2.1 Infections acquired through the Gastrointestinal Tract - Part 2  

In vertebrates, by far the most favourable sites for intestinal parasites are the duodenum, ileum, caecum and large intestine. To survive to reproduce in the gastrointestinal tract the parasites have to adapt to continuous physiological changes relative to the feeding habits of the host, the battery of protein, fat and carbohydrate-splitting enzymes, pH changes and the almost oxygen-free environment. Despite these features many parasites, both protozoa and helminths are capable of colonising the gastrointestinal tract successfully.

Protozoa are single-celled animals which resemble a single cell of a higher organism. However, the protozoan cell is capable of carrying out vital functions such as reproduction, feeding and locomotion. Intestinal protozoa include species which can live in the lumen of the intestine and others which additionally live and reproduce in the cells of the intestinal walls.

The protozoa make up a wide spectrum of organisms which have different life cycles and variable characteristics.

The Ciliates

The ciliates belong to the family Ciliophora. They possess simple cilia or compound ciliary organelles, 2 types of nuclei and a large contractile vacuole. The only member of the ciliate family to cause human disease is Balantidium coli.

Balantidium coli

Introduction

Balantidium coli is widely distributed in warmer climates, which is where human infections most commonly occur. The organisms inhabit the large intestine, caecum and terminal ileum where they feed on bacteria. The most common hosts being humans, pigs and rodents. Human infection is usually from pigs and is rare. Diagram 1 illustrates the life cycle of Balantidium coli.

Diagram 1. Life cycle of Balantidium coli. (Professor JH. Cross)

Morphology of cyst

The cyst is spherical or ellipsoid and measures from 30-200m m by 20-120m m. It contains 1 macro and 1 micronucleus. The cilia are present in young cysts and may be seen slowly rotating, but after prolonged encystment, the cilia disappear. (Diagram 2 & Fig 1) Cysts form when diarrhoea subsides and the rectal contents become formed. The cyst, ingested by a fresh host, excysts to liberate the trophozoite. Diagram 2 illustrates a B. coli cyst.

Diagram 2. Systematic diagram of a Balantidium coli cyst. 40 x 60m m (Dr. AJ. Frisby)

Morphology of trophozoite

Trophozoites of B. coli measure approximately 30-150m m in length x 25-120m m in width but have been known to attain lengths of up to 200m m. They are oval in shape and covered in short cilia. A funnel shaped cytosome can be seen near the anterior end. Multiplication is by binary fission in the trophozoite stage. In an unstained preparation, the organisms are easily recognised because of their size and rapid revolving rotation. In a stained preparation, the characteristic macro and micronuclei may be observed. (Diagram 3 & Fig 2)

Clinical Disease

Severe B.coli infections may resemble amoebiasis. Symptoms include diarrhoea, nausea, vomiting and anorexia. The diarrhoea may persist for long periods of time resulting in acute fluid loss. Balantidium coli also has the potential to penetrate the mucosa resulting in ulceration just as those of Entamoeba histolytica, but perforation is more common. Metastatic lesions do not occur. Extra-intestinal disease has also been reported, but is rare.

Laboratory Diagnosis

Wet preparations of fresh and concentrated stool samples reveal the characteristic cysts and motile trophozoites. (Fig 2) They are easier to identify in direct-smear saline preparations than permanently stained faecal smears.

                                                  Fig. 2 wet preparation of B.coli trophozoite

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The Coccidia

The Coccidia are a group of organisms which parasitise the epithelial cells of the intestinal tract. This group includes Cryptosporidium parvum, Cyclospora cayetanensis and Isospora belli. Most of the coccidian infections in man are zoonoses (having the potential to infect animals or arise from animals). In immunocompetent individuals, they usually produce mild, self-limiting infections.

Introduction

Cryptosporidium species, are coccidian protozoa, which are cosmopolitan in distribution, occurring in both developed and underdeveloped countries and causing infection in both humans and their live stock. Cryptosporidium parvum is the species responsible for human infection.

Life cycle and Morphology

Development of Cryptosporidia occurs in a parasitophorous vacuole located on the microvillous surface of the epithelial cells. Diagram 4 illustrates the life cycle of Cryptosporidia.

Diagram 4. Life cycle of Cryptosporidium sp. (www.dpd.cdc.gov)

Sporulated oocysts, containing 4 sporozoites, are excreted by the infected host through faeces (and possibly other routes such as respiratory secretions). Following ingestion (and possibly inhalation) by a suitable host, excystation (a) occurs. The sporozoites are released and parasitize epithelial cells (b, c) of the gastrointestinal tract (or other tissues such as the respiratory tract). In these cells the parasites undergo asexual multiplication (schizogony or merogony)(d, e, f) and then sexual multiplication (gametogony)(g). Upon fertilization of the macrogamonts (female) by the microgametes (male)(i), oocysts (j, k) develop that sporulate in the infected host, and are excreted. Because the oocysts sporulate inside the infected host, autoinfection can occur. Oocysts (measuring 4-5m m in diameter and containing 4 sporozoites) are infective upon excretion, thus permitting direct and immediate faecal-oral transmission. (Fig 3 & 4)

Clinical Disease

C. parvum is now widely recognised as a cause of acute gastro-enteritis, particularly in children. The infection produces a persistent, watery, offensive diarrhoea often accompanied by abdominal pain, nausea, vomiting and anorexia.

In immunocompetent persons, symptoms are usually short lived (1 to 2 weeks). The small intestine is the site most commonly affected, symptomatic Cryptosporidium infections have also been found in other organs including other digestive tract organs, the lungs, and possibly conjunctiva.

Fig 4. Cryptosporidium parvum oocysts (Acid-fast stain) The oocysts are variable in size (4-5µm) and in colour ranging from pale pink to vermillion red. The oocyst are easily identifiable. The slide background is blue-green even though the counter colour uses was malachite green. This is not unusual if the stools have been preserved in 10% formalin soultion (x825)

Cryptosporidiosis in immunocompromised individuals, especially in HIV patients, can be life threatening, as many as 10% may pass oocysts of C. parvum. Infections are characterised by the production of frequent, large volume watery stools and sometimes there is invasion of the pancreas, biliary or respiratory tract.

Oocyst excretion and symptoms may fluctuate during the course of infection. Asymptomatic infections are commonly found in developing countries with poor hygiene, where there is close contact with livestock.

Laboratory Diagnosis

Definitive diagnosis of cryptosporidiosis is by finding the characteristic spherical oocysts in faecal samples. They do not concentrate well using standard concentration techniques and are identified using various staining techniques.

Using the modified Ziehl-Neelsen staining method (fuschin followed by methylene blue), the oocysts are acid fast. (Fig 3) However, staining results within a smear and between specimens are diverse, varying from unstained to partial red staining and complete staining. Fully sporulated forms can be seen in which the red staining sporozoites are within an unstained oocyst wall. When staining the faecal smear with phenol-auramine/carbol-fuchsin, the oocysts appear as bright yellow discs with an "erythrocyte" pattern of staining against a dark red background.

Detection of the oocysts can also be achieved by using specific polyclonal or monoclonal antibodies conjugated to fluorescein. (Fig 5) These tests are now commercially available and offer a high degree of sensitivity. However, caution must be exercised when they are used to detect oocysts in the faecal smears distributed by NEQAS parasitology. Such specimens are preserved in formalin, which interferes with the fluorescent staining of the parasites, and are thus difficult to detect.

Oocysts in stool specimens (fresh or in storage media) remain infective for extended periods. Thus stool specimens should be preserved in 10% buffered formalin or sodium acetate-acetic acid-formalin (SAF) to render oocysts non-viable. (Contact time with formalin necessary to kill oocysts is not clear; we suggest at least 18 to 24 hours).

Fig 5. Staining of Cryptosporidium parvum oocysts in a stool smear with monoclonal antibodies conjugated to fluorescein. The Cryptosporidium oocysts appear with a peripheral green fluorescence. This technique could be of interest in epidemiological inquiries. (x 670)

Isospora belli

Introduction

Isospora belli is a coccidian protozoan of cosmopolitan distribution, occurring especially in warm regions of the world infecting both humans and animals.

Life cycle and morphology

The life cycle of I. belli involves an asexual (schizogonic stage) and a sexual (sporogonic stage). (Diagram 5)

Infection with I. belli occurs in both immunocompetent and immunocompromised patients and begins when the mature oocyst is ingested in water or food.

Morphology of oocysts The mature oocyst contains 2 sporocysts, each containing 4 sporozoites measure on average 35 x 9m m.

Diagram 5. Diagrammatic illustration of Isospora belli life cycle. (Adapted and redrawn from NCDC.)

Diagram 6 illustrates diagrammatically an immature and a mature oocyst with each sporocysts containing 4 sporozoites. Fig 6 and 7 demonstrate faecal smears of oocysts. The sporulated oocyst is the infective stage of the parasite and they excyst in the small intestine releasing sporozoites which penetrate the epithelial cells, thus initiating the asexual stage of the life cycle. The sporozoite develops in the epithelial cell to form a schizont, which ruptures the epithelial cell containing it, liberating merozoites into the lumen. These merozoites will then infect new epithelial cells and the process of asexual reproduction in the intestine proceeds. Some of the merozoites form macrogametes and microgametes (sexual stages) which fuse to form a zygote maturing finally to form an oocyst.

Diagram 6. Isospora belli immature oocysts showing a central individual mass of protoplasm. The mature oocysts illustrates the 2 sporocysts each containing 4 sporozoites. (10 x 16m m)

Fig 6. Isospora belli oocyst with 2 sporoblasts. (Imature oocyst) (Direct wet preparation without staining). The sporoblast divides itself into 2 identical round masses x990.

Fig 7. Isospora belli oocyst. with its elongated rugby ball shape. (28µm).The wall is smooth and refractive. One end is tapered and the other often shows a slight striction. The rounded granular sporoblast ocupies the central area, this is the most prominent feature. x660

Fig 8. Isospora belli mature oocyst. The oocyst keeps its elongated shape but the striction at one end is very pronounced. The sporoblasts are transformed into 2 sporocysts each containing 4 sporozoites and a residual body. (Direct wet preparation with no staining). (x660)

In the immunocompetent, infection is generally asymptomatic or a self-limiting gastro-enteritis. However, in chronic infections, severe non-bloody diarrhoea with cramp-like abdominal pain can last for weeks and result in fat malabsorption and weight loss. Eosinophilia may be present (atypical of other protozoal infections).

In immunocompromised individuals, infants and children, infection ranges from self-limiting enteritis to severe diarrhoeal illness resembling that of cryptosporidiosis.

Laboratory Diagnosis

Oocysts are thin walled, transparent and ovoid in shape. They can be demonstrated in faeces after a formal ether concentration where they appear as translucent, oval structures.

Alternatively, oocysts can be seen in a faecal smear stained by a modified Ziehl-Neelsen method, where they stain a granular red colour against a green background, or by phenol-auramine.

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Cyclospora cayetanensis

Introduction

Cycloclospora cayetanensis, a coccidian protozoan, has been described in association with diarrhoeal illness in various countries, in particular Nepal, Pakistan and India. Infection results in a disease with non-specific symptoms. Quite often the disease is the cause of unexplained summer diarrhoeal illness and similar illness following travel to tropical areas.

Life Cycle

The life cycle of this organism is unknown, however environmental data suggest that Cyclospora, like Cryptosporidium species, is a water-borne parasite. The oocysts of C. cayetanensis are spherical, measuring 8-10m m in diameter and the mature oocyst contains 2 sporocysts. Oocysts of Cyclospora cayetanensis, are twice as large in comparison with C. parvum and are not sporulated (do not contain sporocysts - upon excretion). Fig 9, 10 and 11 illustrate immature and mature oocysts of Cyclospora species

Fig 9 and 10. Cyclospora sp. immature oocysts. In the centre of each picture is a rounded body, 9µm in diameter. The outer wall is distinct and thick. Each body contains several refractive granules. (Direct wet preparation with no staining) (x660, x1650)

Clinical Disease

Patients from whose stools the organism has been isolated have reported nausea, vomiting, weight loss and explosive watery diarrhoea. Flatulence and bloatedness, nausea and vomiting, myalgia, low-grade fever, and fatigue are associated symptoms. The site of infection is the small bowel. The disease is usually self-limiting to three to four days but untreated infections can last from several days to a month or longer, and may follow a relapsing course. Some infections are asymptomatic.

Fig. 11. Cyclospora sp. oocyst. If the examination is not done immediately after stool emission some oocsts can start sporulation. You then observe globular bodies which vary in size and are very refractive. Thediagnosis is therefore very diffcult. (Direct wet preparation with no staining) (x1650).

Laboratory Diagnosis

The oocysts of C. cayetanensis are spherical as can be seen in formol-ether concentrated stool samples by light microscopy. They are refractile spheres which exhibit blue autofluorescence under ultraviolet light. It is important to note that UV microscopes set up for FITC and auramine microscopy only (450-500nm) will fail to detect the autofluorescence of the oocyst. Iodine-quartz microscopes do not produce UV wavelength below 400nm, while both mercury vapour and xenon vapour microscopes must be fitted with a 340-380nm excitation filter to demonstrate autofluorescence.

The oocysts are variably acid-fast when stained by the modified Ziehl-Neelsen method. Some cysts are acid-fast whereas others appear as round holes against a green background. They do not stain well with phenol-auramine.

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Microsporidia species

Introduction

The term microsporidia is also used as a general nomenclature for the obligate intracellular protozoan parasites belonging to the phylum Microsporidia. To date, more than 100 genera and 1,000 species have been described as parasites infecting a wide range of vertebrate and invertebrate hosts. There are at least 7 microsporidian species that are well characterised as human pathogens. (Table 1)

Microsporidia are characterised by the production of resistant spores and the polar tubule (or polar filament) which is coiled inside the spore as demonstrated by its ultrastructure.

They have recently come to medical attention as opportunistic pathogens in humans with Acquired Immune Deficiency Syndrome (AIDS) and have been implicated in conditions ranging from enteritis to keratoconjunctivitis.

Morphology and Life cycle

Microsporidia are primitive organisms. They possess no mitochondria and have prokaryotic like ribosomes. Classification is based on the ultrastructural features, which include the number of coils in the polar tubes, the configuration of nuclei and the spore size 1-4m m, depending on the species.

Diagram 7. The general intracellular cycle of Microsporidia.    

The spores are the infective stage of the organism (1), spores can survive for a long time in the environment. The spore hatches by extruding its polar tubule and infects the host cell (2). Infection occurs when the infective sporoplasm within the organism is injected into the host cell through the polar tube (3). Microsporidia multiply rapidly within the cytoplasm of the cell to produce sporoblasts (merogony) (4), followed by sporogony (5). This development can occur either in direct contact with the host cell cytoplasm (A) or inside a vacoule termed a parasitophorous vacoule (B). This results in the production of infective, in the case of Enterocytozoon and Encephalitozoon species thick walled spores which are released into the intestinal lumen. (6)

Table 1. Microsporidia found in humans and their associated disease.

In addition to the species in Table 1 above there are other, not well-characterised microsporidian human pathogens. These are designated as Microsporidum, a collective taxon, that includes Microsporidium africanum and Microsporidium ceylonensis.

Clinical disease

The most common microsporeans found in patients with AIDS are Enterocytozoon bieneusi, Encephalitozoon intestinalis and Encephalitozoon hellem. Patients with these infections tend to be severely immuno-deficient with a CD4 count less than 100 x 10⁶/L. Additionally, cases of microsporidiosis have been reported in immunocompromised persons not infected with HIV and in immunocompetent individuals. The clinical manifestations of microsporidiosis are very diverse, varying according to the causal species, with diarrhoea being the most common. (Table 1)

Enterocytozoon bieneusi

Infections with E. bieneusi are restricted to the enterocytes of the small intestine, resulting in villous atrophy and malabsorption. Clinical symptoms include chronic watery, non-bloody diarrhoea, malaise and weight loss.

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Encephalitozoon intestinalis

Infection with Encephalitozoon intestinalis occurs in the enterocytes of the small intestine but is more widely disseminated than E. bieneusi and has been found in the colon, liver and kidney.

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Encephalitozoon hellem   and  Encephalitozoon cuniculim

These organisms have also been found in disseminated microsporidiosis. Clinical symptoms may include sinusitis, nephritis, hepatitis, keratoconjunctivitis and peritonitis.

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Nosema corneum

This organism has been detected in AIDS patients with keratoconjunctivitis.

^{Fig 12. Microsporidia spore. (Electron microscopy). In this tiny, oval-shaped spore can be seen the polar filament rolled up in a number of coils, the exact number of which will vary according to the genus and species of the microsporidia under study.}

^{Fig. 13. Microsporidia spores in stool smear. (Modified Weber staining technique). The spores appear as minute bodies (1.5-2.0µm), oval shaped and coloured bright pink. An uncoloured vacuole can be seen in several of them. (x1650)}

Laboratory Diagnosis

Initially, the diagnosis of intestinal microsporidiosis depended on tissue biopsies which were stained with Gram’s stain and examined by light microscopy. However, in order that ill patients were not subjected to unnecessary invasive procedures, non-invasive diagnostic procedures were developed. The modified Trichrome stain and the Fungiqual fluorescent stain are the stains of choice. Immunofluorescence assays (IFA) using monoclonal and / or polyclonal antibodies are being developed for the identification of microsporidia in clinical samples.

2.6 Examination of faecal specimens for Parasites

Introduction

Many intestinal disorders are due to intestinal parasites which cannot be diagnosed symptomatically. Laboratory investigation is therefore required and the staff responsible should have adequate expertise in examining faecal specimens for parasitic organisms.

2.7 Relevant information required

The request form should always state the patients clinical symptoms and signs and whether the patient is resident in the UK, or had recent overseas travel. If the patient has had no recent history of overseas travel, examination for Cryptosporidium, Giardia and Microsporidia, if immunocompromised should be considered. If overseas travel has been undertaken, it is important to note is the patient ill or whether a routine post-tropical screen is requested. The Geographical location is also important as it may indicate these parasites which could be present.

2.8 Collection of samples

If a faecal sample is not properly collected and taken care of before examination, it will be of little or no value for accurate diagnosis. This is especially true if protozoa are present. Amoebic trophozoites begin to degenerate 1-2 hours after passage, as do flagellate trophozoites. Cysts will deteriorate if the faecal specimen is left standing for many hours or overnight, especially at high temperatures.

Helminth eggs and larvae are less affected by the age of the specimen than are protozoa. Nevertheless, changes may occur that could affect their identification e.g. hookworm larvae may become embryonated and larvae may hatch from the eggs risking confusion with Strongyloides larvae. Larvae themselves may disintegrate thus making their identification difficult.

To ensure that good specimens are provided for examination, it is important to note the following points.

1. A clean dry container must be used for the collection of faecal samples. Urine and water will destroy trophozoites, if present, and the presence of dirt also causes identification problems.
1. Ideally the specimen should be brought to the lab as soon as it is passed, to avoid deterioration of protozoa and alterations of the morphology of protozoa and helminths.
2. The specimen container should be clearly labelled with the patient’s name, date, and time of passage of the specimen.
3. An amount of stool adequate for parasite examination should be collected and a repeat sample requested if too little is supplied. The smallest quantity that should be accepted is about the size of a pigeon’s egg.
4. Diarrhoeal specimens, or those containing blood and mucus, should be examined promptly on arrival in the laboratory. The specimens may contain motile amoebic or flagellate trophozoites and may round up and thus be missed if examination is delayed. Where amoebic dysentry is suggested, the laboratory should be informed that a "hot stool" is being supplied so that it can be examined within twenty minutes of being passed.
5. With the exception of "hot stools" if specimens cannot be examined as soon as they arrive, they should be put in the refrigerator.

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2.9 Visual observation of the faecal sample

It is important to observe the macroscopic appearance of the stool as this can give a clue to the type of organisms present. Therefore the consistency; formed, unformed or liquid; the colour and the presence or absence of the exudate are reported. The presence of adult worms can also be seen in a freshly passed stool e.g. adult stage of Ascaris lumbricoides and Enterobius vermicularis. Proglottids of Taenia species can also be seen.

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1. Direct microscopy should be done on all unformed and liquid samples by mixing a small amount of the specimen in 0.9% sodium chloride solution. This permits detection of trophozoites of Entamoeba histolytica and Giardia lamblia. It can also provide information on the content of the stool i.e. the presence of leucocytes and red blood cells.
2. A formol-ether concentrate should be done on all faecal samples examined for parasites. This reveals the presence of most protozoan cysts, eggs of nematodes, cestodes and trematodes and also the larval stages of some nematodes.
3. A permanently stained direct faecal smear should be used for all bloody, liquid or semi-formed stools. The smear can reveal the presence of intestinal parasites that can be either destroyed or missed by the formol-ether concentration method e.g. Dientamoeba fragilis.
4. Specimens from patients with HIV should be left in 10% formalin for one hour before proceeding with parasite examination.

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2.11 Principals of diagnostic methods for the identification of parasites

The principal of the successful identification of faecal parasites is based upon,

1. Measurement - The use of an eyepiece graticule is of the utmost importance, especially for cyst identification.
2. Morphology - In protozoan cysts, the number of nuclei and the presence of inclusions e.g. glycogen mass and chromidial bar, aid the identification of protozoa. In trophozoites, the presence of red cells in amoebae is diagnostic of Entamoeba histolytica and flagella also aid identification of some protozoan trophozoites.
3. Appearance - In helminth eggs, the shape of the egg, the thickness of the shell, the colour of the ovum and the presence or absence of features such as an operculum, spine or hooklets are diagnostic pointers to the identity of the parasite.
4. Stains also aid in identification of the parasite.

The addition of iodine to formol ether concentrates highlights the internal

structure of cysts and helps distinguish between vegetable matter and

cysts. Permanently stained faecal smears are useful in demonstrating the

nuclear pattern of cysts.

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2.12 Problems of identification

Many things in stool specimens look like parasites but are not.

Epithelial cells and macrophages can be confused with amoebic trophozoites, especially macrophages that show slight amoeboid movement and may contain red blood cells. Pus cells can be confused with amoebic cysts. The nuclei appear as 3 or 4 rings and usually stain heavily. The cytoplasm is ragged and the cell membrane is often not seen. Amoebic cysts have a distinct cell wall.

Hair and fibres may be confused with larvae, but they do not have the same internal structure as larvae.

Plant cells can be confused with cysts or eggs. Though plant cells usually have a thick wall and cysts have a thin wall.

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2.13 Reporting of parasites

Ideally, the presence of all parasites should be reported, whether they are pathogens or non-pathogens. This particularly applies to the presence of cysts. However, if it is laboratory practice to report all cysts, the report should state whether they are pathogenic or non-pathogenic.

The stage of the parasite should always be reported. For the protozoa, whether cysts or trophozoites are present; the stage of larvae as in Strongyloides; and whether adult stages or eggs of helminths are present.

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References

Murray, PR, Drew, WL, Koyayashi, 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)

Brisdon, E: M, Moody, A, Edwards, H & Chiodini, PL: Evaluation of the Parasep® (faecal parasite concentrator).

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

Samways, KL et al: Assessments of ParasepÒ , 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 Cryptospridium parvum and Giardia lamblia in faecal samples. St Mary’s Hospital Portsmouth.

Juranek, DD. Cryptosporidiosis. In: Hunter’s Tropical Medicine. 8^th edition, Strickland, GT (editor).

Brown, VC. A Longitudinal study of the prevalence of intestinal helminths in baboons (Papio doguera) from Tanzania. (1994) Thesis, Liverpool.

Acknowledgements

We would like to thank the authors of the following web sites:

www.cdfound.to.it/atlas.htm

www.aisr.li.tju.edu,

www.medlib.med.utah.eduparasitology

www.ferris.edu

www.jeffline.tju.edu,

www.dpd.cdc.gov

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