Diagnosis

DIAGNOSIS

Specimens

  • PBS containing 1%BSA is used as transport medium
    • Heparinised blood for culture
    • Whole blood/serum for serological tests (blood must be collected during early febrile stage)
    • Mid-stream urine for dark filed examination
  • Leptospires can disintegrate quite quickly in a urine sample, especially if it is acidic.
  • So, if the urine cannot be examined within 20 mts, it should be neutralized with N/10 Hcl, or N/10 NaOH.
  • To preserve the morphology of the leptospires for several days, 20 ml of midstream urine should be added immediately to 1.5ml of 10% formalin.
  • By adding formalin, the leptospires will be killed but will retain their morphology for several days and can be examined by dark field microscopy.
  • Kidneys for both darkfield microscopy and culture. Kidneys/or liver sections in 10% formalin for histopathology.
  • Foetal kidney smears (cryostat sections or smears) for FAT. Foetal abomasal contents, cotyledons and uterine discharge for differential diagnosis.
  • CSF and semen from bull can also be useful for demonstration of leptospires.

Direct microscopy

  • Leptospires can be demonstrated in urine, other body fluids, and tissues by darkfield microscopy (DFM) and by FAT.
  • To detect under DFM, one probably needs 10,000 to 20,000 leptospires/ml in the sample to view atleast one Leptospira in the high power field.
  • Urine is centrifuged to concentrate the leptospires. Unclotted blood is centrifuged at low speed to sediment the RBC after which the plasma can be removed and centrifuged at high speed.

Isolation in culture media

  • After inoculating the suitable media incubate at 30ºC for upto 8 weeks. A drop of the culture is examined by DFM.

Animal inoculation

  • Guineapigs, hamsters and weaning gerbils can be inoculated i/p with 0.5 to 1 ml of neutralized urine, unclotted blood or a 10% tissue suspension in EMJH or 1% BSA.
  • Cardiac blood is taken aseptically when a temperature rise is detected or at 5,8,10 and 14 days post infection.
  • Media are inoculated with 2-3 drops of the freshly collected blood.

By serology

  • Macroscopic agglutination test: it is a screening test and uses dead antigen (lack of specificity).
  • Microscopic agglutination test (MAT): it uses live leptospires as antigen and is highly sensitive and serovar specific.
  • CFTand ELISA are also useful for detection of leptospiral antibodies in serum.
  • To identify the serovar, MAT, restriction endonucleae, DNA analysis and monoclonal antibodies are useful.
Last modified: Monday, 4 June 2012, 5:42 AM