Volume 1. The Amoebae 

Volume 1. The Amoebae

Parasitology is the study of parasites and as such does not include bacterial, fungal or viral parasites. Human parasites are separated into intestinal and blood borne parasites. For a parasite to be defined as intestinal it must have an intestinal life cycle stage, though it may have life-cycle stages in the heart, circulation, lung, tissue, other animals or the environment.

Parasites found in the intestines can be categorised into two groups: Protozoa and Helminths.

Protozoa are single celled organisms. There are four classes of Protozoa commonly found in concentrated faecal samples. These are differentiated by the method of motility. Protozoa include Entamoeba, Giardia, Trichomonas, Cryptosporidium, Isospora, Pneumocystis and Balantidium. There are two diagnostic life-cycle stages commonly seen in parasites - the cyst and the adult trophozoite stage. The trophozoite stage is analysed directly on a slide without concentration. Cysts require concentration. The key diagnostic factor is that Protozoan cysts are typically 5-30 microns in diameter, and as such are smaller than most Helminth eggs. Due to the size they are particularly difficult to see under the microscope if the sample clarity is bad.

The medically important Helminths are nematodes (roundworms), cestodes (tapeworms) and trematodes (flukes). Genera include: Fasciola, Schistosoma, Ascaris, Hookworm, Trichuris, Taenia and Enterobius. The normal stage for examination is the egg stage, although larvae may develop in some organisms (Strongyloides). The diameter of the eggs range from 30-150 microns.

The other major grouping of parasites are known as blood-borne parasites where they are transmitted through an arthropod vector. By far the most important arthropod for transmitting parasitic infections are the mosquitoes. Mosquitoes are known to carry malaria and filarial nematodes. Different types of biting flies transmit African trypanosomiasis, leishmaniasis and several kinds of filariasis.

Most protozoan and helminthic infections that are transmitted by arthropods can readily be diagnosed, on clinical grounds alone, but are usually identified by fairly simple techniques designed to present the presence of the causative parasite by microscopy. Sophisticated techniques are also being employed including highly sensitive and specific simple monoclonal antibody tests, DNA probes and PCR primers.

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1.2 Infections acquired through the Gastrointestinal Tract

Many of the infections of the Gastrointestinal tract (GI) are caused by parasites that are cosmopolitan in distribution. Protozoa can be directly infectious for man when they are passed in the faeces into the environment, but helminths require a period of maturation whilst in the soil, where they become infectious. Others such as Taenia saginata require the involvement of an intermediate host during their life cycle.

Infections of the GI tract account for a high proportion of deaths in infants where the standards of hygiene and nutrition are low.

Faecal-oral transmission of the pathogens is the most common mode of GI infections, whereby water, food and hands become contaminated with faecal material which then come in contact with the mouth.

A number of GI infections can reach epidemic proportion, protozoal pathogen Cryptosporidium parvum, has been known to cause the severe water-borne epidemics, even in 1^st world countries such as the United States and the UK. Other infections such as amoebiasis or enterobiasis can be more localised, infecting households or institutions.

Some of the rarer, protozoal infections such as the microsporidia are only now being understood as they are appearing as concomitant infections in people with depressed immune responsiveness, e.g. AIDS.

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Amoebae are characterised by possessing clear protoplasm which form pseudopodia. These pseudopodia are the means by which these organisms move and engulf bacteria and red blood cells for feeding purposes. The most common amoebae seen in the intestinal tract are Entamoeba histolytica / dispar,

Entamoeba coli, Entamoeba hartmanni, Endolimax nana and Iodamoeba

bütschlii. All but Entamoeba histolytica are thought to be non-pathogenic. The cysts can be identified in an ethyl acetate concentrate by the addition of iodine to reveal the characteristic inclusions and also by measuring the cyst using an eyepiece graticule. The trophozoites can be seen in a fresh saline preparation of the stool although accurate identification is on a permanently stained faecal smear.

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Entamoeba histolytica

Introduction

There are a large number of species of amoebae which parasitise the human intestinal tract. Of these Entamoeba histolytica / dispar is the only species found to be associated with intestinal disease. Although many people harbour this organism world wide, only about 10% develop clinically invasive disease thus the parasite has been shown to present as two very differing clinical presentations.

1. The commensal or non-invasive luminal form where the parasite causes no signs or symptoms of disease.
2. The pathogenic or invasive form where the parasite invades the intestinal mucosa and produces dysentery or amoebomas and may give rise to extra-intestinal lesions via the blood, mainly to the liver.

Sargeaunt and Williams (1978) conclusively proved that invasive and non-invasive strains of E. histolytica could be differentiated by isoenzyme electrophoresis and the application of molecular biology has confirmed the presence of two distinct species with the same morphological features. The pathogenic or invasive species has retained the name E. histolytica and the non-pathogenic, non-invasive species has been named E. dispar. 

Morphology of Trophozoites

The trophozoites of E. histolytica / dispar recovered from dysenteric stools exhibit ingested red blood cells and clear pseudopodia. Those of E. dispar will have no ingested red blood cells. They can be up to 60m in diameter and motility is rapid and unidirectional. On a permanently stained faecal smear e.g. Trichrome or Iron haematoxylin, the morphological features are more visible. When using Trichrome stain nuclei, chromidial bars, chromatin, red cells and bacteria stain red cytoplasm stains blue-green and background and yeasts stain green. The presence of a small centrally placed karyosome is clearly visible. With Iron haematoxylin, nuclear chromatin and the karyosome will be stained immensely black. The remainder will be varying shades of grey/black. (Fig 1)

Morphology of cysts

Cysts of E. histolytica / dispar are 10 - 15m in diameter and contain 1 - 4 nuclei. Chromatoid bodies are usually present in young cysts as elongated bars with bluntly rounded ends. Glycogen is usually diffuse, but in young cysts it is often present as a concentrated mass, staining reddish brown with iodine. (Fig 2)

Fig 1. Entamoeba histolytica trophozoites in a caecum biopsy, each with pseudopods. (<30µm)

Fig 2. Entamoeba histolytica cyst. Present with large nucleus and central (>30m ) Karyosome. Direct examination of bloody mucus stool. (> 30m m)

Clinical Disease

Amoebiasis is an infection usually caused by the pathogenic Entamoeba histolytica / dispar, and is commonly an infection of the colon. It has a world wide distribution where environmental sanitation is poor. The parasite may behave as a commensal (causing no harm to the host) or it may act as a parasite (harming the host). It is a disease of human beings, although some monkeys can become infected and the infection is then transmissable to humans.

Intestinal disease

Patients with intestinal disease may exhibit a number of symptoms including profuse diarrhoea with blood and mucus, fever and dehydration. Amoebic ulcers may develop in the large colon and can also be found in the rectal area. The ulcers are usually "flask shaped" with a small opening on the mucosal surface and a larger area below the surface. Fig 3 illustrates E. histolytica trophozoites in the intestine, resulting in amoebiasis.

Fig 3. Entamoeba histolytica trophozoite present in the intestine causing Intestinal amoebiasis. (HES stain). Note in the centre of the picture trophozoites stained red. The nucleus is situated on the right in the amoeba. The central karyosome and the nucleus membrane with its chromatin are distinct.

Hepatic Disease

Trophozoites are transported from the intestine to the liver and liver disease is characterised with abdominal pain, fever, hepatomegaly and tenderness. If the abscess ruptures, there is spreading to the brain, pericardium and other sites. If left untouched the abscess will grow normally until it reaches a surface where it can discharge, e.g. the skin, the peritoneum, the pleural cavity or the pericardium. The stretching of the liver is presumably the main source of the pain.

Microscopy

Where amoebic dysentery is suspected, the laboratory should be informed that a "hot stool" is being supplied so that it can be examined within twenty minutes of being passed. On cooling the amoebae stop moving which then become very difficult to identify. Direct microscopy should be done by mixing a small amount of the specimen in 0.9% sodium chloride solution. This permits detection of motile trophozoites of Entamoeba histolytica / dispar and can also provide information on the content of the stool i.e. the presence of leucocytes and red blood cells. One search e.g. primarily for cysts, not for amoebae, several stool samples are required to be examined, by direct microscopy and a sensitive concentration technique. Three negative stool samples are required before is can be accepted that there is no amoebic infection. Microscopic examination of an amoebic abscess aspirate e.g. in the liver or lungs, may reveal haematophagous trophozoites. It must be examined immediately by mixing a drop of warm saline with some aspirated pus on a microscope slide.

Serology

If visceral or hepatic amoebiasis is suspected serological tests should be done as microscopic methods do not always reveal the characteristic trophozoites. The tests of choice are indirect fluorescent antibody test (IFAT), counter immunoelectrophoresis (CIEP) and enzyme linked immunosorbent assay (ELISA)

The search for E. histolytica / dispar is mainly carried out in Europe and North America, as there is a natural concern to ensure that patients, even in the absence of symptoms are not harbouring parasites that may lead to serious complications later on.

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Entamoeba coli

Introduction

Entamoeba coli is a non-pathogenic amoeba with world wide distribution. Its life cycle is similar to that of E. histolytica but it does not have an invasive stage and does not ingest red blood cells.

Morphology of Trophozoite

The trophozoite is larger than that of E. histolytica ranging from 15-50m in diameter. It exhibits blunt pseudopodia with sluggish movement. A permanently stained preparation shows a nucleus with a moderately large eccentric karyosome with the chromatin clumped on the nuclear membrane. The cytoplasm appears granular containing vacuoles with ingested bacteria and other food particles. (Fig 4)

Morphology of Cysts

Cysts of E. coli are 15 - 30m in diameter and contain 1 - 8 nuclei with irrecgular peripheral chromatin: karyosomes not central. Chromatoid bodies are not frequently seen but when present they are usually splinter-like with pointed ends. Glycogen is usually diffuse but in young cysts is occasionally found as a well-defined mass, which stains reddish brown with iodine. (Fig 5)

Laboratory Diagnosis

Laboratory diagnosis is made by finding the characteristic cysts in an iodine stained, formol-ether concentration method or by detecting the characteristic trophozoites in a wet preparation or a permanent stained preparation.

Fig 4. Entamoeba coli Trophozoite

Fig 5. Entamoeba coli cysts.The cell wall is not visible as it is a trophozoite, Number of cysts with distinct walls and so will be changing shape continuously nuclei. (18 - 30m m) (Junod technique) to form pseudopods. (17m m) (MIF Stain)

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Entamoeba hartmanni

Introduction

Entamoeba hartmanni is a non-pathogenic amoeba with world wide distribution. Its life cycle is similar to that of E. histolytica but it does not have an invasive stage and does not ingest red blood cells.

Morphology of trophozoites

Morphology of the trophozoites is similar to those of E. histolytica / dispar but they do not contain ingested red blood cells and the motility is less rapid. (Fig 6)

Morphology of cysts

Cysts of E. hartmanni 7-9m m in diameter and contain 1 - 4 nuclei. Chromatoid bodies are usually present in young cysts as elongated bars with bluntly rounded ends. Glycogen is usually diffuse, but in young cysts it is often present as a concentrated mass, staining reddish brown with iodine. (Fig 7)

Laboratory Diagnosis

Laboratory diagnosis is made by finding the characteristic cysts in an iodine stained, formol-ether concentration method or by detecting the characteristic trophozoites in a wet preparation or a permanent stained preparation.

Fig 6. Entamoeba hartmanni trophozoite

in stool smear.(Gomori-Wheatley stain). No distinct wall, cytoplasm is poorly coloured. . Distinct nucleus showing a central karyosome

and a thick chromatin.

Fig7. Entamoeba hartmanni cysts (Junod technique) 2 small cysts present (7µm) with distinct walls and vacuolizedcytoplasm.

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Introduction

Endolimax nana is a small non-pathogenic amoeba with world wide distribution. Its life cycle is similar to that of E. histolytica but is non-invasive.

Morphology of trophozoite

Trophozoites of E. nana measures from 6-12m m. Motility is sluggish with blunt hyalin pseudopodia. In a permanently stained preparation, the nucleus exhibits a large karyosome with no peripheral chromatin on the nuclear membrane. (Fig 8)

Morphology of cysts

Cysts of E. nana are 6 - 9m in diameter. They can be spherical or ovoid in shape and contain 4 pinpoint nuclei, which are highlighted by the addition of iodine. Chromatoid bodies are not found and glycogen is diffuse. (Fig 9)

Laboratory Diagnosis

Laboratory diagnosis is made by finding the characteristic cysts in an iodine stained, formol-ether concentration method or by detecting the characteristic trophozoites in a wet preparation or a permanent stained preparation.

Fig 8. Endolimax nana trophozoites in a stool smear (Gomori-Wheatley stain). (10µm). Nucleus is formed of a huge karyosome surrounded by a white halo.

Fig 9. Endolimax nana cysts (Junod   Technique) very small and round (oval). Nuclei grouped together at one end. (<10µm)

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Introduction

Iodamoeba bütschlii is a non-pathogenic amoeba with world wide distribution although not as common as E. coli or E. nana. Its life cycle is similar to that of E. histolytica but is non-invasive.

Morphology of trophozoites

Trophozoites of I. butschlii are 8 - 20m and are actively motile. On a permanently stained faecal smear, a nucleus with a large karyosome is evident. Chromatin bodies form striations around the karyosome. The cytoplasm appears granular containing vacuoles with ingested bacteria and debris. (Fig 10)

Morphology of cysts

Cysts of I. bütschlii are 9 - 15m in diameter and have one nucleus in mature cysts usually eccentrically placed. Chromatoid bodies are not present. Glycogen is present as a compact well defined mass staining dark brown with iodine. (Fig 11)

Laboratory Diagnosis

Laboratory diagnosis is made by finding the characteristic cysts in an iodine stained, formol-ether concentration method. Trophozoites are difficult to detect in a wet preparation.

Fig 10. Iodamoeba bütschlii trophozoite in a stool smear with 2 active trophozoites emitting a rounded refractive pseudopod. They can produce several pseudopods at the same time limiting their movement. A concentration technique will confirm that they are in fact I.bütschlii trophozoites.

Fig 11. Iodamoeba bütschlii cysts, easy to diagnose due to their size (13m m), the presence of a large well defined vacuole in the cytoplasm. By refocusing you can see  the karyosome which appears like a large black spot. These elements allow the diagnosis to be made.

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Blastocystis hominis

Introduction

B. hominis is an inhabitant of the human intestinal tract. It is capable of both sexual and asexual reproduction by binary fission and of pseudopod extension and retraction.

Morphology

The classic form that is usually seen in stool specimens varies in size from 6 - 40m m nd is characterised by a large membrane bound central body which occupies 90% of the cell. It has no internal nuclear structure and a rim of peripheral granules the function of which is not known. (Fig 12)

Clinical disease

The pathogenic potential of B. hominis is unclear, though this organism has been associated with nausea, fever, vomiting, diarrhoea and abdominal pain.

Diagnosis

Permanently stained preparations of faecal smears is the procedure of choice for identification, although the organism can be seen in wet preparations. The recommended stains are Fields’ and Giemsa.

Fig12. Cyst of Blastocystis hominis stained with Romanowski’s stain demonstrating its vacuolated cell wall.

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1.2 DIRECT FAECAL PARASITE CONCENTRATION METHODS

Diagnosis of intestinal parasites is confirmed by the recovery of protozoan trophozoites and cysts, helminth eggs and larvae in the clinical parasitology laboratory. Microscopic examination of faeces is essential for the recognition and identification of intestinal parasites. Due to the low density of the parasites in the faeces, direct microscopy is useful for the observation of motile protozoan trophozoites and the examination of cellular exudate, is not recommended solely for the routine examination of suspected parasitic infections. It is essential to increase the probability of finding the parasites in faecal samples to allow for an accurate diagnosis. Therefore, a concentration method is employed. (Direct wet mount examination should not be entirely excluded as the trophozoites are usually destroyed during the concentration procedure and therefore, microscopic examination of wet mounts should be performed).

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Ridley-Allen Method

The concentration procedure used in hospitals requires the use of ether or ethyl acetate as a lipid removing agent and formalin as a fixative. The process involves the use of either expensive brass seives or the use of tea strainers as the filter element.

Tea strainers have a very open pore structure of at least 600 micron and due to the shape of the strainer it is a non-linear pore size.

The faecal matter is filtered directly through these meshes in a dead stop manner, and hence there is the tendency for occlusion of the filter. There is also a formation of a secondary filter layer, which retains eggs and allows the extrusion of particles (particularly fibres) into the sediment. The net result is a reduction in egg yield and in sample clarity.

1. Using orange sticks, select a quantity of faeces (approx. 1g) to include external and internal portions.
2. Place in a centrifuge containing 7ml of 10% formalin.
3. Emulsify the faeces in the formalin and filter through the brass/plastic filter into the dish.
4. Wash the filter and discard any lumpy residue.
5. Transfer the filtrate to a boiling tube-add 3ml of ether and mix well on a vortex mixer for 15 seconds or mix by hand for 1 minute.
6. Transfer back to the centrifuge tube and centrifuge at 3,000 rpm for 1 min.
7. Loosen that fatty plug with an orange stick and pour the supernatant away by quickly inverting the tube.
8. Allow the fluid on the side of the tube to drain onto the deposit – mix well and transfer a drop to a slide for examination under a coverslip. (WHO Basic lab methods in Medical Parasitology)

The advantages of this method are that it will recover most ova, cysts and larvae and retain their morphology (thus facilitating identification). It has the disadvantage of destroying trophozoite stages and distorting cellular exudate. Liquid faeces do not concentrate well, thus it is necessary in these cases to examine the stool by direct microscopy. Since the sieves are not disposable there is a problem with cleaning for re-use. The system is also open so there is a biohazard and odour issue.

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Flotation method

This technique is predominantly used in veterinary laboratories. By exploiting the density of the parasites, particularly eggs, it allows the parasites to float to the top of a dense solution (final specific gravity of about 1.20) and can then be skimmed from the top of the tube. The most commonly used reagent is zinc sulphate. Operculated eggs as well as schistosome and infertile Ascaris eggs are not easily recovered by this method. Also trophozoites are killed due to the high specific gravity and certain other fragile eggs such as Hymenolepis nana become distorted.

1. Crush 10-20g (about 1 teaspoon) of faeces with applicator sticks and mix well with 10-12ml of saline. Filter the mixture through two layers of dampened surgical gauze into a 15ml conical centrifuge tube.
2. Centrifuge the suspension at 1500 rpm for 5 minutes. Decant the supernatant into disinfectant. Resuspend the sediment and recentrifuge in saline if there is excessive debris in the sample.
3. Resuspend and thoroughly mix the sediment in 12ml of zinc sulphate solution (specific gravity, 1.18 to 1.20, as verified with a hydrometer).
4. Centrifuge for 1 minute at 2500 rpm. Place tube in a rack in a vertical position and slowly add enough zinc sulphate with a dropper pipette to fill the tube so that an inverted meniscus forms.
5. Without shaking the tube, carefully place a 22 x 22 mm coverslip on top of the tube so that its underside rests on the meniscus. The meniscus should not be so high that fluid runs down the side of the tube carrying parasites away from the cover glass.
6. Allow the tube to stand vertically in a rack with the coverslip suspended on top for 10 minutes.
7. Carefully lift the coverslip with its hanging drop containing parasite eggs and cysts on the underside and mount on a clean slide, liquid side down. A small drop of iodine stain may be placed on the slide prior to adding the coverslip. The slide is gently rotated after adding the coverslip to ensure a uniform mixture. The slide is then thoroughly examined microscopically. (Clinical Laboratory procedures)

PARASEP    Ò  METHOD

Parasep Ò  is a commercial kit for faecal parasite concentration, it is a rapid, single use, disposable module for the clean and efficient concentration of helminth ova, protozoal cysts and oocysts. The methodology is a modification of the Ridley-Allen method.

The ParasepÒ incorporates into the device a patented filter thimble presenting a large surface area. The 3-dimensional structure prevents blocking of the filtration surface, therefore, large particles are prevented from passing through the filter and are held by the horizontal bars of the primary screen. The vertical orientation of the second screen means that the filtration is tangential, again preventing build up of rejected particles on the filter mesh. At the base of the filter thimble is a debris trap, where the rejected particles gather and hence are prevented from occluding the pores, (pore size 425m m) allowing for more ova and cysts to filter through. The large surface area retains more faecal exudate and hence yields very high clarity microscopic mounts. (Diagram 2)

Diagram 2. Parasep Ò  Faecal Parasite Concentrator product description

Parasep Ò Instructions for use

 Advantages of Parasep Ò , compared with conventional method

An in-house study was carried out where, the conventional Ridley-Allen method, was compared with the Parasep Ò , which is an enclosed, single use, disposable system. During the study the observations that they took into consideration included parasite recovery, the density of the deposit, ease of handling, health and safety aspects and cost.

100 faecal samples were carried out in duplicate by both techniques to allow for comparable results. The parasite recovery by both techniques was comparable. Although the Ridley-Allen technique is cheaper it is labour intensive and has inherent health and safety hazards.

The Parasep Ò however is:

Simple and has four rapid steps.

• Health and safety advantages over all other disposable units.
• Totally enclosed/sealed device preventing operator bio-exposure.
• Exploits a unique patented filter configuration.
• Reduced reagent volumes.
• No cleaning required.
• Single use, no sample contamination.
• Optimum sample recovery.
• Enhanced sample clarity/identification.
• Human resources optimised.
• Designed to fit all 50ml centrifuge buckets.

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1.3 References

Murray, PR, Drew, WL, Kobayashi, GS & Thomson, JH. Medical Microbiology. Mosby Books Inc., New York (1990)

Peters, W & Gilles, HM. Tropical Medicine & Parasitology. Wolfe Medical Publications Ltd.

Jeffrey & Leach.  Atlas of Medical Helminthology and Protozoology. E & S Livingstone Ltd.

Ash, LR & Orihel, TC.  Atlas of Human Parasitology. ASCP Press, Chicago.

Garcia, LS & Bruckner, DA. Diagnostic Medical Parasitology. Elsevior Science Publishing Co. Inc.

Muller, R & Baker, JR. Medical Parasitology. Gower Medical Publishing.

Snell, JJS, Farrell, ID & Roberts, C. Quality Control, Principles and Practice in the Microbiology Laboratory. Public Health Laboratory Service. ISBN 0 901 144 312.

World Health Organisation. Basic Laboratory Methods in Medical Parasitology. ISBN 92 4 154410 4. (1991)

Bridson, E. Vade-Mecum of Microbiology. Oxoid Ltd., Basingstoke, UK.

Kettlehut, M, Moody, A, Edwards, H & Chiodini, PL. Evaluation of the ParasepÒ (faecal parasite concentrator).

Pennell, DR et al. Advances in Giardia Research. p211-213. University of Calgary Press.

Samways, KL et al.  Assessment of Parasep Ò , a novel parasite egg retrieval system; use in faecal and waste water testing. Presented to Royal. Soc. Trop.Med.

Howells, H. A critical evaluation of methods available for diagnosis of Cryptosporidium parvum and Giardia lamblia in faecal samples. St Mary’s Hospital Portsmouth.

Deluol, AM.  Colour Atlas of Parasitology. Vol .1. Editions Varia, France (1998)

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© 2000       M. Arcari ¹, A. Baxendine¹ and C. E. Bennett  ²