Stool analysis using concentration methods

Introduction

Diagnosis of intestinal parasite is confirmed by the recovery of protozoan, trophozoite and cyst, helminthes eggs and larvae in the chemical parasitology laboratory. Microscopic examination of faeces is essential for the recognition and identification of the intestinal parasites.

Due to the low density of the parasites in the faeces, direct microscopy is used for the observation of motile protozoan trophozoite and examination of cellular exudates (discharge) is not recommended solely for the routine examination of suspected parasite infections. It is essential to increase the probability of finding parasite in faecal samples to allow for an accurate diagnosis. Therefore a concentration method is employed.

Application/clinical significant of stool concentration techniques

• Diagnosis of digestive system infection disease; bacterial, parasites, virus and fungi.
• Diagnosis of pancreas disorders (inflammation) which associated with malabsorption of nutrients
• Primary screening test for some types of digestive system malignancy such as; colon cancer
• Primary screening test for peptic ulcer disease and some types of anaemia.

The following are the main tasks involve in the faecal sample analysis.

• Collection of faecal samples
• Separation of eggs/larva from faecal material and their concentration.
• Microscopical examination of prepared specimens.

It is important to understand the following basic limitation of faecal examination in the diagnosis of gastro-intestinal parasite.

• The demonstration of parasitic eggs or larvae in the faeces provides positive evidence that a man is infected but does not indicate the degree of an infection.
• The failure to demonstrate eggs or larvae does not necessarily mean that no parasites are present they may be present in an immature stage or the test used may not be sufficiently sensitive.
• There is generally no correlation between the number of eggs/larvae per gram of faeces and the numbers of adult nematodes present in man.

However, various factors can limit the accuracy and significance of a faecal egg count

• There is fairly regular fluctuation in faecal egg output.
• Eggs are not evenly distributed throughout the feaces.
• The quantity of faeces passed will affect the number of eggs per unit weight.
• The eggs output is influenced by the season of the spear (more in rainy seasons)
• Immature worms do not indicate their presence by producing eggs.
• The resistance of the host depress or entirely inhabit the eggs production of parasites.
• Immunity may result in a marked extension of the prepatient period and a lower eggs output by female parasite
• Eggs may not be detected due to low number of them or to low test sensitivity.

Collection of faecal samples

Fresh faecal samples may be collected from the subject and should be dispatched as soon as possible to the laboratory in a suitable container such as screw cap bottles, plastic containers with lid gloves.

If samples are to be dispatched, it should be in a cool box to avoid eggs developing and hatching. If prolonged in delay transport is expected, the following will help prevent development hatching.

• Fill the container to capacity to avoid air
• Add 3% formal-saline in the ratio of 1:3 ratio
• Store in the refrigerator at 4⁰C until they are process.

Direct method technique procedure

1. Place a drop of saline and iodine respectively at each end of a clean, dry, grease free slide
2. Using an applicator stick or wire loop, take a small amount of specimen and emulsify first in saline and in iodine make smooth this preparation.
3. Cover each preparation with a cover glass
4. Examine the saline preparation under microscope starting with x10 and x40 objectives for larva, cillate, eggs of helminthes, cyst and occyts and examine the iodine preparation for cysts.

Materials and method

A number of different methods are available for separation, concentration and demonstrating of eggs, cyst and larvae in faecal samples. Several methods are available.

The sedimentation techniques

Principle for using formal-ether

Faeces are emulsified in form of solution and suspension is strained to remove large faecal particles. Ether or ethyl acetate is added and the mixture is centrifuged. The parasites are concentrated at the bottom of the tube by gravitational or centrifugal force.

Materials

Centrifuge, applicator stick, microscope, centrifuge tube, gauze, cotton wool, swab, 10% formaldehyde, diethyl ether, glass slides, cover slip, normal saline and lugol’s iodine.

Procedure

• Place a small quantity about 1g of faeces in 10ml of 10% formol solution in a tube.
• Shake vigorously to mix.
• Filter with a wire sieve into a centrifuge tube
• Add 3-5ml of diethyl ether to the supernatant and mix vigorously.
• Centrifuge at 2000rpm for 5 minutes.
• Use a stick to loosen the layer of faecal debris from the side of the tube, invert the tube to discard the ether, faecal debris and the formal-solution.
• Tap the bottom of the tube to re-suspend the sediment.
• Place a drop of the sediment on a clean, grease free glass slide and place a cover glass.
• Examine microscopically.

Advantages

1. Cysts, certain helminth, eggs and larva are concentrated about 30 times.
2. All protozoa, eggs and larva are recovered
3. It is easy to perform in the side room of urban health care units
4. Ova and cysts are fixed in formal saline
5. It is particularly effective in the concentration of schistosome eggs.

Disadvantage

The preparation has more faecal debris than flotation method.

The floatation techniques

The floatation method is particularly used for the recovery of protozoan cysts and helminthes eggs such as the cysts of G. Lambia and E. Histolytica and the eggs of T. Trichuria. The parasites float on the surface of the liquid and the debris concentrate at the bottom of the tube.

Floatation method using zinc sulphate principle

Faeces are emulsified in the zinc sulphate solution and the suspension is left undisturbed for the eggs and cyst to float to the surface. The cysts and eggs are recovered with a cover glass from the surface of the suspension.

Materials

Microscope, applicator stick, glass slide, cover slip, normal saline, 33% zinc sulphate, test tube, centrifuge, cotton wool and gauze.

Procedure

1. Mix about a half teaspoon of stool (about 0.5g) with 1 to 2ml of normal saline in a test tube
2. Pass the faecal suspension through a strainer into a test tube to remove the large particles
3. Fill the tube with the zinc sulphate solution
4. Place a clean, dry, and grease free cover glass on top of the tube, avoid trapping air-bubbles.
5. Leave undisturbed for 35-40 minutes for the cysts and eggs to float.
6. Carefully remove the cover glass from the tube by a straight pull upwards. Place the cover glass face downwards on a slide containing a drop of iodine and examine microscopically.

Brine floatation method

The brine solution method is an in-expensive one, used significantly in the concentration of eggs of Hookworm or Ascaris, especially in field’s survey.

Materials

Saturated sodium chloride solution, stir excess amount of sodium chloride (NaCl), in a hot clean, distilled boil filtered water, place in a conical flask until a few gms of undisturbed salt remain at the bottom of the container. Mix well and leave the undisolved salt to sediment. Allow to cool before use, filter the solution; burner flame / bottle, test tube, cover slip, applicator stick, sieve, microscope.

Procedure

1. Place about 1g of faeces in a glass tube.
2. Add saturated salt solution till the tube is about ¼ filled.
3. Close the tube tightly and shake vigorously and add more saturated solution of salt (NaCl) until the tube is ¾ fill and shake vigorously again and place in the bench.
4. Using a pipette, add more salt solution carefully until the meniscus of the fluid is level with the mouth of the tube.
5. Allow the tube to stand for about 20 minutes and most of the eggs in the faecal sample will float on the surface of the fluid. The fluid must not over flow or else, all or some of the eggs may be lost.
6. Place a slide horizontally over the tube with the surface touching the meniscus of the fluid.
7. Lift the slide immediately and turn over. Place a cover slip over the fluid on the slide and examine as a saline and iodine mount.

Advantages

1. Cheap to perform
2. Easy to perform
3. It concentrates nematodes ova well.

Disadvantages

1. It does not concentrate cysts
2. Delay in examination can result in distortion
3. Larvae and some flukes eggs do not concentrate
4. Frequent checking of specific gravity

Beermann techniques

This technique depends on the migration of active larvae out of the faecal material through a wire guaze. The larvae enter the water where they settle out. The fluid can be then be removed, centrifuged and the deposit examined for larvae.

Materials

Microscope, centrifuge tube, centrifuge, funnel, gauze, cotton wool, glass slide.

Procedure

1. Attach a rubber-tubing with a pinch clamp to the bottom of a funnel.
2. Fix the funnel on a stand and place a beaker or flask under the stem of the funnel.
3. Fill the funnel with luke-warm (37-40 ^oC) water
4. Place about 5g of fresh faeces at the bottom of a wire gauze strainer and keep it over the funnel in such a way that it touches the water in the funnel.
5. Allow the apparatus to stand undisturbed for at least two hours, so that the larvae migrate from the faeces into the water.
6. Drain off about 10ml of the fluid from the funnel by releasing the pinch clamp.
7. Centrifuge at 500g for one minute
8. Examine the sediment for larvae.

Kato-katze Technique

It is used for recovery of schistosomes eggs in faeces. It is a semi-quantitative method used mostly for field survey.

Principle

When faeces are pressed through a mesh screen, it removes large particles; the rest of sieve sample is transferred to a template. This is then covered with a piece of cellophane soaked in glycerol which clears the faecal materials around the eggs which are counted and calculated per gram of faeces.

Materials

Template with hole, cellophane as cover slip, soaked in glycerol-malachite-green solution, screen, nylon plastic spatula, gloves, newspaper or glazed tile, microscope slides.

Procedure

1. Prepare the layer, newspaper or glazed tile
2. Place the template with hole in the centre of a slide.
3. Place a small faecal material on the newspaper
4. Press the screen on top so that a faeces filters through.
5. Collect the faeces across the upper surface with spatula.
6. Add the collected faeces in the hole of the template so that it is completely filled.
7. Remove the template carefully so that the cylinder of faeces is left on the slide.
8. Cover the faecal material with the pre-soaked cellophane strip
9. Invert the microscope slide and press firmly the faecal sample against the cellophane strip to spread evenly.
10. Carefully remove the slide by gently sliding it sideways.
11. Place the slide with cellophane upwards
12. Examine systematically and report the parasite seen.

Advantages

1. Kato-katze is used for the recovery of schistomas eggs in faeces
2. It is used for field survey.

Disadvantages

1. It is less sensitive
2. It is not suitable for fluid/hard specimens
3. It can alter the morphological appearance of eggs
4. It is less safe and hygienic.

Clinical importance of concentration technique in laboratory over direct method

• It is necessary for the detection of faecal parasites in light infection in addition to wet preparation. This is because some parasites such as the larvae of strongyloides, the eggs of tenia, cysts of G. lambia, and small parasites may be missed in direct wet preparation.
• Cysts, certain helminthes, eggs and larva are concentrated about 30 times.
• All protozoa, eggs and larva are recovered.
• It is particularly effective in the concentration of schistosome eggs.

Conclusion

Concentration methods in parasitology depends on several reason but one of the widely acceptable reason for its use is that they are 25% more sensitive than direct smears. Concentration method help to recover all cyst, trophozoite of protozoa and egg or ova of heminth. It is therefore important to make one of the concentration methods a routine use in the laboratory.

References

Adeoye A. (2007): A textbook for medical laboratory practice

Bowman’s Georgi Parasitology for veterinaries ovassay plus symbiotic corp. publishers and Diego (App 100-126)

Manual of basic technique for health laboratory (WHO) World Health Organisation publication. England pp. 162-170.

Cheesebrough, M. (2005). District laboratory practice London: Cambridge University press.

Ochei J. and Kolhatkar, A. (2007). Medical laboratory Science theory and practice. Tata MCGraw Hill Publishing Company Limited.

Piekarski, G. (2005). A textbook of medical parasitology. Feirbenfabriken Bayer, AG. Leverkusan Germay publisher pp. 163-172.