerobic cocci

Bacteriods

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Aseptic Meningitis

� In these conditions CSF is clear or only slightly turbid contain moderate number of leucocytes 10 – 500 / mm3

Majority of cells are lymphocytes, except in early stages. majority are caused by viruses

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Etiological agents of Aseptic Meningitis

� Enteroviruses

ECHO viruses

Coxsackie virus

Polio virus

Mumps virus moderately infective

Herpes simplex

Varicella zoster

Measles –Adenovirus

Arboviruses

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CSF resembles - Aseptic Meningitis in

Several other Infections

� Few conditions associated with other etiological agent resemble aseptic meningitisLeptospirosis

( Serovars Canicola icterohaemorrhagea ) Fungi ( Cryptococcus neoformans ) Amoeba – Naegleria, Harmanella.

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Confusing CSF appearance

� When early treatment is given in Bacterial meningitis the Clinico pathological appearance appears as Viral meningitis

� In viral Encephalitis moderate Lymphocyte exduate is found as it in Viral meningitis

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Tuberculosis Meningitis

� On many occasions Tuberculosis present as Aseptic meningitis, results from Pulmonary or mesenteric tuberculosis

� Can be associated with Miliary tuberculosis.

� Cell counts on CSF will reveal 100 – 500 leucocytes / mm3

� Majority are Lymphocytes� May form veil clot when CSF is

allowed to stand in a undisturbed state.

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WHY MICROBIOLOGICAL DIAGNOSIS IS

LIFE SAVINGInformation derived from the results has impact on :

�Diagnosis of infectious diseases

�Antibiotic prescribing

�Formulation of local antibiotic policy

�Public health impact eg Meningococcal infection.

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Cerebrospinal fluid

examination

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Why specimen collection is Important

in Microbiology

Specimen collection in Microbiology to isolate and identify the causative agents forms back bone of the investigative procedures.

In developing world, lack of awareness and casual attitude among junior staff hampers the definitive diagnosis.

Specific procedures in collecting specimens will certainly improve the quality of services of Microbiology Departments

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Some tips better Diagnosis

� Laboratory investigation should start as early as possible

� Specimens obtained early, preferably prior to antimicrobial treatment likely to yield the infective pathogen

� Before doing anything, explain the procedure to patient and relatives

� When collecting the specimen, avoid contamination

� Take a sufficient quantity of material

� Follow the appropriate precautions for safety19

An Ideal Request formAn Ideal Request formAn Ideal Request formAn Ideal Request form

� Name xxxx Age Sex

� IP/ OP No xyz Time Date

� Ward xx123 Urgent / Routine

� Nature of specimen CSF � Investigation needed xxxx

� xxxx

Doctor/StaffContact No 1234567

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Why Proper written RequestWhy Proper written RequestWhy Proper written RequestWhy Proper written Request

� Your request is a legal document.

� Identifies all the outcome of test.

� No interchange of results.

� Short forms are dangerous

� Signature of the Doctor / Nurse is essential in legible form, can help to contact in case of results which can save a patient.

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Specimen collection for

CSF Examination

� Lumbar puncture to collect the CSF for examination to be collected by Physician trained in procedure with aseptic precautions to prevent introduction of Infection.

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Procedure to collect CSF

� The trained physician will collect only 3-5 ml into a labeled sterile container

� Removal of large volume of CSF lead to headache,

� The fluid to be collected at the rate of 4-5 drops per second.

� If sudden removal of fluid is allowed may draw down cerebellum into the Foramen magnum and compress the Medulla of the Brain

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CSF needs a New and Sterile container

� Fresh sterile screw capped container to be used.

� Reused containers, not to be used, contamination from the previous specimens misrepresent the present specimen.

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Lumbar puncture for CSF collection

� The best site for puncture is inter space between 3 and 4 lumbar vertebrae

( Corresponds to highest point of iliac crest )

The Physician should wear sterile gloves and conduct the procedure with sterile precautions, The site of procedure should be disinfected and sterile occlusive dressing applied to the puncture site after the procedure.

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Transportation to Laboratory

� The collected specimen of CSF to be dispatched promptly to Laboratory , delay may cause death of delicate pathogens, eg Meningococci and disintegrate leukocytes

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Preservation of CSF

� It is important when there is delay in transportation of specimens to Laboratory do not keep in Refrigerator, which tends to kill H. Influenza

� If delay is anticipated leave at Room Temperature.

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Laboratory Examination of CSF

� The specimens should be examined with naked eye

Look for Turbidity

Contamination with Blood

Normal CSF appears like water

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Specimen Examination

� CSF to be examined for

Cell counts

Gram staining

Culturing

Estimation of protein and glucose

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Cell counts in CSF

� Microscopic examination of uncentrigured, well mixed CSF is done in slide counting chamber.

� Count the number of

Polymorphs

Lymphocytes

Erythrocytes

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Normal cell counts31

� CSF normally contains 0- 5 leucocytes / mm3

Mainly LymphocytesNewly born children contain up to 30/mm3

Mainly polymorphsIn purulent Meningitis there are usually 100 – 300

leucocytes/mm3In aseptic meningitis there are usually 10 – 500

leucocytes/mm3

Mostly lymphocytes, though polymorphs may predominate in the earliest stage of the illness.

In Tuberculosis meningitis there are usually 100 – 500 leucocytes/mm3

Care in Counting the Cells

When counting the cells, care must be taken to identify the RBC and

rare presence of yeasts, amoeba should not be mistaken for leukocytes

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Differential Leukocyte counts

� If there is any difficulty in differentiating polymorphs and lymphocytes in the counting chamber Make a film of cellular deposit after specimen has been centrifuged Stain with Methylene blueleishmans or Carol thionine and examined under oil immersion to asses the relative number of two types of leucocytes

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Gram Staining of CSF

� The CSF to be centrifuged to deposit the cells and bacteria

� The film made from the deposit to be stained with Gram’s method

� Make a thick smear with of area spread 10 mm in diameter encircle by a scratch on the surface of the slide

� If the CSF appears turbid make a thin film� All the smears are dried and fixed on heat

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Gram Staining technique35

Gram Staining Procedure

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Examination of Gram Stained smear

� A careful search for Bacteria to be made in particular where there are plenty of leucocytes

� At least keen observation to be done for 10 mt before reporting a negative smear.

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Observe for the Presence of

One should be familiar with the following bacteria for successful reportingMeningococciPneumococciHaemophilusColi form bacilliStreptococciListeria

All the results are promptly reported to treating Physician

When variety of bacteria are found specimens may be contaminated.May need a fresh specimen for examination

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Culturing of CSF

� The deposited sediment plated on culture mediaBlood agar,Chocolate agar

incubated with 5-10% Carbon dioxide

A part of the specimen inoculated into Robertson's cooked medium

In suspected cases of Brain abscess Bacteroides and anaerobic cocci are cultured in anaerobic medium

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Direct antibiotic sensitivity detection

� When the organisms are numerous on Gram stained film CSF can be directly inoculated into Blood agar and Chocolate agar

� The commonly used effective antibiotic disks are tested with sensitivity pattern,

� Commonly we can test Benzyl Penicillin, and Chloramphenicol

� The antibiotic sensitivity pattern can be reported at the earliest

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Biochemical testing for Infections

� CSF should be tested for quantization of Glucose and Protein

Normal CSF contain 2.2 to 4mmol/liter correlates to 60% of the plasma levelsProtein is present at concentration of 0.15 to 0.4 grams/literIt can be higher in neonates can be up to 1.5 grams / literIn pyogenic meningitis Protein concentration is increased and Glucose concentration decreased.In aseptic meningitis Glucose concentration is normal and protein concentration raised

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Tests for Bacterial antigen Detection

Co agglutination Tests

There are several test kits available commercially for detection antigens ofMeningococci PneumococciH influenzae

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Diagnosis of Viral Meningitis

� The virus are to be isolated from CSF

� Presence of Viral antibodies by paired sampling of serum

� In few viral infections the virus can be isolated from

Throat swabsSpecimens of feces

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Tuberculosis Meningitis -Diagnosis

�CSF should be tested for presence of Acid fast bacilli by simple Ziehl Neelsen method

� The deposit of the concentrate can be inoculated onto Lowenstein Jensen’s Medium

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AFB Staining Methods

�Zeihl Neelsen’s-

hot stain

�Kinyoun’s-cold

stain

Modifications

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Ziehl- Neelsen Procedure

Make a smear. Air Dry. Heat Fix.2. Flood smear with Carbol Fuchsin stain

� Carbol Fuchsin is a lipid soluble, phenolic compound, which is able to penetrate the cell wall

3. Cover flooded smear with filter paper4. Steam for 10 minutes. Add more Carbol Fuchsin stain as needed5. Cool slide6. Rinse with DI water7. Flood slide with acid alcohol (leave 15 seconds). The acid alcohol

contains 3% HCl and 95% ethanol, or you can declorase with 20% H2 S04� The waxy cell wall then prevents the stain from being removed by the acid alcohol

(decolorizer) once it has penetrated the cell wall. The acid alcohol decolorizer will remove the stain from all other cells.

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Ziehl- Neelsen Procedure (continued)47

8. Tilt slide 45 degrees over the sink and add acid alcohol drop wise (drop by drop) until the red color stops streaming from the smear

9. Rinse with DI water

10. Add Loeffler’s Methylene Blue stain (counter stain). This stain adds blue color to non-acid fast cells!! Leave Loeffler’s Blue stain on smear for 1 minute

11. Rinse slide. Blot dry.

12. Use oil immersion objective to view.

Ziehl-Neelsen

stain

4 5 6

7

1 2 3

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How the Acid fast bacteria appear49

Leptospiral Meningitis - Diagnosis

� On few occasions in endemic areas Leptospira can produce meningitis

� Rarely Leptospira can be seen in CSF under Dark ground microscopy

� Cane cultured on Korthoff other Leptospiral medium

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Viruses - Meningitis

� The following viruses can cause Aseptic meningitis

1 Echovirus

2 Coxsackie

3 Herpes virus

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Tele contact is crucial in serious patients

� When the patient is

serious, write a

Tele contact

number which can help in prompt

delivery of results

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�The Programme is created by Dr.T.V.Rao MD for ‘e’ learning for Young Resident

Doctors in the Developing World� Email

doctortvrao@gmail.com

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