AMir's back...and its week 12!!!

How is everyone doing?.. wah 3 months of attachment already..thats fast.. 2 more months and we can meet up already..ok what shall i say about this week.. hmmmmmm


PARASITOLOGY!!


For last week I was posted at parasite lab.. In this lab, they mainly deal with protozoans and helminths(i hope you guys remember what we learn in BMic).. if not nvm i shall give a brief intro...


Parasite are organisms that live on or in a host organism, usually causing it some harm. They are relatively smaller in size and are dependent on their host for nourishment. There are many forms of parasites like, arthropod parasites (ticks), plant parasites (mistletoe), single-celled protozoan (amoebas) and even viruses. Numerous parasites live in the gastrointestinal tract, known as intestinal parasites which are common human pathogens. Parasite are subdivided into protozoans (unicellular) and helminths ova/larva (multicellular)


Eg of protozoans include: Amoeba histolytica, Giardia lamblia, Cryptosporidium
Eg of helminths include: Roundworms (Ascaris lumbricoides), tapeworm, flatworms

Parasite infections in Singapore is not as common as bacterial infections. So, we do not recieve much specimens. There are many different tests carried out: Ova and Parasite Exam, Silver stain for Pneumocystis jirovecii (PCP), leukocytes examination and occult blood. For this blog i will focus more on Ova and Parasite exam


Ova & Parasite exam

Ova and parasite exam is basically analysis of stool to check for the presence of a parasite or worm-like infection of the intestine. Most of the specimens, or i shall say ALL of the specimens are stool specimens, as intestinal parasites there will be released in the stool of infected humans. There will be a large population of parasites in the stool, there it will be easier to detect the presence of this parasites.

Principle

• Ova and parasite exam is the most common performed procedure in diagnostic parasitology
• For identification of intestinal protozoan parasites, it is based on the recognition of their cyst or trophozoites stages or even both
• Cysts are spherical with smooth uniform walls formed during the dormant resting stage
• Trophozoites have thin limiting membrane with variations in size and shape, they are formed during active feeding stage
  
  
image taken from: http://www.stanford.edu/group/parasites/ParaSites2006/Giardiasis/images/Giardia_LifeCycle%20cdc.gif



• For Helminthic infections, it is based on identification of eggs, larvae or proglottids in faeces
• Eggs or ova are usually found in stool specimen as it is passed out in faeces
• Ova and parasite exam consists of 3 separate protocols: Direct wet mount, Concentration and Permanent stained smear
• Direct wet mount allows detection of motile protozoans tryphozoites and helminth worms
• Concentration is designed to facilitate recovery of protozoan cysts, coccidian oocysts, microsporidial spores, and helminth eggs and larvae.
• Formalin-ethyl acetate sedimentation method is used to prepare concentrated specimen for direct wet mount.
• Permanent stained smear is used to identify intestinal protozoa, using the common Trichrome stain method
• It is the most important procedure as it aids in the confirmation of any suspicious objects seen in the direct wet mount

Materials

• Para-Pak® ULTRA Zn-PVA fixative


• Para-Pak® Macro-Con® Kit
  - Surfactant bottle (30ml)
  - Conical 50ml centrifuge tubes with filtration units and screw caps


• Disposable ice-cream stick and applicator sticks


• Disposable Pasteur’s pipette


• Paper towel


• Microscopic slides


• Timer


• 10% Formalin


• Ethyl acetate (Clearance)
  
• Disposable cotton swabs
  
• Normal saline


• Trichrome stain
  · For destaining: 10% acetic acid in water, 50% and 70% ethyl alcohol

Procedures

A. Preparation of material for Trichrome staining

1. Using a disposable ice-cream stick, place fresh faeces into the PVA fixative in the
Slides ready to be stained ratio of 3 parts PVA fixatives to 1 part faeces.
2. Mix thoroughly so that it appears homogenous
3. Allow the faeces to fix for a minimum of 30mins; overnight fixation is acceptable
4. Pipette out some of the well-mixed PVA-faecal mixture onto a paper towel using a disposable Pasteur’s pipette and allow to stand for 3mins to absorb out the excess PVA (This step is critical to obtain the best possible stain)
5. With an applicator stick, apply some of the faeces from the paper towel onto a clean slide (Spread the material onto the edge of the slide for better adherence)
6. Dry the slides for 3 hours or overnight at room temperature (Slide must be dried thoroughly to prevent material from being washed off during staining)

B. Concentration procedure
1. Add 10 drops of surfactant into the ParaPak specimen vial
2. Insert the filtration tightly into the specimen vial
3. Invert assembled unit, tapping sharply to force the solution into the conical tube
4. Unscrew the filtration unit and discard appropriately
5. Add 10% Formalin to the dotted line, plus 5 ml of Ethyl Acetate (Clearance)
6. Tightly screw the cap and shake vigorously for 1min
7. Centrifuge at 1500xg for 10mins
8. After centrifugation the specimen is clearly separated into four layers
9. Using an applicator stick, rim the debris layer. Pour off the debris and supernatant fluid, leaving the sediment
10. With the tube still inverted, use a cotton swab to clean and remove the remaining debris
11. Return the tube to upright position and add a few drops of saline to mix the sediment

C. Wet Mount
1. Using an applicator stick, prepare a coverslip preparation from the sediment
2. Examine microscopically

D. Trichrome Staining
1. Place slide in Trichrome stain solution for 10mins
2. Destain by placing the slide in acidified alcohol for 1-3sec (Do not allow slides to remain in this solution)
3. Dip slide several times in 100% ethyl alcohol
4. Place slide in 2 additional changes of 100% ethyl alcohol for 5mins each
5. Place slide in 2 changes of xylene substitute for 5mins each
6. Mount in immersion oil, using a No.1 thickness cover glass
7. Examine microscopically

Conclusion



In Trichrome stain, cytoplasm of cysts and trophozoites will stain blue-green tinged with purple. Parasite eggs and larvae usually stain red. All ova, pathogenic protozoan trophozoites or cysts seen must be reported. The reports will be sent to the doctors, and they will decide whether to do any further confirmatory tests depending on the patient's clinical symptoms. image taken from: http://www.med-chem.com/Para/prob%20of%20month/IMAGES/Giardia,%20trophs%20(2)%20in%20mucus%20string,fixed%20great.jpg

So, thats about all!!!.. I shall be back sooon!!.. take care peeps!!

AmiR


TG02