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  1. 1. Laboratory Diagnosis of Meningitis Dr.T.V.Rao MD
  2. 2. What is Meningitis <ul><li>Meningitis is an infection of the coverings around the brain and spinal cord. </li></ul><ul><li>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. </li></ul>
  3. 3. Why Diagnosing Meningitis is Important <ul><li>Diagnosing Meningitis is top priority in clinical Medicine, in particular Bacterial meingitis, can be a life threatening condition , the need for appreciate antibiotic therapy at the earliest is a priority. </li></ul><ul><li>Minimal Diagnostic faculties if done with precision can reduce morbidity and mortality </li></ul>
  4. 4. On suspicion of Meningitis <ul><li>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 . </li></ul>
  5. 5. Basic Understanding on Meningitis <ul><li>On a broad basis Meningitis is classified as </li></ul><ul><li>1 Purulent Meingitis </li></ul><ul><li>2 Aseptic Meingitis </li></ul>
  6. 6. What is Purulent Meningitis <ul><li>The CSF appears typically turbid due to the presence of Leucocytes 100 to several thousands / mm 3 most of which are Polymorphonuclear leucocytes </li></ul>
  7. 7. Major Aetiological agents of Meningitis <ul><li>1 Meningococcus </li></ul><ul><li>2 Pneumococcus </li></ul><ul><li>3 Haemophilus influenzae </li></ul><ul><li>On majority of the occasions the pathogens pass from Respiratory tract via blood stream and infect Meningtis </li></ul><ul><li>Can occur at any age </li></ul>
  8. 8. Neonates and Infants Meningitis <ul><li>There is specific affinity of some pathogens infecting Neonates and Infants </li></ul><ul><li>1 Coli forms </li></ul><ul><li>2 ß hemolytic streptococci </li></ul><ul><li>3 Pseudomonas </li></ul><ul><li>4 Salmonella and Listeria Monocytogenes </li></ul>
  9. 9. Iatrogenic Meningitis <ul><li>Carelessly performed Lumbar puncture </li></ul><ul><li>Accidental wound infection in neurosurgical wounds </li></ul><ul><li>Pyogenic Staphylococcus </li></ul><ul><li>Streptococci </li></ul><ul><li>Coli form bacilli </li></ul><ul><li>Anaerobic cocci </li></ul><ul><li>Bacteriods </li></ul>
  10. 10. CSF infection in Venous shunts <ul><li>When venous shunts are implanted for therapeutic purposes </li></ul><ul><li>Staphylococcus epidermidis </li></ul><ul><li>or some other Saprophytic bacteria which rarely cause infection in normal people can infect meningis </li></ul>
  11. 11. Aseptic Meingitis <ul><li>In these conditions CSF is clear or only slightly turbid contain moderate number of leucocytes </li></ul><ul><li>10 – 500 / mm 3 </li></ul><ul><li>Majority of cells are lymphocytes, except in early stages. </li></ul><ul><li>majority are caused by viruses </li></ul>
  12. 12. Aetiological agents of Aseptic Meningitis <ul><li>Enteroviruses </li></ul><ul><li>ECHO viruses </li></ul><ul><li>Coxsackie virus </li></ul><ul><li>Polio virus </li></ul><ul><li>Mumps virus moderately infective </li></ul><ul><li>Herpes simplex </li></ul><ul><li>Varicella zoster </li></ul><ul><li>Measles – Adenovirus </li></ul><ul><li>Arboviruses </li></ul>
  13. 13. CSF resembles - Aseptic Meningitis <ul><li>Few conditions associated with other etiological agent resemble aseptic meingitis </li></ul><ul><li>Leptospirosis </li></ul><ul><li>( Serovars Canicola icterohaemorrhagea ) </li></ul><ul><li>Fungi ( Cryptococcus neofroms ) </li></ul><ul><li>Amoeba – Naegleria, Harmanella. </li></ul>
  14. 14. Confusing CSF appereance <ul><li>When early treatment is given in Bacterial meingitis the Clinico pathological apperance appears as Viral meingitis </li></ul><ul><li>In viral Encephalitis modereate Lympoyte exduate is found as it in Viral meingitis </li></ul>
  15. 15. Tuberculosis Meningitis <ul><li>On many occasions Tuberculosis present as Aseptic meingitis, results from Pulmonary or mesentric tuberculosis </li></ul><ul><li>Can be associated with Miliary tuberculosis. </li></ul><ul><li>Cell counts on CSF will reveal 100 – 500 leucocytes / mm 3 </li></ul><ul><li>Majority are Lymphocytes </li></ul><ul><li>May form veil clot when CSF is allowed to stand in a undisturbed state. </li></ul>
  16. 16. Specimen collection for CSF Examination <ul><li>Lumbar puncture to collect the CSF for examination to be collected by Physician trained in procedure with aseptic precautions to prevent introduction of Infection. </li></ul>
  17. 17. Procedure to collect CSF <ul><li>The trained physician will collect only 3-5 ml into a labelled sterile container </li></ul><ul><li>Removal of large volume of CSF lead to headache, </li></ul><ul><li>The fluid to be collected at the rate of 4-5 drops per second. </li></ul><ul><li>If sudden removal of fluid is allowed may draw down cerebellum into the Foramen magnum and compress the Medulla of the Brain </li></ul>
  18. 18. CSF needs a New and Sterile container <ul><li>Fresh sterile screw capped container to be used. </li></ul><ul><li>Reused containers not to be used contamination from the previous specimens misrepresent the present specimen . </li></ul>
  19. 19. Lumbar puncture for CSF collection <ul><li>The best site for puncture is inter space between 3 and 4 lumbar vertebrae </li></ul><ul><li>( Corresponds to highest point of iliac crest ) </li></ul><ul><li>The Physcian 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. </li></ul>
  20. 20. Transportation to Laboratory <ul><li>The collected specimen of CSF to be dispatched promptly to Laboratory , delay may cause death of delicate pathogens eg Meningococci and disintegrate leukocyte </li></ul>
  21. 21. Preservation of CSF <ul><li>It is important when there is delay in transportation of specimens to Laboratory do not keep in Refrigerator, which tends to kill H.influenzae. </li></ul><ul><li>If delay is anticipated leave at Room Temperature. </li></ul>
  22. 22. Blood Culturing <ul><li>A simultaneous blood culture should be collected in all suspected cases of Meningitis before the Antibiotics are started </li></ul><ul><li>Many cases of Bacterial ( Pyogenic ) </li></ul><ul><li>meningitis are associated with Bacteriaemia </li></ul>
  23. 23. Septic spots in Meningtis <ul><li>Patients having Meiningococcal Meningtis present with several septic spots. </li></ul><ul><li>The spots should be scraped and fluid examined for demonostration of Gram - ve cocci, on several occaions bacteria are not seen in CSF </li></ul>
  24. 24. Laboratory Examination of CSF <ul><li>The specimens should be examined with naked eye </li></ul><ul><li>Look for Turbidity </li></ul><ul><li>Contamination with Blood </li></ul><ul><li>Normal CSF appears like water </li></ul>
  25. 25. Specimen Examination <ul><li>CSF to be examined for </li></ul><ul><li>Cell counts </li></ul><ul><li>Gram staining </li></ul><ul><li>Culturing </li></ul><ul><li>Estimation of protein and glucose </li></ul>
  26. 26. Cell counts in CSF <ul><li>Microscopic examination of uncentrigured, well mixed CSF is done in slide counting chamber. </li></ul><ul><li>Count the number of </li></ul><ul><li>Polymorphs </li></ul><ul><li>Lymphocytes </li></ul><ul><li>Erythrocytes </li></ul>
  27. 27. Normal cell counts <ul><li>CSF normally contains 0- 5 leucocytes / mm 3 </li></ul><ul><li>Mainly Lymphocytes </li></ul><ul><li>Newly born children contain upto 30/mm 3 </li></ul><ul><li>Mainly polymorphs </li></ul><ul><li>In purulent Meningtis there are usually 100 – 300 leucocytes/mm3 </li></ul><ul><li>In aseptic meningitis there are usually 10 – 500 leucocytes/mm 3 </li></ul><ul><li>Mostly lymphocytes, though polymorphs may predominate in the earliest stage of the illness. </li></ul><ul><li>In Tuberculosis meingitis there are usually 100 – 500 leucocytes/mm 3 </li></ul>
  28. 28. Care in Counting the Cells <ul><li>When counting the cells, care must be taken to identify the RBC and rare presence of yeasts, amoeba should not be mistaken for leukocytes </li></ul>
  29. 29. Differential Leukocyte counts <ul><li>If there is any difficulty in differentiating polymorphs and lymphocytes in the counting chamber </li></ul><ul><li>Make a film of cellular deposit after specimen has been centrifuged </li></ul><ul><li>Stain with </li></ul><ul><li>Methylene blue </li></ul><ul><li>leishman or Carol thionine and examined under oil immersion to asses the relative number of two types of leucocytes </li></ul><ul><li>Dr.T.V.Rao MD </li></ul>
  30. 30. Gram Staining of CSF <ul><li>The CSF to be centrifuged to deposit the cells and bacteria </li></ul><ul><li>The film made from the deposit to be stained with Gram’s method </li></ul><ul><li>Make a thick smear with of area spread 10 mm in diameter encircle by a scratch on the surface of the slide </li></ul><ul><li>If the CSF appears turbid make a thin film </li></ul><ul><li>All the smears are dried and fixed on heat </li></ul>
  31. 31. Examination of Gram Stained smear <ul><li>A careful search for Bacteria to be made in particular where there are plenty of leucocytes </li></ul><ul><li>At least keen observation to be done for 10 mt before reporting a negative smear. </li></ul>
  32. 32. Observe for the Presence of <ul><li>One should be familiar with the following bacteria for successful reporting </li></ul><ul><li>Meningococci </li></ul><ul><li>Pneumococci </li></ul><ul><li>Haemophili </li></ul><ul><li>Coli form bacilli </li></ul><ul><li>Streptococci </li></ul><ul><li>Listeria </li></ul><ul><li>All the results are promptly reported to treating Physician </li></ul><ul><li>When variety of bacteria are found specimens may be contaminated. </li></ul><ul><li>May need a fresh specimen for examination </li></ul>
  33. 33. Culturing of CSF <ul><li>The deposited sediment plated on culture media </li></ul><ul><li>Blood agar, </li></ul><ul><li>Chocolate agar </li></ul><ul><li>incubated with 5-10% </li></ul><ul><li>Carbon dioxide </li></ul><ul><li>A part of the specimen inoculated into Robertson's cooked medium </li></ul><ul><li>In suspected cases of Brain abscess Bacteriods and anaerobic cocci are cultured in anaerobic medium </li></ul>
  34. 34. Direct antibiotic sensitivity detection <ul><li>When the organisms are numerous on Gram stained film CSF can be directly inoculated into Blood agar and Chocolate agar </li></ul><ul><li>The commonly used effective antibiotic disks are tested with sensitivity pattern, </li></ul><ul><li>Commonly we can test Benzyl Pencillin, and Choramphenicol </li></ul><ul><li>The antibiotic sensitivity pattern can be reported at the earliest </li></ul><ul><li>Dr.T.V.Rao MD </li></ul>
  35. 35. Dealing with Growth in Robertson's Cooked medium <ul><li>If the turbidity devlops in RCM the broth should be filmed and subcultured on to Blood agar and Chocolate agar plates and incubated both aerobically and aerobically. </li></ul><ul><li>Dr.T.V.Rao MD </li></ul>
  36. 36. Biochemical testing for Infections <ul><li>CSF should be tested for quantization of </li></ul><ul><li>Glucose and Protein </li></ul><ul><li>Normal CSF contain </li></ul><ul><li>2.2 to 4mmol/liter correlates to 60% of the plasma levels </li></ul><ul><li>Protein is present at concentration of 0.15 to 0.4 grams/liter </li></ul><ul><li>It can be higher in neonates can be upto 1.5 grams / liter </li></ul><ul><li>In pyogenic meningitis Protein concentration is increased and Glucose concentration decreased. </li></ul><ul><li>In aseptic meningitis Glucose concentration is normal and protein concentration raised </li></ul>
  37. 37. Tests for Bacterial antigen Detection <ul><li>Co agglutination Tests </li></ul><ul><li>There are several test kits avialble commercially for detection antigens of </li></ul><ul><li>Meningococci </li></ul><ul><li>Pneumococci </li></ul><ul><li>H influenzae </li></ul><ul><li>Dr.T.V.Rao MD </li></ul>
  38. 38. Diagnosis of Viral Meningitis <ul><li>The virus are to be isolated from CSF </li></ul><ul><li>Presence of Viral antibodies by paired sampling of serum </li></ul><ul><li>In few viral infections the virus can be isolated from </li></ul><ul><li>Throat swabs </li></ul><ul><li>Specimens of feces </li></ul><ul><li>Dr.T.V.Rao MD </li></ul>
  39. 39. Viruses - Meningitis <ul><li>The following viruses can cause Aseptic meningitis </li></ul><ul><li>1 Echovirus </li></ul><ul><li>2 Coxsackie </li></ul><ul><li>3 Herpes virus </li></ul>
  40. 40. Tuberculosis Meningitis -Diagnosis <ul><li>CSF should be tested for presence of Acid fast bacilli by simple Ziehl Neelsen method </li></ul><ul><li>The deposit of the concentrate can be inoculated onto Lowenstein Jensens Medium </li></ul><ul><li>Other tests which support diagnosis are Reduction of Glucose, Protein is increased </li></ul><ul><li>Dr.T.V.Rao MD </li></ul>
  41. 41. Leptospiral Meningtis - Diagnosis <ul><li>On few occasions in endemic areas Leptospira can produce meningitis </li></ul><ul><li>Rarely Leptospira can be seen in CSF under Dark ground microscopy </li></ul><ul><li>Cane cultured on Korthoff other Leptospiral medium </li></ul>
  42. 42. Antibiotic Selection in Meningitis <ul><li>In the past Benzyl Pencillin or Choramphenicol are given in majority of cases suspected with meningitis. </li></ul><ul><li>In the recent past several new generation of Cephalosporins are replacing past theraputic ideas. </li></ul><ul><li>It is important to isolate and identify the causative agent. </li></ul><ul><li>However it is essential to start a theraputic trail with best clinical guess before the laboratory results are available. </li></ul>
  43. 43. Problems in Devloping World <ul><li>It is much difficult to deal with precision in Devloping countries as many several infrastructural facilities are not available and Physicians have work their best of the previous clinical experiences. </li></ul><ul><li>The wisdom of treating Physicians still save several lives in the third world countries. </li></ul><ul><li>Dr.T.V.Rao. MD </li></ul>
  44. 44. Created for benefit of Medical and paramedical Students in Devloping World Dr.T.V.Rao MD Email [email_address]