Laboratory Diagnosis Laboratory Diagnosis of Meningitisof Meningitis

Dr.T.V.Rao MDDr.T.V.Rao MD

What is MeningitisWhat is Meningitis

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

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

Why Diagnosing Meningitis is Why Diagnosing Meningitis is ImportantImportant

Diagnosing Meningitis is top priority in Diagnosing Meningitis is top priority in clinical Medicine, in particular Bacterial clinical Medicine, in particular Bacterial

meingitis, can be a life threatening meingitis, can be a life threatening condition , the need for appreciate condition , the need for appreciate

antibiotic therapy at the earliest is a antibiotic therapy at the earliest is a priority.priority.

Minimal Diagnostic faculties if done with Minimal Diagnostic faculties if done with precision can reduce morbidity and precision can reduce morbidity and

mortalitymortality

On suspicion of MeningitisOn suspicion of Meningitis

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

infection and to rule out infection and to rule out infectioninfection..

Basic Understanding on Basic Understanding on MeningitisMeningitis

On a broad basis Meningitis is On a broad basis Meningitis is classified as classified as

1 Purulent Meingitis1 Purulent Meingitis

2 Aseptic Meingitis2 Aseptic Meingitis

What is Purulent MeningitisWhat is Purulent Meningitis

The CSF appears typically turbid The CSF appears typically turbid due to the presence of Leucocytes due to the presence of Leucocytes 100 to several thousands / mm100 to several thousands / mm3 3 most most of which are Polymorphonuclear of which are Polymorphonuclear leucocytesleucocytes

Major Aetiological agents of Major Aetiological agents of MeningitisMeningitis

1 Meningococcus1 Meningococcus 2 Pneumococcus2 Pneumococcus 3 Haemophilus influenzae3 Haemophilus influenzae

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

Can occur at any ageCan occur at any age

Neonates and Infants Neonates and Infants MeningitisMeningitis

There is specific affinity of some There is specific affinity of some pathogens infecting Neonates and pathogens infecting Neonates and InfantsInfants

1 Coli forms1 Coli forms

2 ß hemolytic streptococci 2 ß hemolytic streptococci

3 Pseudomonas3 Pseudomonas

4 Salmonella and Listeria 4 Salmonella and Listeria MonocytogenesMonocytogenes

Iatrogenic MeningitisIatrogenic Meningitis

Carelessly performed Carelessly performed Lumbar puncture Lumbar puncture

Accidental wound Accidental wound infection in infection in neurosurgical woundsneurosurgical wounds

Pyogenic Pyogenic StaphylococcusStaphylococcus

StreptococciStreptococci Coli form bacilliColi form bacilli Anaerobic cocciAnaerobic cocci Bacteriods Bacteriods

CSF infection in Venous shuntsCSF infection in Venous shunts

When venous shunts When venous shunts are implanted for are implanted for therapeutic purposestherapeutic purposes

Staphylococcus Staphylococcus epidermidis epidermidis

or some other or some other Saprophytic bacteria Saprophytic bacteria which rarely cause which rarely cause infection in normal infection in normal people can infect people can infect meningismeningis

Aseptic MeingitisAseptic Meingitis

In these conditions CSF is clear or only In these conditions CSF is clear or only slightly turbid contain moderate number slightly turbid contain moderate number of leucocytesof leucocytes

10 – 500 / mm10 – 500 / mm33

Majority of cells are lymphocytes, Majority of cells are lymphocytes, except in early stages.except in early stages.

majority are caused by virusesmajority are caused by viruses

Aetiological agents of Aseptic Aetiological agents of Aseptic MeningitisMeningitis

EnterovirusesEnteroviruses ECHO virusesECHO viruses Coxsackie virusCoxsackie virus Polio virusPolio virus Mumps virus Mumps virus

moderately moderately infectiveinfective

Herpes simplexHerpes simplex Varicella zosterVaricella zoster Measles – Measles –

AdenovirusAdenovirus ArbovirusesArboviruses

CSF resembles - Aseptic CSF resembles - Aseptic MeningitisMeningitis

Few conditions Few conditions associated with other associated with other etiological agent etiological agent resemble aseptic resemble aseptic meingitismeingitis

LeptospirosisLeptospirosis ( Serovars Canicola ( Serovars Canicola

icterohaemorrhagea )icterohaemorrhagea ) Fungi ( Cryptococcus Fungi ( Cryptococcus

neofromsneofroms ) ) Amoeba – Naegleria, Amoeba – Naegleria,

Harmanella.Harmanella.

Confusing CSF appereanceConfusing CSF appereance

When early When early treatment is given in treatment is given in Bacterial meingitis Bacterial meingitis the Clinico the Clinico pathological pathological apperance appears apperance appears as Viral meingitisas Viral meingitis

In viral Encephalitis In viral Encephalitis modereate Lympoyte modereate Lympoyte exduate is found as exduate is found as it in Viral meingitisit in Viral meingitis

Tuberculosis MeningitisTuberculosis Meningitis

On many occasions On many occasions Tuberculosis present as Tuberculosis present as Aseptic meingitis, results Aseptic meingitis, results from Pulmonary or from Pulmonary or mesentric tuberculosismesentric tuberculosis

Can be associated with Can be associated with Miliary tuberculosis. Miliary tuberculosis.

Cell counts on CSF will Cell counts on CSF will reveal 100 – 500 reveal 100 – 500 leucocytes / mmleucocytes / mm33

Majority are LymphocytesMajority are Lymphocytes May form veil clot when May form veil clot when

CSF is allowed to stand in CSF is allowed to stand in a undisturbed state.a undisturbed state.

Specimen collection for Specimen collection for CSF ExaminationCSF Examination

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

Procedure to collect CSFProcedure to collect CSF

The trained physician will The trained physician will collect only 3-5 ml into a collect only 3-5 ml into a labelled sterile containerlabelled sterile container

Removal of large volume Removal of large volume of CSF lead to headache,of CSF lead to headache,

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

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

CSF needs a New and Sterile CSF needs a New and Sterile containercontainer

Fresh sterile screw Fresh sterile screw capped container capped container to be used.to be used.

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

Lumbar puncture for CSF Lumbar puncture for CSF collectioncollection

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

( ( Corresponds to highest point of iliac crest )Corresponds to highest point of iliac crest )

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

Transportation to LaboratoryTransportation to Laboratory

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

Preservation of CSFPreservation of CSF

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

If delay is anticipated If delay is anticipated leave at Room leave at Room Temperature.Temperature.

Blood CulturingBlood Culturing

A simultaneous blood A simultaneous blood culture should be culture should be collected in all collected in all suspected cases of suspected cases of Meningitis before the Meningitis before the Antibiotics are startedAntibiotics are started

Many cases of Many cases of Bacterial ( Pyogenic )Bacterial ( Pyogenic )

meningitis are meningitis are associated with associated with BacteriaemiaBacteriaemia

Septic spots in MeningtisSeptic spots in Meningtis

Patients having Patients having Meiningococcal Meiningococcal Meningtis present with Meningtis present with several septic spots.several septic spots.

The spots should be The spots should be scraped and fluid scraped and fluid examined for examined for demonostration of demonostration of Gram - ve cocci, on Gram - ve cocci, on several occaions several occaions bacteria are not seen bacteria are not seen in CSFin CSF

Laboratory Examination of CSFLaboratory Examination of CSF

The specimens should be examined The specimens should be examined with naked eyewith naked eye

Look for TurbidityLook for Turbidity

Contamination with BloodContamination with Blood

Normal CSF appears like Normal CSF appears like waterwater

Specimen ExaminationSpecimen Examination

CSF to be CSF to be examined for examined for

Cell countsCell counts

Gram stainingGram staining

CulturingCulturing

Estimation of Estimation of protein and protein and glucoseglucose

Cell counts in CSFCell counts in CSF

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

Count the number ofCount the number of

PolymorphsPolymorphs

LymphocytesLymphocytes

ErythrocytesErythrocytes

Normal cell countsNormal cell counts

CSF normally contains 0- 5 CSF normally contains 0- 5 leucocytes / mmleucocytes / mm33

Mainly LymphocytesMainly Lymphocytes Newly born children contain Newly born children contain

upto 30/mmupto 30/mm33

Mainly polymorphsMainly polymorphs In purulent Meningtis there are In purulent Meningtis there are

usually 100 – 300 usually 100 – 300 leucocytes/mm3 leucocytes/mm3

In aseptic meningitis there are In aseptic meningitis there are usually 10 – 500 leucocytes/mmusually 10 – 500 leucocytes/mm33

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

In Tuberculosis meingitis there In Tuberculosis meingitis there are usually 100 – 500 are usually 100 – 500 leucocytes/mmleucocytes/mm33

Care in Counting the CellsCare in Counting the Cells

When counting the When counting the cells, care must be cells, care must be taken to identify taken to identify the RBC and rare the RBC and rare presence of presence of yeasts, amoeba yeasts, amoeba should not be should not be mistaken for mistaken for leukocytesleukocytes

Differential Leukocyte countsDifferential Leukocyte counts

If there is any difficulty in If there is any difficulty in differentiating polymorphs differentiating polymorphs and lymphocytes in the and lymphocytes in the counting chamber counting chamber

Make a film of cellular deposit Make a film of cellular deposit after specimen has been after specimen has been centrifuged centrifuged

Stain with Stain with Methylene blueMethylene blue leishman or Carol leishman or Carol

thionine and examined thionine and examined under oil immersion to under oil immersion to asses the relative number asses the relative number of two types of leucocytesof two types of leucocytes

Dr.T.V.Rao MDDr.T.V.Rao MD

Gram Staining of CSFGram Staining of CSF

The CSF to be centrifuged The CSF to be centrifuged to deposit the cells and to deposit the cells and bacteriabacteria

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

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

If the CSF appears turbid If the CSF appears turbid make a thin filmmake a thin film

All the smears are dried All the smears are dried and fixed on heatand fixed on heat

Examination of Gram Stained Examination of Gram Stained smearsmear

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

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

Observe for the PresenceObserve for the Presence ofof

One should be familiar with the One should be familiar with the following bacteria for following bacteria for successful reportingsuccessful reporting

MeningococciMeningococci PneumococciPneumococci HaemophiliHaemophili Coli form bacilliColi form bacilli StreptococciStreptococci Listeria Listeria All the results are promptly All the results are promptly

reported to treating reported to treating PhysicianPhysician

When variety of bacteria are When variety of bacteria are found specimens may be found specimens may be contaminated.contaminated.

May need a fresh specimen May need a fresh specimen for examinationfor examination

Culturing of CSFCulturing of CSF The deposited The deposited

sediment plated on sediment plated on culture mediaculture media

Blood agar,Blood agar, Chocolate agarChocolate agar incubated with 5-10% incubated with 5-10% Carbon dioxideCarbon dioxideA part of the specimen inoculated A part of the specimen inoculated

into Robertson's cooked mediuminto Robertson's cooked mediumIn suspected cases of Brain abscess In suspected cases of Brain abscess

Bacteriods and anaerobic cocci Bacteriods and anaerobic cocci are cultured in anaerobic mediumare cultured in anaerobic medium

Direct antibiotic sensitivity Direct antibiotic sensitivity detectiondetection

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

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

Commonly we can test Benzyl Commonly we can test Benzyl Pencillin, and Choramphenicol Pencillin, and Choramphenicol

The antibiotic sensitivity pattern The antibiotic sensitivity pattern can be reported at the earliestcan be reported at the earliest

Dr.T.V.Rao MDDr.T.V.Rao MD

Dealing with Growth in Robertson's Dealing with Growth in Robertson's Cooked mediumCooked medium

If the turbidity devlops If the turbidity devlops in RCM the broth in RCM the broth should be filmed and should be filmed and subcultured on to subcultured on to Blood agar and Blood agar and Chocolate agar plates Chocolate agar plates and incubated both and incubated both aerobically and aerobically and aerobically.aerobically.

Dr.T.V.Rao MDDr.T.V.Rao MD

Biochemical testing for Biochemical testing for InfectionsInfections

CSF should be tested for CSF should be tested for quantization of quantization of

Glucose and ProteinGlucose and Protein Normal CSF contain Normal CSF contain 2.2 to 4mmol/liter correlates 2.2 to 4mmol/liter correlates

to 60% of the plasma levelsto 60% of the plasma levels Protein is present at Protein is present at

concentration of 0.15 to 0.4 concentration of 0.15 to 0.4 grams/litergrams/liter

It can be higher in neonates It can be higher in neonates can be upto 1.5 grams / litercan be upto 1.5 grams / liter

In pyogenic meningitis Protein In pyogenic meningitis Protein concentration is increased concentration is increased and Glucose concentration and Glucose concentration decreased.decreased.

In aseptic meningitis Glucose In aseptic meningitis Glucose concentration is normal and concentration is normal and protein concentration raisedprotein concentration raised

Tests for Bacterial antigen Tests for Bacterial antigen DetectionDetection

Co agglutination TestsCo agglutination Tests

There are several test There are several test kits avialble kits avialble commercially for commercially for detection antigens ofdetection antigens of

Meningococci Meningococci

PneumococciPneumococci

H influenzaeH influenzae

Dr.T.V.Rao MDDr.T.V.Rao MD

Diagnosis of Viral MeningitisDiagnosis of Viral Meningitis

The virus are to be isolated from CSFThe virus are to be isolated from CSF Presence of Viral antibodies by paired Presence of Viral antibodies by paired

sampling of serumsampling of serum In few viral infections the virus can be In few viral infections the virus can be

isolated fromisolated from Throat swabsThroat swabs Specimens of fecesSpecimens of feces

Dr.T.V.Rao MDDr.T.V.Rao MD

Viruses - MeningitisViruses - Meningitis

The following The following viruses can cause viruses can cause Aseptic meningitisAseptic meningitis

1 1 EchovirusEchovirus

2 Coxsackie2 Coxsackie

3 Herpes virus3 Herpes virus

Tuberculosis Meningitis -Tuberculosis Meningitis -DiagnosisDiagnosis

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

The deposit of the The deposit of the concentrate can be concentrate can be inoculated onto Lowenstein inoculated onto Lowenstein Jensens MediumJensens Medium

Other tests which support Other tests which support diagnosis are Reduction of diagnosis are Reduction of Glucose, Protein is Glucose, Protein is increasedincreased

Dr.T.V.Rao MDDr.T.V.Rao MD

Leptospiral Meningtis - Leptospiral Meningtis - DiagnosisDiagnosis

On few occasions in On few occasions in endemic areas endemic areas Leptospira can Leptospira can produce meningitisproduce meningitis

Rarely Leptospira can Rarely Leptospira can be seen in CSF under be seen in CSF under Dark ground Dark ground microscopymicroscopy

Cane cultured on Cane cultured on Korthoff other Korthoff other Leptospiral mediumLeptospiral medium

Antibiotic Selection in MeningitisAntibiotic Selection in Meningitis

In the past Benzyl Pencillin or In the past Benzyl Pencillin or Choramphenicol are given in majority of Choramphenicol are given in majority of cases suspected with meningitis.cases suspected with meningitis.

In the recent past several new generation In the recent past several new generation of Cephalosporins are replacing past of Cephalosporins are replacing past theraputic ideas.theraputic ideas.

It is important to isolate and identify the It is important to isolate and identify the causative agent.causative agent.

However it is essential to start a theraputic However it is essential to start a theraputic trail with best clinical guess before the trail with best clinical guess before the laboratory results are available.laboratory results are available.

Problems in Devloping WorldProblems in Devloping World It is much difficult to deal It is much difficult to deal

with precision in Devloping with precision in Devloping countries as many several countries as many several infrastructural facilities are infrastructural facilities are not available and Physicians not available and Physicians have work their best of the have work their best of the previous clinical previous clinical experiences.experiences.

The wisdom of treating The wisdom of treating Physicians still save Physicians still save several lives in the third several lives in the third world countries.world countries.

Dr.T.V.Rao. MDDr.T.V.Rao. MD

Created for benefit of Created for benefit of Medical and Medical and

paramedical Students paramedical Students in Devloping Worldin Devloping World

Dr.T.V.Rao MDDr.T.V.Rao MD

EmailEmail

doctortvrao@gmail.comdoctortvrao@gmail.com