Diagnosis of Meningitis , Basic skills in diagnostic Microbiologu
1DIAGNOSIS OF MENINGITIS SKILLS FOR RESIDENTS Dr.T.V.Rao MD
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 Meningitis3 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.
Why Diagnosing Meningitis is4 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
On suspicion of Meningitis5 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.
Basic Understanding on6 Meningitis On a broad basis Meningitis is classified as 1 Purulent Meningitis 2 Aseptic Meningitis
What is Purulent Meningitis7 The CSF appears typically turbid due to the presence of Leucocytes 100 to several thousands / mm3 most of which are Polymorph nuclear leucocytes
Major Etiological agents of8 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
Neonates and Infants9 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
Iatrogenic Meningitis10 Carelessly performed Lumbar puncture Accidental wound infection in neurosurgical wounds Pyogenic Staphylococcus Streptococci Coli form bacilli Anaerobic cocci Bacteriods
Aseptic Meningitis11 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
CSF resembles - Aseptic Meningitis in Several other Infections13 Few conditions associated with other etiological agent resemble aseptic meningitis Leptospirosis ( Serovars Canicola icterohaemorrhagea ) Fungi ( Cryptococcus neoformans ) Amoeba – Naegleria, Harmanella.
Confusing CSF appearanceWhen early treatment isgiven in Bacterialmeningitis the Clinicopathological appearanceappears as Viral meningitisIn viral Encephalitismoderate Lymphocyteexduate is found as it inViral meningitis 14
Tuberculosis Meningitis15 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.
WHY MICROBIOLOGICAL DIAGNOSIS IS LIFE SAVING Information derived from the results has impact on : Diagnosis of infectious diseases Antibiotic prescribing Formulation of local antibiotic policy Public health impact eg Meningococcal infection.16
Why specimen collection is Important18 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
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 form20 Name xxxx Age Sex IP/ OP No xyz Time Date Ward xx123 Urgent / Routine Nature of specimen CSF Investigation needed xxxx xxxx Doctor/Staff Contact No 1234567
Why Proper written Request21 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.
Specimen collection for CSF ExaminationLumbar puncture tocollect the CSF forexamination to becollected by Physiciantrained in procedurewith asepticprecautions to preventintroduction ofInfection. 22
Procedure to collect CSFThe trained physician willcollect only 3-5 ml into alabeled sterile containerRemoval of large volume ofCSF lead to headache,The fluid to be collected at the rateof 4-5 drops per second.If sudden removal of fluid isallowed may draw downcerebellum into the Foramenmagnum and compress the Medullaof the Brain 23
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. 24
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. 25
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 26
Preservation of CSFIt is important when there isdelay in transportation ofspecimens to Laboratorydo not keep inRefrigerator, which tendsto kill H. InfluenzaIf delay is anticipated leaveat Room Temperature. 27
Laboratory Examination of CSF28 The specimens should be examined with naked eye Look for Turbidity Contamination with Blood Normal CSF appears like water
Specimen Examination29 CSF to be examined for Cell counts Gram staining Culturing Estimation of protein and glucose
Cell counts in CSFMicroscopic examination ofuncentrigured, well mixedCSF is done in slidecounting chamber.Count the number of Polymorphs Lymphocytes Erythrocytes 30
Normal cell counts31 CSF normally contains 0- 5 leucocytes / mm3 Mainly Lymphocytes Newly born children contain up to 30/mm3 Mainly polymorphs In purulent Meningitis there are usually 100 – 300 leucocytes/mm3 In 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 Cells32 When counting the cells, care must be taken to identify the RBC and rare presence of yeasts, amoeba should not be mistaken for leukocytes
Differential Leukocyte countsIf there is any difficulty indifferentiating polymorphs andlymphocytes in the countingchamberMake a film of cellular depositafter specimen has beencentrifuged Stain withMethylene blueleishmans or Carol thionineand examined under oilimmersion to asses therelative number of twotypes of leucocytes 33
Gram Staining of CSF34 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
Examination of Gram Stained smear37 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.
Observe for the Presence ofOne should be familiar with the following bacteria for successful reporting Meningococci Pneumococci Haemophilus Coli form bacilli Streptococci ListeriaAll the results are promptly reported to treating PhysicianWhen variety of bacteria are found specimens may be contaminated. May need a fresh specimen for examination 38
Culturing of CSF39 The deposited sediment plated on culture media Blood agar, Chocolate agar incubated with 5-10% Carbon dioxide A part of the specimen inoculated into Robertsons cooked medium In suspected cases of Brain abscess Bacteroides and anaerobic cocci are cultured in anaerobic medium
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 40
Biochemical testing for Infections CSF should be tested for quantization of Glucose and ProteinNormal CSF contain2.2 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 41
Tests for Bacterial antigen Detection Co agglutination TestsThere are several test kits available commercially for detection antigens of Meningococci Pneumococci H influenzae 42
Diagnosis of Viral Meningitis43 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 swabs Specimens of feces
Tuberculosis Meningitis -Diagnosis44 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
Ziehl- Neelsen Procedure46 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 paper 4. Steam for 10 minutes. Add more Carbol Fuchsin stain as needed 5. Cool slide 6. Rinse with DI water 7. 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.
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.
Leptospiral Meningitis - Diagnosis50 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