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 D...
What is Meningitis3    Meningitis is an infection    of the coverings around    the brain and spinal cord.    The infectio...
Why Diagnosing Meningitis is4                Important        Diagnosing Meningitis is top priority in clinical    Medicin...
On suspicion of Meningitis5        Every patient suspected of    having Meningitis should have a    specimen of CSF examin...
Basic Understanding on6                Meningitis    On a broad basis    Meningitis is    classified as     1 Purulent    ...
What is Purulent Meningitis7    The CSF appears    typically turbid due to    the presence of    Leucocytes 100 to    seve...
Major Etiological agents of8                 Meningitis    1 Meningococcus    2 Pneumococcus    3 Haemophilus influenza   ...
Neonates and Infants9                 Meningitis    There is specific affinity of some pathogens infecting    Neonates and...
Iatrogenic Meningitis10     Carelessly performed Lumbar     puncture     Accidental wound infection     in neurosurgical w...
Aseptic Meningitis11      In these conditions CSF is     clear or only slightly     turbid contain moderate     number of ...
Etiological agents of Aseptic Meningitis12     Enteroviruses        ECHO viruses        Coxsackie virus        Polio virus...
CSF resembles - Aseptic Meningitis in               Several other Infections13      Few conditions associated      with ot...
Confusing CSF appearanceWhen early treatment isgiven in Bacterialmeningitis the Clinicopathological appearanceappears as V...
Tuberculosis Meningitis15     On many occasions Tuberculosis     present as Aseptic meningitis,     results from Pulmonary...
WHY MICROBIOLOGICAL DIAGNOSIS IS                 LIFE SAVING     Information derived from the results has impact on :     ...
17Cerebrospinal fluid   examination
Why specimen collection is Important18              in Microbiology     Specimen collection in Microbiology to isolate and...
Some tips better Diagnosis     Laboratory investigation should start as early as possible     Specimens obtained early, pr...
An Ideal Request form20     Name      xxxx       Age       Sex     IP/ OP No xyz      Time       Date     Ward       xx123...
Why Proper written Request21     Your request is a legal document.     Identifies all the outcome of test.     No intercha...
Specimen collection for               CSF ExaminationLumbar puncture tocollect the CSF forexamination to becollected by Ph...
Procedure to collect CSFThe trained physician willcollect only 3-5 ml into alabeled sterile containerRemoval of large volu...
CSF needs a New and Sterile container                    Fresh sterile screw capped                    container to be use...
Lumbar puncture for CSF collection  The best site for puncture is inter  space between 3 and 4 lumbar  vertebrae ( Corresp...
Transportation to Laboratory                The collected specimen                of CSF to be dispatched                p...
Preservation of CSFIt is important when there isdelay in transportation ofspecimens to Laboratorydo not keep inRefrigerato...
Laboratory Examination of CSF28     The specimens should be     examined with naked eye     Look for Turbidity            ...
Specimen Examination29     CSF to be examined for       Cell counts        Gram staining        Culturing        Estimatio...
Cell counts in CSFMicroscopic examination ofuncentrigured, well mixedCSF is done in slidecounting chamber.Count the number...
Normal cell counts31     CSF normally contains 0- 5 leucocytes / mm3         Mainly Lymphocytes         Newly born childre...
Care in Counting the Cells32     When counting the cells,     care must be taken to     identify the RBC and     rare pres...
Differential Leukocyte countsIf there is any difficulty indifferentiating polymorphs andlymphocytes in the countingchamber...
Gram Staining of CSF34     The CSF to be centrifuged to deposit the cells and     bacteria     The film made from the depo...
35     Gram Staining technique
Gram Staining Procedure36
Examination of Gram Stained smear37     A careful search for     Bacteria to be made in     particular where there     are...
Observe for the Presence ofOne should be familiar with the  following bacteria for successful  reporting Meningococci Pneu...
Culturing of CSF39        The deposited sediment        plated on culture media         Blood agar,         Chocolate agar...
Direct antibiotic sensitivity detection When the organisms are numerous on Gram stained film CSF can be directly inoculate...
Biochemical testing for Infections CSF should be tested for quantization of  Glucose and ProteinNormal CSF contain2.2 to 4...
Tests for Bacterial antigen Detection      Co agglutination TestsThere are several test  kits available  commercially for ...
Diagnosis of Viral Meningitis43     The virus are to be     isolated from CSF     Presence of Viral     antibodies by pair...
Tuberculosis Meningitis -Diagnosis44     CSF should be     tested for presence     of Acid fast bacilli     by simple Zieh...
45     AFB Staining MethodsZeihl Neelsen’s-hot stainKinyoun’s-coldstainModifications
Ziehl- Neelsen Procedure46     Make a smear. Air Dry. Heat Fix.     2. Flood smear with Carbol Fuchsin stain          Carb...
Ziehl- Neelsen Procedure (continued)47     8. Tilt slide 45 degrees over the sink and add acid alcohol       drop wise (dr...
1   2          348     4       5          6     7             Ziehl-Neelsen             stain
How the Acid fast bacteria appear49
Leptospiral Meningitis - Diagnosis50     On few occasions in endemic     areas Leptospira can produce     meningitis     R...
Viruses - MeningitisThe following viruses cancause Aseptic meningitis1 Echovirus2 Coxsackie3 Herpes virus                 ...
Tele contact is crucial in serious patientsWhen the patient isserious, write aTele contactnumber whichcan help in promptde...
53     The Programme is created by Dr.T.V.Rao      MD for ‘e’ learning for Young Resident        Doctors in the Developing...
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Diagnosis of Meningitis , Basic skills in diagnostic Microbiologu

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Diagnosis of Meningitis , Basic skills in diagnostic Microbiologu

  1. 1. 1DIAGNOSIS OF MENINGITIS SKILLS FOR RESIDENTS Dr.T.V.Rao MD
  2. 2. 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
  3. 3. 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.
  4. 4. 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
  5. 5. 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.
  6. 6. Basic Understanding on6 Meningitis On a broad basis Meningitis is classified as 1 Purulent Meningitis 2 Aseptic Meningitis
  7. 7. 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
  8. 8. 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
  9. 9. 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
  10. 10. Iatrogenic Meningitis10 Carelessly performed Lumbar puncture Accidental wound infection in neurosurgical wounds Pyogenic Staphylococcus Streptococci Coli form bacilli Anaerobic cocci Bacteriods
  11. 11. 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
  12. 12. Etiological agents of Aseptic Meningitis12 Enteroviruses ECHO viruses Coxsackie virus Polio virus Mumps virus moderately infective Herpes simplex Varicella zoster Measles – Adenovirus Arboviruses
  13. 13. 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.
  14. 14. Confusing CSF appearanceWhen early treatment isgiven in Bacterialmeningitis the Clinicopathological appearanceappears as Viral meningitisIn viral Encephalitismoderate Lymphocyteexduate is found as it inViral meningitis 14
  15. 15. 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.
  16. 16. 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
  17. 17. 17Cerebrospinal fluid examination
  18. 18. 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
  19. 19. 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
  20. 20. 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
  21. 21. 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.
  22. 22. Specimen collection for CSF ExaminationLumbar puncture tocollect the CSF forexamination to becollected by Physiciantrained in procedurewith asepticprecautions to preventintroduction ofInfection. 22
  23. 23. 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
  24. 24. 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
  25. 25. 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
  26. 26. 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
  27. 27. 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
  28. 28. Laboratory Examination of CSF28 The specimens should be examined with naked eye Look for Turbidity Contamination with Blood Normal CSF appears like water
  29. 29. Specimen Examination29 CSF to be examined for Cell counts Gram staining Culturing Estimation of protein and glucose
  30. 30. Cell counts in CSFMicroscopic examination ofuncentrigured, well mixedCSF is done in slidecounting chamber.Count the number of Polymorphs Lymphocytes Erythrocytes 30
  31. 31. 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
  32. 32. 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
  33. 33. 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
  34. 34. 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
  35. 35. 35 Gram Staining technique
  36. 36. Gram Staining Procedure36
  37. 37. 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.
  38. 38. 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
  39. 39. 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
  40. 40. 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
  41. 41. 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
  42. 42. Tests for Bacterial antigen Detection Co agglutination TestsThere are several test kits available commercially for detection antigens of Meningococci Pneumococci H influenzae 42
  43. 43. 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
  44. 44. 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
  45. 45. 45 AFB Staining MethodsZeihl Neelsen’s-hot stainKinyoun’s-coldstainModifications
  46. 46. 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.
  47. 47. 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.
  48. 48. 1 2 348 4 5 6 7 Ziehl-Neelsen stain
  49. 49. How the Acid fast bacteria appear49
  50. 50. 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
  51. 51. Viruses - MeningitisThe following viruses cancause Aseptic meningitis1 Echovirus2 Coxsackie3 Herpes virus 51
  52. 52. Tele contact is crucial in serious patientsWhen the patient isserious, write aTele contactnumber whichcan help in promptdelivery of results 52
  53. 53. 53 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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