Kato-Thick and Kato-Katz Smear

• Kato and Miura introduced the "cellophane thick smear technique"


  1954
• Cellophane thick smear technique

  Principle of direct fecal sampling
• The Kato thick technique was adopted in control program in Japan
• Difference between DFS and Kato-thick/Kato-Katz

  • Larger amount of fecal sample is employed
  • Cellophane strips are used as cover slips instead of glass
• Kato-Thick Smear

  • Simple and economical
  • Useful in mass stool examination of STH (soil-transmitted helminths)
  • Eggs with thick shells are very visible (Ascaris and Trichuris) but not eggs with thin shells (e.g. Hookworm)
  • Not useful for diarrhea or watery sample; cellophane as cover is not suitable for the watery sample
  • Not used for detecting larvae
  • Used for eggs of common STH but not for the detection of protozoan cysts/trophozoites and not for watery samples
• Materials and Reagents for Kato-Thick Smear

  • Stool sample - dry, soft to formed
  • Scrap paper, newspaper
  • Wooden applicator . tongue depressor
  • Glass slides
  • Rubber stopper
  • Microscope
  • Plastic spatula
  • Hydrophilic cellophane (25x30 mm or 25x35mm)
  • Forceps
  • Glycerol-Malachite green solution or Glycerol-Methylene blue solution
• Glycerin

  Used as clearing solution for the stool; enhances the settlement of parasites
• Malachite Green


  Used to give color to cellophane to give a pale green background to the eggs and minimize the brightness of the microscopic field
• Procedure for Kato-Thick Smear

  1. Label slide properly and place approximately 50-60 mg of stool (size of a soybean) at the center of a glass slide

  2. Cover the stool with pre-treated cellophane

  3. With the aid of a rubber stopper, press the cellophane gently to spread the stool specimen evenly to the circumference of the stopper

  4. Leave the prepared slide for 10-20 minutes to allow the field to become clear due to the action of glycerin

  5. Examine the smear under microscope - start with LPO and then HPO
• Do not allow slides to stand for too long = hookworm eggs has thin shell; will not visible after 30-60 minutes (10-20 minutes is recommended)
• Kato-Katz Smear

  • Egg counting procedure that aids in determining the intensity of infection with Schistosoma and common STH such as Ascaris, Trichuris, and Hookworm
  • Quantitative procedure
  • Also serves as a tool for assessment of the efficacy of anti-helminthic drugs
  • Specific for fresh, formed, and preferably drier stool samples
  • Main determinant for sensitivity: consistency of stool (fresh stool)
  • Not preferred for liquid-preserved sample
  • Dry sample - yields higher egg count than moist or wet stools
  • A. lumbricoides and T. trichiura eggs will remain visible and recognizable for many months
  • Schistosoma spp. may be recognizable for up to several months, it is best observed within 24 hours from smear preparation in endemic areas
• Materials and Reagents for Kato-Katz Smear

  • Stool sample - dry, soft to formed
  • Scrap paper, newspaper
  • Wooden applicator . tongue depressor
  • Glass slides
  • Rubber stopper
  • Microscope
  • Plastic spatula
  • Hydrophilic cellophane (25x30 mm or 25x35mm)
  • Forceps
  • Glycerol-Malachite green solution or Glycerol-Methylene blue solution
  • Mesh (stainless steel, nylon, or plastic: 60-105 micrometer)
  • Template (stainless steel, plastic, or cardboard)
• Template sizes

  • 1 mm thick template with a 9 mm hole = 50 mg of feces
  • 1.5 mm thick template with a 6 mm hole = 41.7 mg of feces
  • 0.5 mm thick template with a 6.5 mm hole = 20 mg of feces
• The same size of template should always be used to ensure repeatability and comparability of prevalence or intensity of infection
• Procedure for Kato-Katz Smear

  1. Place a small amount of fecal sample on a newspaper and press a piece of nylon screen (mesh) on top. Using a spatula, scrape the sieved material from the screen

  2. Label a glass slide with the sample number and place a template with a hole on the center of a microscope slide

  3. Fill the hole in the template with the fecal material, avoiding air bubbles and leveling the feces off to remove any excess material

  4. Carefully life the template and place it in a bucket of water mixed with concentrated detergent and disinfectant so that it can be reused

  5. Place one piece of cellophane, which has been soaked overnight in glycerol solution, over the fecal sample

  6. Invert the microscope slide and firmly press the sample against the cellophane strip on another microscope slide or on a smooth hard surface to spread the feces in a circle (rubber stopper)

  7. Carefully pick up the slide again by gently sliding it sideways to avoid separating the cellophane strip or lifting it off. Place the slide on the bench with the cellophane upwards. Water evaporates while glycerol clears the feces. When clarified it should be possible to read newspaper through the stool smear

  8. Examine the smear in a systematic manner - LPO and then HPO
• Clearing action can be easily made in two ways: (1) Placing in an incubator at 40 degrees C and (2) exposing to intense light (incandescent, fluorescent, and bright/direct sunlight)
• Egg Counting Procedure

  • May help correlate the severity of clinical disease with the intensity of infection or worm burden
  • Also done to assess the efficacy of anthelmintics and the reduction of worm burden following treatment
• Reporting and Interpretation

  1. The eggs of different species of helminths may be counted

  2. To compute the number of eggs per gram of feces (EPG), multiply the number of eggs seen in the whole smear with factor based on the type of template used

  3. Template with 9 mm hole: Number of eggs counted x 20

  4. Template with 6 mm hole: Number of eggs counted x 24

  5. Template with 6.5 mm hole: Number of eggs counted x 50
• Reporting and Interpretation Examples

  • 300 eggs seen and 9 mm hole used: 300 x 20 = 6,000 epg
  • 300 x 24 = 7,200 epg
  • 300 x 50 = 15,000 epg
  • 214 A. lumbricoides eggs using 6 mm hole: 214 x 24 = 5,136 epg = classification: moderate intensity
  • 214 T. trichiura eggs using 6 mm hole: 214 x 24 = 5,136 epg = classification: moderate intensity
  • 214 hookworm eggs using 6 mm hole: 214 x 24 = 5,136 epg = heavy intensity
  • 19 S. japonicum eggs using 9 mm: 19 x 20 = 380 epg = moderate intensity
• Identify all numerical values

  A) identify all the ff numerical values

  
• The egg count is expressed as EPG (eggs per gram).
• To calculate the EPG value, we need to know the size of the template used.